人脐带间充质干细胞生物学特性及脉冲电磁场促进其成骨分化研究
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摘要
间充质干细胞是一群中胚层来源的具有自我更新和多向分化潜能的多能干细胞。来自于胎儿期的间充质干细胞具有更强大的增殖能力、更广谱的分化潜能、更低的免疫原性等。有望作为组织工程或细胞替代治疗优良的种子细胞。
本实验从流产胎儿(胎龄12～18周)脐带沃顿胶中分离人脐带间充质干细胞,对其形态学、细胞周期、生长动力学、免疫表型及多分化潜能(向脂肪细胞、软骨细胞、成骨细胞等分化)等生物学特性进行研究,证实所分离的细胞为间充质干细胞。
以脉冲电磁场作为刺激hUCMSCs成骨分化的手段,比较脉冲电磁场方式和化学诱导方式对hUCMSCs分化为成骨细胞的速度、基因表达等方面的差异,明确PEMFs刺激hUCMSCs体外成骨分化能力;建立兔挠骨缺损模型,hUCMSCs与羟基磷灰石混合后注入兔挠骨缺损处,PEMFs每天刺激3h,观察骨缺损再生情况,探讨PEMFs促进hUCMSCs体内成骨分化能力。从而为骨组织工程和细胞替代治疗提供新的种子细胞来源;证实PEMFs促进hUCMSCs成骨分化的可行性。本实验研究结果为实现hUCMSCs的常规化分离培养提供了有价值的实验依据;为验证hUCMSCs具有多分化潜能提供有力依据;为证实PEMFs是促进hUCMSCs成骨分化的有效手段提供了实验依据;为PEMFS诱导hUCMSCs向成骨细胞分化的临床应用提供理论依据和技术支持。
Bone defection or bone fracture delaying to heal which caused by different reasons are a hard work to solution for orthopedics clinical workers. Developments of bone tissue engineering and regenerative medicine bring a new hope for the treatment to those diseases. human umbilical cord mesenchymal stem cells(hUCMSCs )are a promising source of stem cells for regenerative medicine and tissue engineering applications by virtue of their great capacity for self-renewal ,deficiency of immunogenicity and potential for differentiation into cells of various types of tissues, such as bone, cartilage, and adipose.
However, the methods of induction bone tissue engineering seed cells to osteoblasts adopted more chemical regents or growth factors inducement currently. The chemical regents or growth factors can’t work by stable density in the body. The growth factors in super concentration will intoxicate the cells. Exceed physiology quantity of inducement factors to osteogenesis will result in the heterotopic bone formation.
Pulsed electromagnetic field used of electric and magnetic forces to treat disease has fascinated the orthopedics scientists more than 30 years. PEMFs has obtained affirmation effects in many orthopedics diseases such as fractures and osteoporosis.
Making the PEMFs to be effective means that induces MSCs to osteoblasts will avoid the use of the chemical inducement that is not physiological. The mesenchymal stem cells are precursor cells of osteoblasts during restoration of bone injury. It has been proved that PEMFs can promote proliferation and differentiation of osteoblasts cell line by means of various frequencies. The biological characteristics of human umbilical cord mesenchymal stem cells are not comprehensive understanding as yet. There is no report on the influence of biological character to hUCMSCs after stimulus by PEMF at present. Whether PEMFs can accelerate differentiation to osteoblasts from hUCMSCs or not is awaiting further study.
The task one is to establish a suitable cell growth and expanded of hUCMSCs in vitro conditions, expounded its basal biological characteristics, identification to their adipogenic, osteogenic and Chondrogenic differentiation potential. The task two is to confirm PEMFs accelerating Osteogenesis in hUCMSCs and repairing bone defect with the compound of hUCMSCs and Hydroxyapatite .
1、Studies on the basic biological characteristics of hUCMSCs
Fresh human umbilical cord which were obtained after abortion and stored in balanced salt solution for 1-12hours before tissue processing to isolate cells. After removal of blood vessels, hUCMSCs were isolated off from the Wharton’s jelly with collagenase-Ⅱ(0.01%)and hyaluronidase (0.05%) for 12 hours at 37℃. Finally, the cells were washed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum in 5% CO2 in a 37℃incubator.Using flow cytometric analysis, we found that mesenchymal cells isolated from the Wharton’s jelly from the umbilical cord express mesenchymal stem cells markers CD29, CD44, CD73, CD105, CD166 but not hematopoietic lineage markers CD34, CD45, and not costimulatory molecules CD40, CD40L, CD80, CD86, HLA-DR is also negative expressed.DNA content analysis showed that cells were normal diploid cells, and cell cycle analysis showed that exponential phase of growth cells were over 80% in G0 to G1period.
2、Studies on cell differentiation potency of hUCMSCs
Currently mesenchymal stem cells have not highly recognized specific molecular marker, there are no more separation and cultured standard methods to get mesenchymal stem cells. These experiments aim to prove that the mesenchymal stem cells have the potency to differentiate into 3 cells types included osteocyte, chondrocyte, adipocyte.
(1)Adipogenic differentiation
hUCMSCs with adipogenic medium resulted in expanded cell morphology and a time-dependent increase in intracellular Oil Red O staining; an established lipid dye. When total RNA was isolated and analyzed by Real-time PCR, the adipocyte marker PPARγwas not expressed in untreated cells but was expressed in adipogenic formula-treated cells.
(2)Osteogenic differentiation
hUCMSCs with osteogenic medium (10-7mol/L dexame thasone, 50mg/L vitamin C, 10mmol/Lβ-glycerophosphate, 10% FBS , DMEM/F12) were Induced into osteogenic cells. To confirm osteogenesis, cells were examined by Real-time PCR for the expression of osteopontin genes after induction. Using Von Kossa staining and alizarin red staining detected the formation of calcium nodus.
(3)Chondrogenic differentiation
Chondrogenic differentiation of hUCMSCs was achieved by adding the chondrogenic medium with serum-free medium supplemented with ITS+10ng/ml of TGF-β. toluidine blue staining of an aggrecan-rich extracellular matrix was evident. A typeⅡcollagen-rich extracellular matrix was demonstrated by immunohistochemically, and collagenⅡgenes was detected by Real-time PCR.
3、Acceleration of Osteogenesis in hUCMSCs by PEMF Stimulation
The third generation of hUCMSCs are devided into 4 groups: No.1: The basal medium culture group; No.2 The basal medium culture combine with PEMFs group; No.3: The basal medium culture combine with chemical reagents group; No.4: The chemical reagents combine with PEMFs group. The production of alkaline Phosphatase(ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMFs treatment in both basal and chemical reagents cultures as compared to untreated controls. Furthermore, the expressions of other osteogenic genes, including Runx2 ,collagen-Ⅰand osteopontin were also modulated by PEMFs confirmed by Real-time PCR and western-blot. Our results indicate that extremely advisable frequency PEMFs stimulation may play a modulating role in hUCMSCs osteogenesis.
4、PEMFs accelerate repairing bone defect with the compound of hUCMSCs and Hydroxyapatite
First, we made the animal models of bilateral radius bone defect: we selected 40 New Zealand white rabbits. In the middle of bilateral radius, 1.5 cm bone defects were made. Randomly-selected 40 rabbits from the models of bilateral radius defects were implanted with the compound of hUCMSCs and HA or HA only. PEMFs stimulated the radius defects part 3 hours everyday. In the 2th, 4th, 8th and 12th weeks, all the rabbits had X-ray examination to observe the reparation of bone defect and growth of bone callus.
Conclusively, to our knowledge, this is the first study in the literature regarding the following aspects. (a) Isolation of hUCMSCs from the Wharton’s jelly which were obtained from abortion fetus in gestational age from 12 to 18 weeks with collagenase-Ⅱand hyaluronidase . (b) PEMFs accelerate Osteogenesis in hUCMSCs and repairing bone defect with the compound of hUCMSCs and Hydroxyapatite .The findings provide insights on the development of PEMFs as an effective technology for bone tissue engineering and regenerative medicine.
本实验从流产胎儿(胎龄12～18周)脐带沃顿胶中分离人脐带间充质干细胞,对其形态学、细胞周期、生长动力学、免疫表型及多分化潜能(向脂肪细胞、软骨细胞、成骨细胞等分化)等生物学特性进行研究,证实所分离的细胞为间充质干细胞。
以脉冲电磁场作为刺激hUCMSCs成骨分化的手段,比较脉冲电磁场方式和化学诱导方式对hUCMSCs分化为成骨细胞的速度、基因表达等方面的差异,明确PEMFs刺激hUCMSCs体外成骨分化能力;建立兔挠骨缺损模型,hUCMSCs与羟基磷灰石混合后注入兔挠骨缺损处,PEMFs每天刺激3h,观察骨缺损再生情况,探讨PEMFs促进hUCMSCs体内成骨分化能力。从而为骨组织工程和细胞替代治疗提供新的种子细胞来源;证实PEMFs促进hUCMSCs成骨分化的可行性。本实验研究结果为实现hUCMSCs的常规化分离培养提供了有价值的实验依据;为验证hUCMSCs具有多分化潜能提供有力依据;为证实PEMFs是促进hUCMSCs成骨分化的有效手段提供了实验依据;为PEMFS诱导hUCMSCs向成骨细胞分化的临床应用提供理论依据和技术支持。
Bone defection or bone fracture delaying to heal which caused by different reasons are a hard work to solution for orthopedics clinical workers. Developments of bone tissue engineering and regenerative medicine bring a new hope for the treatment to those diseases. human umbilical cord mesenchymal stem cells(hUCMSCs )are a promising source of stem cells for regenerative medicine and tissue engineering applications by virtue of their great capacity for self-renewal ,deficiency of immunogenicity and potential for differentiation into cells of various types of tissues, such as bone, cartilage, and adipose.
However, the methods of induction bone tissue engineering seed cells to osteoblasts adopted more chemical regents or growth factors inducement currently. The chemical regents or growth factors can’t work by stable density in the body. The growth factors in super concentration will intoxicate the cells. Exceed physiology quantity of inducement factors to osteogenesis will result in the heterotopic bone formation.
Pulsed electromagnetic field used of electric and magnetic forces to treat disease has fascinated the orthopedics scientists more than 30 years. PEMFs has obtained affirmation effects in many orthopedics diseases such as fractures and osteoporosis.
Making the PEMFs to be effective means that induces MSCs to osteoblasts will avoid the use of the chemical inducement that is not physiological. The mesenchymal stem cells are precursor cells of osteoblasts during restoration of bone injury. It has been proved that PEMFs can promote proliferation and differentiation of osteoblasts cell line by means of various frequencies. The biological characteristics of human umbilical cord mesenchymal stem cells are not comprehensive understanding as yet. There is no report on the influence of biological character to hUCMSCs after stimulus by PEMF at present. Whether PEMFs can accelerate differentiation to osteoblasts from hUCMSCs or not is awaiting further study.
The task one is to establish a suitable cell growth and expanded of hUCMSCs in vitro conditions, expounded its basal biological characteristics, identification to their adipogenic, osteogenic and Chondrogenic differentiation potential. The task two is to confirm PEMFs accelerating Osteogenesis in hUCMSCs and repairing bone defect with the compound of hUCMSCs and Hydroxyapatite .
1、Studies on the basic biological characteristics of hUCMSCs
Fresh human umbilical cord which were obtained after abortion and stored in balanced salt solution for 1-12hours before tissue processing to isolate cells. After removal of blood vessels, hUCMSCs were isolated off from the Wharton’s jelly with collagenase-Ⅱ(0.01%)and hyaluronidase (0.05%) for 12 hours at 37℃. Finally, the cells were washed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum in 5% CO2 in a 37℃incubator.Using flow cytometric analysis, we found that mesenchymal cells isolated from the Wharton’s jelly from the umbilical cord express mesenchymal stem cells markers CD29, CD44, CD73, CD105, CD166 but not hematopoietic lineage markers CD34, CD45, and not costimulatory molecules CD40, CD40L, CD80, CD86, HLA-DR is also negative expressed.DNA content analysis showed that cells were normal diploid cells, and cell cycle analysis showed that exponential phase of growth cells were over 80% in G0 to G1period.
2、Studies on cell differentiation potency of hUCMSCs
Currently mesenchymal stem cells have not highly recognized specific molecular marker, there are no more separation and cultured standard methods to get mesenchymal stem cells. These experiments aim to prove that the mesenchymal stem cells have the potency to differentiate into 3 cells types included osteocyte, chondrocyte, adipocyte.
(1)Adipogenic differentiation
hUCMSCs with adipogenic medium resulted in expanded cell morphology and a time-dependent increase in intracellular Oil Red O staining; an established lipid dye. When total RNA was isolated and analyzed by Real-time PCR, the adipocyte marker PPARγwas not expressed in untreated cells but was expressed in adipogenic formula-treated cells.
(2)Osteogenic differentiation
hUCMSCs with osteogenic medium (10-7mol/L dexame thasone, 50mg/L vitamin C, 10mmol/Lβ-glycerophosphate, 10% FBS , DMEM/F12) were Induced into osteogenic cells. To confirm osteogenesis, cells were examined by Real-time PCR for the expression of osteopontin genes after induction. Using Von Kossa staining and alizarin red staining detected the formation of calcium nodus.
(3)Chondrogenic differentiation
Chondrogenic differentiation of hUCMSCs was achieved by adding the chondrogenic medium with serum-free medium supplemented with ITS+10ng/ml of TGF-β. toluidine blue staining of an aggrecan-rich extracellular matrix was evident. A typeⅡcollagen-rich extracellular matrix was demonstrated by immunohistochemically, and collagenⅡgenes was detected by Real-time PCR.
3、Acceleration of Osteogenesis in hUCMSCs by PEMF Stimulation
The third generation of hUCMSCs are devided into 4 groups: No.1: The basal medium culture group; No.2 The basal medium culture combine with PEMFs group; No.3: The basal medium culture combine with chemical reagents group; No.4: The chemical reagents combine with PEMFs group. The production of alkaline Phosphatase(ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMFs treatment in both basal and chemical reagents cultures as compared to untreated controls. Furthermore, the expressions of other osteogenic genes, including Runx2 ,collagen-Ⅰand osteopontin were also modulated by PEMFs confirmed by Real-time PCR and western-blot. Our results indicate that extremely advisable frequency PEMFs stimulation may play a modulating role in hUCMSCs osteogenesis.
4、PEMFs accelerate repairing bone defect with the compound of hUCMSCs and Hydroxyapatite
First, we made the animal models of bilateral radius bone defect: we selected 40 New Zealand white rabbits. In the middle of bilateral radius, 1.5 cm bone defects were made. Randomly-selected 40 rabbits from the models of bilateral radius defects were implanted with the compound of hUCMSCs and HA or HA only. PEMFs stimulated the radius defects part 3 hours everyday. In the 2th, 4th, 8th and 12th weeks, all the rabbits had X-ray examination to observe the reparation of bone defect and growth of bone callus.
Conclusively, to our knowledge, this is the first study in the literature regarding the following aspects. (a) Isolation of hUCMSCs from the Wharton’s jelly which were obtained from abortion fetus in gestational age from 12 to 18 weeks with collagenase-Ⅱand hyaluronidase . (b) PEMFs accelerate Osteogenesis in hUCMSCs and repairing bone defect with the compound of hUCMSCs and Hydroxyapatite .The findings provide insights on the development of PEMFs as an effective technology for bone tissue engineering and regenerative medicine.
引文
[1] Robert Lanza. Essentials of Stem Cell Biology. 2005,1. Academic Press.
[2] Friedenstein A J, Gorskaja J F, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hhematopoietic organs. Exp Hematol, 1976,4(5):267-274.
[3] Reese JS, Koc ON, Gerson SL.Human mesenchymal stem cells provide stromal support for efficient CD34+ transduction[J].Hematother Stem Cell Res, 1999,8:515-523.
[4] Zuk PA, Zhu M, Mizuno H, et al. Multilieage cells from human adipose tissue:implications for cell based therapies[J]. Tissue Eng, 2001, 7(2): 211-228
[5] Toma JG, Akhavan M, Femandes KJ, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin[J]. Nat Cell Biol, 2001, 3(9): 778-784
[6] Tuli R, Tuli S, Nandi S, et al. Characterization of multipotential mesenchymal progenitor cells derived from human trabecular bone[J].Stem Cells, 2003, 21(60):681-693.
[7] Kwan Chul T,Jong-Won H, Young-Soo K, et al. Effects of human blood mesenchymal stem cells on cutaneous wound healing in lepr db mice[J]. Ann Plast Surg, 2010 Oct 13[Epub ahead of print]
[8] Yumi Fukuchi, Hideake Nakajima,Daisuke Sugiyama. Human placenta-derived cells have mesenchymal stem/progenitor cell potential[J]. Stem Cell, 2004, 22:649-658.
[9] Wexler SA, Donaldson C, Denning-Kendall P, et al. Adult bone marrow is a rich source of human mesenchymal stem cells but umbilical cord and mobilized adult blood are not. Br J Haematol, 2003,121(2):368-374.
[10] Coter D C, Class R, DiGirolanno C M, et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow[J]. Rroc Natl Acad Sci USA, 2000,97:3213-3218.
[11] Karen Bieback,Suanne Kern, Harald Kluter, et al. Critical Parameters for theisolation of mesenchymal stem cells from umbilical cord blood[J]. Stem Cells, 2004,22:625-634.
[12] Wagner W, Wein F, Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue,and umbilical cord blood[J]. Exp Hematol, 2005,33(11):1402-1406.
[13] Romanov YA, Darevskaya AN, Merzlikina NV, et al. Mesenchymal stem cells from human bone marrow and adipose tissue: isolation, characterization and differentiation potentialities[J]. Bull Exp Biol Med, 2005, 140(1):138-143.
[14] Deans R J, Moseley A B. Mesenchymal stem cells: biology and pothntial chlinical uses[J]. Exp Hematol, 2000,28(8):875-884.
[15] Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells[J]. Keio J Med, 2005, 54(3): 132-141
[16] Mastrogiacomo M, derubeis A R, Cancedda R. Bone and cartilage formation by skeletal muscle derived cells[J]. J Cell Physiol, 2005,204(2):594-603.
[17]丹尼尔R,马沙克理查德L,甘德大卫.戈特利布主编,刘景生等译.干细胞生物学.北京:化学工业出版社,2004:302-318.
[18] Pittenger M F, Mackay A M, Beck S C, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284:143-147.
[19] Toma C, Pittenger M F, cahill K S, et al. Human mesenchymal stem cells differentiate a cardiomyocte phenolcyte in thd adult murine heart. Circulation[J]. 2002, 5:93-98.
[20] Ozono S, Fujita T,Matsuo M,et a1.Co-treatment with basic fibroblast growth factor and 17 beta-estradiol in the presence of dexamethasone accelerates bone formation by rat bone marrow stromal cell culture[J].Nihon Hotetsu Shika Gakkai Zasshi,2008,52(3):366-374.
[21] Lin TY, Zhou GD,Wei X, et a1.Influence of transforming growth factor-betaI inducing time on chondrogenesis of bone marrow stromal cells (BMSCs):in vitro experiment with porcine BMSCs[J]. Zhong Hua Yi Xue Za Zhi,2007, 87(31):22 l 8-2222.
[22] Cotherstrom C, ringden O, tammik C, et al. Immunologic properties of human fetal mesenchymal stem cells[J]. Am J Ohstet Gynecol,2004, 190(1):239-245.
[23] Klyshnenkova E, Mosca JD, Zernetkina V, et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression[J]. J Biomed Sci, 2005, 12(1):47-57.
[24] Le Blanc K, Tammik C, Rosendahl K, et al. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells[J]. Exp Hematol, 2003,31:890-896.
[25] Devine SM, Cobbs C, Jennings M, et al. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates[J]. Blood,2003,101(8):2999-3001.
[26] Lee S T, Jang J H, Cheong J W, et al. Treatment of high-risk acute myelogenous leukaemia by myeloabative chemoradiotherapy followed by co-infusion of T cell-depleted haematopoietic stem cells and culture-expanded marrow mesenchymal stem cells from related donor with one fully mismatched human leucocyte antigen haplotype. Br J Haematol, 2002,118:1128-1131
[27] Koc O N, Gerson S L, Cooper B W, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.J Clin Oncol 2000,18:307-316.
[28] Koc O N, Day J Du. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy(MLD) and Hurler syndrome(MPS-IH). Bone Marrow Transplant, 2002,30:215-222.
[29]苗宗宁,潘宇红,祝建中,等.丝素蛋白支架材料复合骨髓间充质干细胞构建组织工程化软骨.中国组织工程研究与临床康复,2008,12(27):5243-5247.
[30] Award HA, Bulter DL, Biovin GP, et al. Autologous mesenchymal stem cells mediated repair of endon[J]. Tissue Engineering, 1999,5(3):267.
[31]周志玲,袁进国,刘英飞,等.自体骨髓干细胞移植治疗骨不连.中国临床康复,2006,10(29):22-23.
[32] Jiang X, Zhang Y, Liu B, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood,2005, 168(1-2):661-669.
[33] Koc O N, Gerson S L, Cooper B W, et al. Rapid hematopoietic recovery after coinfusion of autologousblood stem cells and culture expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000,18(2):307-316.
[34] Cheng L, Qasba P, v anguri P, et al. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34+ hematopoietic progenitor cells[J]. J Cell Physiol, 2000,184(1):58-69.
[35] Wu T, Levay Young B, Heuss N, et al. Inducing tolerance to MHC2 matched allogeneic islet grafts in diabetic NOD mice by simultaneous islet and bone marrow transplantation under non-irradiative and nonmyeloblative conditioning therapy. Transplantation, 2002,74(1):22-27.
[36]王亚平.干细胞衰老与疾病. 2009.北京.科学出版社.
[37] Concise Review: Human Umbilical Cord Stroma with Regard to the Source of Fetus-Derived Stem Cells[J]. Stem Cells,2007,24(11):2886-2895.
[38] Kobayashi K,Kubota T,Aso T.Study on myofibroblast differentiation in the stromal cells of Wharton’s jelly: expression and localization of alpha-smooth muscle actin[J].Early Hum Dev,1998,51(3):223-233.
[39] Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton’s jelly form neurons and glia[J] Stem Cells, 2003, 21(1):50-60.
[40] Romanov YA,Svintsitskaya V A, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord[J]. Stem Cells, 2003,21(1):105-110.
[41] Michell KE, Weiss ML, Mitchell BM, et al. Matrix clls from Wharton’s jelly form neurons and glia[J]. Stem Cells, 2003,21(1):50-60.
[42] Wang H S, Hung S C, Peng S T, et al. Mesenchymal stem cells in the wharton’s jelly of human umbilical cord[J]. Stem cells, 2004,22(7):1330-1337.
[43] Weiss ML, Medicetty S, Bledsoe AR, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of parkinson’s disease[J]. Stem Cells, 2006,24(3):781-792.
[44]吕璐璐,刘拥军,许贞书,等.脐带间充质干细胞的分离和生物学性状[J].福建医科大学学报, 2006,40(2):99-104.
[45] Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors[J]. Stem Cells, 2005,23(2): 220-229.
[46] Karahuseyinoglu S, Cinar O, Kilic E et al. Biology of stem cells in human umbilical cord stroma: In situ and in vitro surveys[J]. Stem Cells, 2007,25:319–331.
[47] Sarugaser R, Lickorish D, Baksh D et al. Human umbilical cord perivascular (HUCPV) cells: A source of mesenchymal progenitors. Stem Cells ,2005,23:220–229.
[48] Lund RD, Wang S, Lu B et al. Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease. Stem Cells ,2007,25:602– 611.
[49]张浪辉,刘拥军,吕璐璐,等.脐带间充质干细胞对异源性脐带血T淋巴细胞激活与增殖抑制作用[J].中国肿瘤生物治疗杂志,2006,13(3):191-195.
[50]马廉,崔冰琳,冯学永等.人脐带间充质干细胞的生物学特性及向神经样细胞分化的研究.中华儿科杂志,2006(7):513-517.
[51]周佳,沈尊理,金羽青,等.人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究.中华显微外科杂志,2009,6(32)210-212.
[52] Kadivar M, Khatami S, Mortazavi Y, et al. In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells[J]. Biochem Biophys Res Commun, 2006,340(2):639-647.
[53] Conconi MT, Burra P, Di Liddo R, et al. CD105(+) cells from Wharton’s jelly show in vitro and in vivo myogenic differentiative potential[J]. Int J Mol Med, 2006,18(6):1089-1096.
[54] Lee OK, Kuo TK, chen WM, et al. Isolation of mulipotent mesenchymal stem cells from umbilical cord blood[J]Blood, 2004,103(5):1669-1675.
[55] Reyes M, verfaillie C M. Characterization of multipotent adult progenitor cells, a subpobulation of mesenchymal stem cells[J]. Ann N Y Acad Sci, 2001,938:231-233.
[56] Fu YS, Cheng YC, Lin MY, et al. Conversion of human umbilical cord mesenchymal stem cells in wharton’s jelly to dopaminergic neurons in vitro: potential therapeutic application for parkinsonism[J]. Stem Cells,2006,24(1): 115-124.
[57]王爱红,何红燕,罗敏洁,等.人脐带间充质干细胞向胰岛样细胞的分化[J].中国组织工程研究与临床康复,2008,12(21):4102-4106.
[58] Friedman R, Betancur M, Boissel L,etal.Umbilical cord mesenchymal stem cells: adjuvants ofr human cell transplantation [J]. Biol Blood Marrow Transplant, 2007,13(12):1477-1486.
[59] Li CD, Zhang WY, Jiang XX, et al. Human-placenta-derived mesenchymal stem cells inhibit proliferation and function of allogeneic immune cells[J]. Cell Tissue Res,2007,330(3):437-446.
[60]漆佩静.脐带间充质干细胞抑制异种移植物抗宿主病的机制研究.清华大学医学院,博士学位论文.2009.
[61] Kim JW, Kim SY, Park SY, et al. Mesenchymal progenitor cells in the human umbilical cord [J].An Hematol, 2004,83(12):733-738.
[62]孙国栋,伍时县,李志忠.人脐带间充质干细胞分离培养及成骨分化原子力显微镜观察细胞膜表面的超微结构变化.中国组织工程研究与临床康复.2010,14(1):33-37.
[63] Schneider RK, Puellen A, Kramann R, et al. The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodeling in three-dimensional collagen scaffolds[J]. Biomaterials, 2010, 31(3):467-480.
[64] Rachakatla R s, Marini F, Weiss M L, et al. Development of human umbilicalcord matrix stem cell-based gene therapy for experimental lung tumors[J]. Cancer Gene Ther, 2007,14(10):828-835.
[65] Sharrard WJ.Adouble-blind trial of pulsed electromagnetic fields for delaye union of tibial fractures[J].Bone Joint Surg(Br),1990,72(3):347-355.
[66] Borsalino G, Bagnacaui L, Fornaciari F, et al. electrical stimulation of human femoral intertrochanteric osteotomies[J].Clin Orthop Relat Res, 1988, 237(1):256-263.
[67]邵雄杰,罗春晓,陈郁鲜.脉冲电磁场对绝经后骨质疏松症的疗效分析.南方医科大学学报, 2008,28(9):1577-1578.
[68] Gupta T D. Comparative study of growth by pulsed electromagnetic fields. Mde Biol Eng Comput[J].1991,29(2):113-120.
[69] Satter S A, Islam M S, rabbani K S. Pulsed electromagneticfields for the treatment of bone fractures[J].Bangladash Mde Res Counc Bull. 1999, 25(1):6-10.
[70] Darendeliler MA, Darendeliler A,Sinclair PM, Effects ofstatic magnetic and pulsed electromagnetic fields on bone healing INT[J].Adult Drthoden Orthognath Surg,1997,12(1):43-53.
[71] Takano-yamamoto T, Kawakam M, Saduda M. Effects of a pulsing electromagnetic fields on demineralized bone matric indced bone formation in a bony defect in the premaxilla of rats[J]. J Dent Res,1992,71(12):1920-1925.
[72] Ibiwoye MO,PowelI KA,Grabiner MD.et a1.Bone mass is preserved in acritical-sized osteotomy by Iow energy pulsed electromagnetic fields as quantitated by in vivo micro-computed tomography.J Orthop Res.2004;22(5):1086-1093.
[73] Yonemori K,Matsunaga S,Ishidou Y. Early effects of eiectricaistimulation on osteogenesis bone,J Orthop Res.1 996,1 9(2):173-180.
[74]罗二平,焦李成,申广浩,等.低强度脉冲电磁场对兔心血管系统的影响[J].第四军医大学学报,2005.26(6):557-561.
[75] Heermeier K.Spann M,Trager J,et a1.Effects of extremely low frequencyelectromagnetic field(EMF)0n collagen type mRNA expression and extracellular matrix synthesis of human osteoblastic cells[J]. Bioelectromagnetics. 1998,19(4):222-231.
[76] Lohmann CH. Schwar Z,Liu Y,et a1.Pulsed electromagnetic field stimulation of MG63 osteoblast like cells affects differentiation and IocaI factor production.J Orthop Res.2000,18(4):637-646.
[77] Stephen M. Combined DC and ELF magnetic fields can alter cell proliferation [J]. Bioelectromagnetics, 1990,11:27-36
[78] Pericles Diniz,Kenji Shomura, Kazuhisa,et al: Effects of Pulsed Electromagnetic Field(PEMF) Stimulation on bone Tissue Like Formation Are Dependent on the Maturation Stages of the Osyeoblasts . J Bioelectromagnetics 2002;23:398-405.
[79] Kyle Chang,Walter Hong -Shong Chang,Ming-Tzu Tssi.Pulsed electromagnetic fields accelerate apoptotic rate in osteodasts.Connective Tissue Research,2006(47):222-228.
[80] Bodamyali T,Bhatt B Hughes F.Pulsed electromagnetic fields simultaneously induce osteogenesis and upregulate transcription of bone morphogenetic proteins-2and-4 in ratosteoblasts in vitro[J].Biochem Biophys Res, 1998,250(2):458-461.
[81] Bassett CAL, Pilla A A, Pawluk R J. A non-operative salavage of surgically- resistant pseudarthrosis and non-unions by pulsing electromagnetic fields. Clin Orthop,1997:124-128.
[82] Inoue N,Ohnishi I,Chen D,et al.Effect of pulsed electromagnetic fields (PEMF) on late-phase osteotomy gap healing in a canine tibial model.J Orthop Res2002,20(4):1106-1114.
[83] Torricelli P,Fini M.et al.Biomimetic PMMA-based bone substitutes: A comparative in vitro evaluation of the effects of pulsed electromagnetic field exposure . J Biomed Mater Res 2003,64(1):182-188.
[84] Rices A, Conget P, Minguell J J. Mesenchymal progenitic or cells in human umbilical cord blood[J]. Br J Haematol, 2000,109(1):235-242.
[85] Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivasclar (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells, 2005, 23(2):220-229.
[86] Kadner A, Zund G, Maurus C, et al. Human umbilical cord cells for cardiovasualar tissue engineering: a comparative study. Eur J Cardiothorac Surg. 2004,25:635-641.
[87] Pereira WC, Khushnooma I, Madkaikar M, et al. Reproducible methodology for the isolation of mesenchymal stem cells from human umbilical cord and its potential for cardiomyocyte generation [J]. J Tissue Eng Regen Med 2008,21(7):394-399.
[88] Jomura S, Uy Michell K, et al. Potential treatment of cerebral global ischemia with Oct-4+ umbilical cord matrix cells[J]. Stem Cells 2007,25:98-106.
[89] Meyer FA, Laver-Rudich Z, Tanenbaum R. Evidence for a mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton’s jelly[J]. Biochim Biophys Acta 1983;755:376-387.
[90] Alp Can, Sepcin Karahuseyinoglu. Concise review: Human umbilical cord stroma with regard to the source of fetus-derived stem cells [J]. Stem Cells 2007;25:2886-2895.
[91]徐若男,施明,王福生.人脐带间充质干细胞的特征及其临床应用前景.中国免疫学杂志,2010,26:82-84.
[92] Alhadlaq A, Mao JJ. Mesenchymal stem cells: isolation and therapeutics [J].Stem Cells Dev, 2004,13(3):436-448.
[93]王凡,邓力,顿爱社,等.大鼠骨髓间充质干细胞的分离培养及表型和功能特点.四川大学学报(医学版),2003,34(4):783-741.
[94] Ogawa R, Mizuno H, Watanabe A, et al. Adipogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice-including relationship of sex differences[J]. Biochem Biophys Res Commun, 2004, 319: 511-517.
[95] Shi X XM, Blair H C, Yang X, et al. Tandern repeat of C/EBP binding sitesmediates PPARgamma2 gene transcription in glucocorticoid induced adipocyte differentiation[J]. Cell Biochem, 2000,76(3):518-527.
[96] Cui Q, Wang GJ, Balian G, Steroid Induced adipogenesis in a pluripotential cell line from bone marrow [J].Bone Joint Surg, 1997, 79(7):1054-1063.
[97] Halvosen YD, Franklin D, Bond AL, et al. Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue derived stromal cells[J]. Tissue Eng, 2001, 7(6): 729-741
[98]段瑞平,吴凌,,林云峰,,等.不同成骨诱导作用下骨髓间充质干细胞的基因表达模式[J].四川大学学报医学版,2006, 37(6): 856-859.
[99] Jorgensen NR, Henriksen Z, Sorensen OH, et al. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids, 2004, 69(4): 219-226
[100] Solchaga L A, Goldberg V M, Caplan A I. Cartilage regeneration using principles of tissue engineering. Clin Orthop Res, 2001,391:161-167.
[101] Prigozhina T B, Khitrin S, Elkin G, et al . Mesenchymal stromal cells lose their immunosuppressive potential after allotransplantation[J]. Exp Hematol, 2008,36(10):1370-1376.
[102] Wakitani S, Imoto K, Yamamot T, et al. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteo-arthritic knees[J].Ostesarthritis Cartilage, 2002,10(3):199-206.
[103] Wakitani S Mitsuoka T, Nakamura N, et al. Autologous bone marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in human patellae: two case reports[J].Cell Transplant, 2004,13(50:595-600.
[104] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteriafor efining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006,8(4):315-317.
[105] Milena Fini Ruggero Cadossi. Et al. The effects of pulsed electromagnetic fields on the osteointegration of hydroxyapatite implants in cancellous bone: a morphologic and microstructural in vivo study[J]. J Orthop. Res. 2002,20(4), 756-763.
[106] Suan Li,Genevieve MC,Michael JM,et al.Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds.J Biomed Mater Res,1997,36:17-28.
[107] Malezadeh R,Jeffrey OH,Dave B,et al.Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering.J Periodontal, 1998,69: 1256- 1262.
[108] Mc Cann J,Dietrich F,Rafferty C.The genotoxic potential of electric and magneticfields :an update.Mutat Res,1998,411:45-86.
[109] Torricelli P,Fini M,Giavaresi G,et a1.Biomimetic PMMA based bone substitutes : A comparative in vitro evaluation of the effects of pulsed electromagnetic field exposure.J Biomed Mater Res,2003,64(1):182
[110] Jones E, McGonagle D. Human bone marrow mesenchymal stem cells in vivo. Rheumatology(Oxford).2008;47(2):126-131.
[111] Pardinsion WC,Hanks CT.Search for cyclotron resonance in cells in vitro. Bioelectromagnetics, 1989,8(2):82.
[112] Muehsam DJ, Pilla AA. The sensitivity of cells and tissusto exogenous fields: effects of target system initial state[J]. Bioelectrochemistry and ioenergetics, 1999,48:35-42.
[113] Sudeepta A, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood.2005;105(4):1815-1822.
[114] Lohmann CH, Schwartz Z, Liu Y, et al . Pulsed electro-magnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production[J]. Orthop Res, 2000,18(40:637-646.
[115] Torricelli Paola, Fini M, Giavaresi G. Biomimetic PMMA-based bone substiutes: A comparative in vitro evaluation of the effects of pulsed electormagnetic fieldexposure[J]. Biomedical Materials Research, 2003, 64(1):182-188.
[116] Pericles Diniz,Kenji Shomura, Kazuhisa,et al: Effects of Pulsed Electromagnetic Field(PEMF) Stimulation on bone Tissue Like Formation Are Dependent on the Maturation Stages of the Osyeoblasts . J Bioelectromagnetics 2002;23:398-405.
[117] Schwartz Z,Simon BJ,Duran MA,et a1.Pulsed electromagnetic fields enhance BMP-2 dependent osteoblastic differentiation of human mesenchymal stem cells.J OrthOD Ras.2008;26(9):1250-1255.
[118] Schmidt-Rohlfing B, Silny J,Woodruff S,et a1.Effects of pulsed and sinusoid electromagnetic fields on human chondrocytes cultivated in a collagen matrix.Rheumatol Int, 2008,28(10):971-977.
[119] Sammons J,Ahmed N,EI Sheemy M,at a1.The role of BMP6,IL 6, and BMP 4 In mesenchymaI stem celI dependent bone development : Effects on osteoblastic differentiation induced by parathyroid hormone and vitarain D3.Stem Cells Dev.2004;1 3(3):273-280.
[120] Sun LY, Hsieh DK, Lin PC, et al. Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation. Bioelectromagnetics. 2010,31(3):209-219.
[121] Tsai MT, Li WJ, Tuan RS, et al. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res. 2009;27(9): 1169-1174.
[122]刘侠,乐以伦.碱性磷酸酶与钙化.中国生物医学工程学报,1997,16:71-82.
[123] Bersani F, Ryaby JT, Magee FP, et al. Electromagnetic fields can affect osteogenesis by the rate of differentiation . Clin Orthop 1997;338-362.
[124] Caprani A, Richert A, Guglielmi JP, et al. Preliminary study of pulsed- electromagnetic fields effects on endothelial (HUVEC) cell secretions- modulation of the thrombo-hemorrhagic balance. Electromagn. Biol Med. 2008; 27(4):386-392.
[125] Nicola C, Emilio B, Simone C, et al. Effect of electromagnetic fields on bone mineral density and biochemical markers of bone turnover in soteoporosis[J]. Radiat Environ Biophys, 2001,62(3):268-271.
[126] Pilla AA, Muehsam DJ, Markov MS, et al. EMF signals and Ion/Ligand binding dinetics: prediction of bioeffective waveform parameters[J]. Bioelectrochem Bioenergetics, 1999,48(1):27-34.
[127] Wang Q, Zhong S, Quyang J, et al. Osteogensis of electricaly stimulated bone cells mediated in part by calcium ions. Clin Orthop, 1998,348:259.
[128]郝海虎,吴华,张海军,等.工频电磁场对骨髓间充质干细胞增殖、分化及其胞浆内钙离子浓度的影响[J].中华物理医学与康复杂志,2007,29(4):217-220.
[129]任凯,吴华,赵文春,等.工频电磁场对体外培养的小鼠骨髓间充质干细胞增殖与分化的影响[J].中华物理医学与康复杂志,2004,26(7):385-387.
[130] Roy K,Wang S. Upregulation of basal TGF-β1 levels by EMF coincident with chondrogenesis-implications for skeletal repair and tissue engineering[J]. J Orthop Res, 2002,20(2):233-240.
[131] Guerkov,H.H,Heckman,J.D.Schwartz,Z.et al.Pulsed Electromagnetic Fields Increase Growth Factor Release by Nonunion Cells.J Clin Orthop 2001, 1(384):265-279
[132]王彤.骨髓间充质干细胞临床研究进展.2010.人民卫生出版社
[2] Friedenstein A J, Gorskaja J F, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hhematopoietic organs. Exp Hematol, 1976,4(5):267-274.
[3] Reese JS, Koc ON, Gerson SL.Human mesenchymal stem cells provide stromal support for efficient CD34+ transduction[J].Hematother Stem Cell Res, 1999,8:515-523.
[4] Zuk PA, Zhu M, Mizuno H, et al. Multilieage cells from human adipose tissue:implications for cell based therapies[J]. Tissue Eng, 2001, 7(2): 211-228
[5] Toma JG, Akhavan M, Femandes KJ, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin[J]. Nat Cell Biol, 2001, 3(9): 778-784
[6] Tuli R, Tuli S, Nandi S, et al. Characterization of multipotential mesenchymal progenitor cells derived from human trabecular bone[J].Stem Cells, 2003, 21(60):681-693.
[7] Kwan Chul T,Jong-Won H, Young-Soo K, et al. Effects of human blood mesenchymal stem cells on cutaneous wound healing in lepr db mice[J]. Ann Plast Surg, 2010 Oct 13[Epub ahead of print]
[8] Yumi Fukuchi, Hideake Nakajima,Daisuke Sugiyama. Human placenta-derived cells have mesenchymal stem/progenitor cell potential[J]. Stem Cell, 2004, 22:649-658.
[9] Wexler SA, Donaldson C, Denning-Kendall P, et al. Adult bone marrow is a rich source of human mesenchymal stem cells but umbilical cord and mobilized adult blood are not. Br J Haematol, 2003,121(2):368-374.
[10] Coter D C, Class R, DiGirolanno C M, et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow[J]. Rroc Natl Acad Sci USA, 2000,97:3213-3218.
[11] Karen Bieback,Suanne Kern, Harald Kluter, et al. Critical Parameters for theisolation of mesenchymal stem cells from umbilical cord blood[J]. Stem Cells, 2004,22:625-634.
[12] Wagner W, Wein F, Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue,and umbilical cord blood[J]. Exp Hematol, 2005,33(11):1402-1406.
[13] Romanov YA, Darevskaya AN, Merzlikina NV, et al. Mesenchymal stem cells from human bone marrow and adipose tissue: isolation, characterization and differentiation potentialities[J]. Bull Exp Biol Med, 2005, 140(1):138-143.
[14] Deans R J, Moseley A B. Mesenchymal stem cells: biology and pothntial chlinical uses[J]. Exp Hematol, 2000,28(8):875-884.
[15] Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells[J]. Keio J Med, 2005, 54(3): 132-141
[16] Mastrogiacomo M, derubeis A R, Cancedda R. Bone and cartilage formation by skeletal muscle derived cells[J]. J Cell Physiol, 2005,204(2):594-603.
[17]丹尼尔R,马沙克理查德L,甘德大卫.戈特利布主编,刘景生等译.干细胞生物学.北京:化学工业出版社,2004:302-318.
[18] Pittenger M F, Mackay A M, Beck S C, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284:143-147.
[19] Toma C, Pittenger M F, cahill K S, et al. Human mesenchymal stem cells differentiate a cardiomyocte phenolcyte in thd adult murine heart. Circulation[J]. 2002, 5:93-98.
[20] Ozono S, Fujita T,Matsuo M,et a1.Co-treatment with basic fibroblast growth factor and 17 beta-estradiol in the presence of dexamethasone accelerates bone formation by rat bone marrow stromal cell culture[J].Nihon Hotetsu Shika Gakkai Zasshi,2008,52(3):366-374.
[21] Lin TY, Zhou GD,Wei X, et a1.Influence of transforming growth factor-betaI inducing time on chondrogenesis of bone marrow stromal cells (BMSCs):in vitro experiment with porcine BMSCs[J]. Zhong Hua Yi Xue Za Zhi,2007, 87(31):22 l 8-2222.
[22] Cotherstrom C, ringden O, tammik C, et al. Immunologic properties of human fetal mesenchymal stem cells[J]. Am J Ohstet Gynecol,2004, 190(1):239-245.
[23] Klyshnenkova E, Mosca JD, Zernetkina V, et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression[J]. J Biomed Sci, 2005, 12(1):47-57.
[24] Le Blanc K, Tammik C, Rosendahl K, et al. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells[J]. Exp Hematol, 2003,31:890-896.
[25] Devine SM, Cobbs C, Jennings M, et al. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates[J]. Blood,2003,101(8):2999-3001.
[26] Lee S T, Jang J H, Cheong J W, et al. Treatment of high-risk acute myelogenous leukaemia by myeloabative chemoradiotherapy followed by co-infusion of T cell-depleted haematopoietic stem cells and culture-expanded marrow mesenchymal stem cells from related donor with one fully mismatched human leucocyte antigen haplotype. Br J Haematol, 2002,118:1128-1131
[27] Koc O N, Gerson S L, Cooper B W, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.J Clin Oncol 2000,18:307-316.
[28] Koc O N, Day J Du. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy(MLD) and Hurler syndrome(MPS-IH). Bone Marrow Transplant, 2002,30:215-222.
[29]苗宗宁,潘宇红,祝建中,等.丝素蛋白支架材料复合骨髓间充质干细胞构建组织工程化软骨.中国组织工程研究与临床康复,2008,12(27):5243-5247.
[30] Award HA, Bulter DL, Biovin GP, et al. Autologous mesenchymal stem cells mediated repair of endon[J]. Tissue Engineering, 1999,5(3):267.
[31]周志玲,袁进国,刘英飞,等.自体骨髓干细胞移植治疗骨不连.中国临床康复,2006,10(29):22-23.
[32] Jiang X, Zhang Y, Liu B, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood,2005, 168(1-2):661-669.
[33] Koc O N, Gerson S L, Cooper B W, et al. Rapid hematopoietic recovery after coinfusion of autologousblood stem cells and culture expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000,18(2):307-316.
[34] Cheng L, Qasba P, v anguri P, et al. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34+ hematopoietic progenitor cells[J]. J Cell Physiol, 2000,184(1):58-69.
[35] Wu T, Levay Young B, Heuss N, et al. Inducing tolerance to MHC2 matched allogeneic islet grafts in diabetic NOD mice by simultaneous islet and bone marrow transplantation under non-irradiative and nonmyeloblative conditioning therapy. Transplantation, 2002,74(1):22-27.
[36]王亚平.干细胞衰老与疾病. 2009.北京.科学出版社.
[37] Concise Review: Human Umbilical Cord Stroma with Regard to the Source of Fetus-Derived Stem Cells[J]. Stem Cells,2007,24(11):2886-2895.
[38] Kobayashi K,Kubota T,Aso T.Study on myofibroblast differentiation in the stromal cells of Wharton’s jelly: expression and localization of alpha-smooth muscle actin[J].Early Hum Dev,1998,51(3):223-233.
[39] Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton’s jelly form neurons and glia[J] Stem Cells, 2003, 21(1):50-60.
[40] Romanov YA,Svintsitskaya V A, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord[J]. Stem Cells, 2003,21(1):105-110.
[41] Michell KE, Weiss ML, Mitchell BM, et al. Matrix clls from Wharton’s jelly form neurons and glia[J]. Stem Cells, 2003,21(1):50-60.
[42] Wang H S, Hung S C, Peng S T, et al. Mesenchymal stem cells in the wharton’s jelly of human umbilical cord[J]. Stem cells, 2004,22(7):1330-1337.
[43] Weiss ML, Medicetty S, Bledsoe AR, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of parkinson’s disease[J]. Stem Cells, 2006,24(3):781-792.
[44]吕璐璐,刘拥军,许贞书,等.脐带间充质干细胞的分离和生物学性状[J].福建医科大学学报, 2006,40(2):99-104.
[45] Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors[J]. Stem Cells, 2005,23(2): 220-229.
[46] Karahuseyinoglu S, Cinar O, Kilic E et al. Biology of stem cells in human umbilical cord stroma: In situ and in vitro surveys[J]. Stem Cells, 2007,25:319–331.
[47] Sarugaser R, Lickorish D, Baksh D et al. Human umbilical cord perivascular (HUCPV) cells: A source of mesenchymal progenitors. Stem Cells ,2005,23:220–229.
[48] Lund RD, Wang S, Lu B et al. Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease. Stem Cells ,2007,25:602– 611.
[49]张浪辉,刘拥军,吕璐璐,等.脐带间充质干细胞对异源性脐带血T淋巴细胞激活与增殖抑制作用[J].中国肿瘤生物治疗杂志,2006,13(3):191-195.
[50]马廉,崔冰琳,冯学永等.人脐带间充质干细胞的生物学特性及向神经样细胞分化的研究.中华儿科杂志,2006(7):513-517.
[51]周佳,沈尊理,金羽青,等.人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究.中华显微外科杂志,2009,6(32)210-212.
[52] Kadivar M, Khatami S, Mortazavi Y, et al. In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells[J]. Biochem Biophys Res Commun, 2006,340(2):639-647.
[53] Conconi MT, Burra P, Di Liddo R, et al. CD105(+) cells from Wharton’s jelly show in vitro and in vivo myogenic differentiative potential[J]. Int J Mol Med, 2006,18(6):1089-1096.
[54] Lee OK, Kuo TK, chen WM, et al. Isolation of mulipotent mesenchymal stem cells from umbilical cord blood[J]Blood, 2004,103(5):1669-1675.
[55] Reyes M, verfaillie C M. Characterization of multipotent adult progenitor cells, a subpobulation of mesenchymal stem cells[J]. Ann N Y Acad Sci, 2001,938:231-233.
[56] Fu YS, Cheng YC, Lin MY, et al. Conversion of human umbilical cord mesenchymal stem cells in wharton’s jelly to dopaminergic neurons in vitro: potential therapeutic application for parkinsonism[J]. Stem Cells,2006,24(1): 115-124.
[57]王爱红,何红燕,罗敏洁,等.人脐带间充质干细胞向胰岛样细胞的分化[J].中国组织工程研究与临床康复,2008,12(21):4102-4106.
[58] Friedman R, Betancur M, Boissel L,etal.Umbilical cord mesenchymal stem cells: adjuvants ofr human cell transplantation [J]. Biol Blood Marrow Transplant, 2007,13(12):1477-1486.
[59] Li CD, Zhang WY, Jiang XX, et al. Human-placenta-derived mesenchymal stem cells inhibit proliferation and function of allogeneic immune cells[J]. Cell Tissue Res,2007,330(3):437-446.
[60]漆佩静.脐带间充质干细胞抑制异种移植物抗宿主病的机制研究.清华大学医学院,博士学位论文.2009.
[61] Kim JW, Kim SY, Park SY, et al. Mesenchymal progenitor cells in the human umbilical cord [J].An Hematol, 2004,83(12):733-738.
[62]孙国栋,伍时县,李志忠.人脐带间充质干细胞分离培养及成骨分化原子力显微镜观察细胞膜表面的超微结构变化.中国组织工程研究与临床康复.2010,14(1):33-37.
[63] Schneider RK, Puellen A, Kramann R, et al. The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodeling in three-dimensional collagen scaffolds[J]. Biomaterials, 2010, 31(3):467-480.
[64] Rachakatla R s, Marini F, Weiss M L, et al. Development of human umbilicalcord matrix stem cell-based gene therapy for experimental lung tumors[J]. Cancer Gene Ther, 2007,14(10):828-835.
[65] Sharrard WJ.Adouble-blind trial of pulsed electromagnetic fields for delaye union of tibial fractures[J].Bone Joint Surg(Br),1990,72(3):347-355.
[66] Borsalino G, Bagnacaui L, Fornaciari F, et al. electrical stimulation of human femoral intertrochanteric osteotomies[J].Clin Orthop Relat Res, 1988, 237(1):256-263.
[67]邵雄杰,罗春晓,陈郁鲜.脉冲电磁场对绝经后骨质疏松症的疗效分析.南方医科大学学报, 2008,28(9):1577-1578.
[68] Gupta T D. Comparative study of growth by pulsed electromagnetic fields. Mde Biol Eng Comput[J].1991,29(2):113-120.
[69] Satter S A, Islam M S, rabbani K S. Pulsed electromagneticfields for the treatment of bone fractures[J].Bangladash Mde Res Counc Bull. 1999, 25(1):6-10.
[70] Darendeliler MA, Darendeliler A,Sinclair PM, Effects ofstatic magnetic and pulsed electromagnetic fields on bone healing INT[J].Adult Drthoden Orthognath Surg,1997,12(1):43-53.
[71] Takano-yamamoto T, Kawakam M, Saduda M. Effects of a pulsing electromagnetic fields on demineralized bone matric indced bone formation in a bony defect in the premaxilla of rats[J]. J Dent Res,1992,71(12):1920-1925.
[72] Ibiwoye MO,PowelI KA,Grabiner MD.et a1.Bone mass is preserved in acritical-sized osteotomy by Iow energy pulsed electromagnetic fields as quantitated by in vivo micro-computed tomography.J Orthop Res.2004;22(5):1086-1093.
[73] Yonemori K,Matsunaga S,Ishidou Y. Early effects of eiectricaistimulation on osteogenesis bone,J Orthop Res.1 996,1 9(2):173-180.
[74]罗二平,焦李成,申广浩,等.低强度脉冲电磁场对兔心血管系统的影响[J].第四军医大学学报,2005.26(6):557-561.
[75] Heermeier K.Spann M,Trager J,et a1.Effects of extremely low frequencyelectromagnetic field(EMF)0n collagen type mRNA expression and extracellular matrix synthesis of human osteoblastic cells[J]. Bioelectromagnetics. 1998,19(4):222-231.
[76] Lohmann CH. Schwar Z,Liu Y,et a1.Pulsed electromagnetic field stimulation of MG63 osteoblast like cells affects differentiation and IocaI factor production.J Orthop Res.2000,18(4):637-646.
[77] Stephen M. Combined DC and ELF magnetic fields can alter cell proliferation [J]. Bioelectromagnetics, 1990,11:27-36
[78] Pericles Diniz,Kenji Shomura, Kazuhisa,et al: Effects of Pulsed Electromagnetic Field(PEMF) Stimulation on bone Tissue Like Formation Are Dependent on the Maturation Stages of the Osyeoblasts . J Bioelectromagnetics 2002;23:398-405.
[79] Kyle Chang,Walter Hong -Shong Chang,Ming-Tzu Tssi.Pulsed electromagnetic fields accelerate apoptotic rate in osteodasts.Connective Tissue Research,2006(47):222-228.
[80] Bodamyali T,Bhatt B Hughes F.Pulsed electromagnetic fields simultaneously induce osteogenesis and upregulate transcription of bone morphogenetic proteins-2and-4 in ratosteoblasts in vitro[J].Biochem Biophys Res, 1998,250(2):458-461.
[81] Bassett CAL, Pilla A A, Pawluk R J. A non-operative salavage of surgically- resistant pseudarthrosis and non-unions by pulsing electromagnetic fields. Clin Orthop,1997:124-128.
[82] Inoue N,Ohnishi I,Chen D,et al.Effect of pulsed electromagnetic fields (PEMF) on late-phase osteotomy gap healing in a canine tibial model.J Orthop Res2002,20(4):1106-1114.
[83] Torricelli P,Fini M.et al.Biomimetic PMMA-based bone substitutes: A comparative in vitro evaluation of the effects of pulsed electromagnetic field exposure . J Biomed Mater Res 2003,64(1):182-188.
[84] Rices A, Conget P, Minguell J J. Mesenchymal progenitic or cells in human umbilical cord blood[J]. Br J Haematol, 2000,109(1):235-242.
[85] Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivasclar (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells, 2005, 23(2):220-229.
[86] Kadner A, Zund G, Maurus C, et al. Human umbilical cord cells for cardiovasualar tissue engineering: a comparative study. Eur J Cardiothorac Surg. 2004,25:635-641.
[87] Pereira WC, Khushnooma I, Madkaikar M, et al. Reproducible methodology for the isolation of mesenchymal stem cells from human umbilical cord and its potential for cardiomyocyte generation [J]. J Tissue Eng Regen Med 2008,21(7):394-399.
[88] Jomura S, Uy Michell K, et al. Potential treatment of cerebral global ischemia with Oct-4+ umbilical cord matrix cells[J]. Stem Cells 2007,25:98-106.
[89] Meyer FA, Laver-Rudich Z, Tanenbaum R. Evidence for a mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton’s jelly[J]. Biochim Biophys Acta 1983;755:376-387.
[90] Alp Can, Sepcin Karahuseyinoglu. Concise review: Human umbilical cord stroma with regard to the source of fetus-derived stem cells [J]. Stem Cells 2007;25:2886-2895.
[91]徐若男,施明,王福生.人脐带间充质干细胞的特征及其临床应用前景.中国免疫学杂志,2010,26:82-84.
[92] Alhadlaq A, Mao JJ. Mesenchymal stem cells: isolation and therapeutics [J].Stem Cells Dev, 2004,13(3):436-448.
[93]王凡,邓力,顿爱社,等.大鼠骨髓间充质干细胞的分离培养及表型和功能特点.四川大学学报(医学版),2003,34(4):783-741.
[94] Ogawa R, Mizuno H, Watanabe A, et al. Adipogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice-including relationship of sex differences[J]. Biochem Biophys Res Commun, 2004, 319: 511-517.
[95] Shi X XM, Blair H C, Yang X, et al. Tandern repeat of C/EBP binding sitesmediates PPARgamma2 gene transcription in glucocorticoid induced adipocyte differentiation[J]. Cell Biochem, 2000,76(3):518-527.
[96] Cui Q, Wang GJ, Balian G, Steroid Induced adipogenesis in a pluripotential cell line from bone marrow [J].Bone Joint Surg, 1997, 79(7):1054-1063.
[97] Halvosen YD, Franklin D, Bond AL, et al. Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue derived stromal cells[J]. Tissue Eng, 2001, 7(6): 729-741
[98]段瑞平,吴凌,,林云峰,,等.不同成骨诱导作用下骨髓间充质干细胞的基因表达模式[J].四川大学学报医学版,2006, 37(6): 856-859.
[99] Jorgensen NR, Henriksen Z, Sorensen OH, et al. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids, 2004, 69(4): 219-226
[100] Solchaga L A, Goldberg V M, Caplan A I. Cartilage regeneration using principles of tissue engineering. Clin Orthop Res, 2001,391:161-167.
[101] Prigozhina T B, Khitrin S, Elkin G, et al . Mesenchymal stromal cells lose their immunosuppressive potential after allotransplantation[J]. Exp Hematol, 2008,36(10):1370-1376.
[102] Wakitani S, Imoto K, Yamamot T, et al. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteo-arthritic knees[J].Ostesarthritis Cartilage, 2002,10(3):199-206.
[103] Wakitani S Mitsuoka T, Nakamura N, et al. Autologous bone marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in human patellae: two case reports[J].Cell Transplant, 2004,13(50:595-600.
[104] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteriafor efining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006,8(4):315-317.
[105] Milena Fini Ruggero Cadossi. Et al. The effects of pulsed electromagnetic fields on the osteointegration of hydroxyapatite implants in cancellous bone: a morphologic and microstructural in vivo study[J]. J Orthop. Res. 2002,20(4), 756-763.
[106] Suan Li,Genevieve MC,Michael JM,et al.Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds.J Biomed Mater Res,1997,36:17-28.
[107] Malezadeh R,Jeffrey OH,Dave B,et al.Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering.J Periodontal, 1998,69: 1256- 1262.
[108] Mc Cann J,Dietrich F,Rafferty C.The genotoxic potential of electric and magneticfields :an update.Mutat Res,1998,411:45-86.
[109] Torricelli P,Fini M,Giavaresi G,et a1.Biomimetic PMMA based bone substitutes : A comparative in vitro evaluation of the effects of pulsed electromagnetic field exposure.J Biomed Mater Res,2003,64(1):182
[110] Jones E, McGonagle D. Human bone marrow mesenchymal stem cells in vivo. Rheumatology(Oxford).2008;47(2):126-131.
[111] Pardinsion WC,Hanks CT.Search for cyclotron resonance in cells in vitro. Bioelectromagnetics, 1989,8(2):82.
[112] Muehsam DJ, Pilla AA. The sensitivity of cells and tissusto exogenous fields: effects of target system initial state[J]. Bioelectrochemistry and ioenergetics, 1999,48:35-42.
[113] Sudeepta A, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood.2005;105(4):1815-1822.
[114] Lohmann CH, Schwartz Z, Liu Y, et al . Pulsed electro-magnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production[J]. Orthop Res, 2000,18(40:637-646.
[115] Torricelli Paola, Fini M, Giavaresi G. Biomimetic PMMA-based bone substiutes: A comparative in vitro evaluation of the effects of pulsed electormagnetic fieldexposure[J]. Biomedical Materials Research, 2003, 64(1):182-188.
[116] Pericles Diniz,Kenji Shomura, Kazuhisa,et al: Effects of Pulsed Electromagnetic Field(PEMF) Stimulation on bone Tissue Like Formation Are Dependent on the Maturation Stages of the Osyeoblasts . J Bioelectromagnetics 2002;23:398-405.
[117] Schwartz Z,Simon BJ,Duran MA,et a1.Pulsed electromagnetic fields enhance BMP-2 dependent osteoblastic differentiation of human mesenchymal stem cells.J OrthOD Ras.2008;26(9):1250-1255.
[118] Schmidt-Rohlfing B, Silny J,Woodruff S,et a1.Effects of pulsed and sinusoid electromagnetic fields on human chondrocytes cultivated in a collagen matrix.Rheumatol Int, 2008,28(10):971-977.
[119] Sammons J,Ahmed N,EI Sheemy M,at a1.The role of BMP6,IL 6, and BMP 4 In mesenchymaI stem celI dependent bone development : Effects on osteoblastic differentiation induced by parathyroid hormone and vitarain D3.Stem Cells Dev.2004;1 3(3):273-280.
[120] Sun LY, Hsieh DK, Lin PC, et al. Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation. Bioelectromagnetics. 2010,31(3):209-219.
[121] Tsai MT, Li WJ, Tuan RS, et al. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res. 2009;27(9): 1169-1174.
[122]刘侠,乐以伦.碱性磷酸酶与钙化.中国生物医学工程学报,1997,16:71-82.
[123] Bersani F, Ryaby JT, Magee FP, et al. Electromagnetic fields can affect osteogenesis by the rate of differentiation . Clin Orthop 1997;338-362.
[124] Caprani A, Richert A, Guglielmi JP, et al. Preliminary study of pulsed- electromagnetic fields effects on endothelial (HUVEC) cell secretions- modulation of the thrombo-hemorrhagic balance. Electromagn. Biol Med. 2008; 27(4):386-392.
[125] Nicola C, Emilio B, Simone C, et al. Effect of electromagnetic fields on bone mineral density and biochemical markers of bone turnover in soteoporosis[J]. Radiat Environ Biophys, 2001,62(3):268-271.
[126] Pilla AA, Muehsam DJ, Markov MS, et al. EMF signals and Ion/Ligand binding dinetics: prediction of bioeffective waveform parameters[J]. Bioelectrochem Bioenergetics, 1999,48(1):27-34.
[127] Wang Q, Zhong S, Quyang J, et al. Osteogensis of electricaly stimulated bone cells mediated in part by calcium ions. Clin Orthop, 1998,348:259.
[128]郝海虎,吴华,张海军,等.工频电磁场对骨髓间充质干细胞增殖、分化及其胞浆内钙离子浓度的影响[J].中华物理医学与康复杂志,2007,29(4):217-220.
[129]任凯,吴华,赵文春,等.工频电磁场对体外培养的小鼠骨髓间充质干细胞增殖与分化的影响[J].中华物理医学与康复杂志,2004,26(7):385-387.
[130] Roy K,Wang S. Upregulation of basal TGF-β1 levels by EMF coincident with chondrogenesis-implications for skeletal repair and tissue engineering[J]. J Orthop Res, 2002,20(2):233-240.
[131] Guerkov,H.H,Heckman,J.D.Schwartz,Z.et al.Pulsed Electromagnetic Fields Increase Growth Factor Release by Nonunion Cells.J Clin Orthop 2001, 1(384):265-279
[132]王彤.骨髓间充质干细胞临床研究进展.2010.人民卫生出版社