摘要
多发性骨髓瘤(MM)是一种浆细胞恶性增殖性疾病,其特点是恶性浆细胞在骨髓微环境中不规则积聚引起溶骨性骨破坏、肾功能损害、贫血、高钙血症和感染等临床表现,占血液科肿瘤的10%-15%左右。目前仍被认为是一种不可治愈的疾病。大量研究表明骨髓微环境在骨髓瘤细胞的生长、存活及耐药的产生中起重要作用。
【目的】
通过共培养方式观察间充质干细胞(MSCs)对MM细胞体外增殖的影响。通过建立浆细胞瘤模型来观察MSCs对MM细胞体内增殖的影响。
【方法】
常规分离培养胎儿骨髓来源的MSCs,流式细胞仪分析其表面标志,与IL-6依赖的人多发性骨髓瘤细胞株XG-7的共培养,通过Annexin V/PI双标记法、MDC(单丹磺酰尸胺)及PI法检测共培养前后XG-7凋亡、自噬及细胞周期变化情况,并用Calcein AM(钙黄绿素)及DiI(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)双标记法观察两种细胞间的间隙连接通讯(gap junction intercellular communication ,GJIC)情况,通过transwell小室远距离与近距离共培养来阻断GJIC,观察细胞增殖的变化。为观察间充质细胞对MM细胞的体内促增殖作用,我们用三种方法来建立浆细胞瘤模型:采用16-18周龄人女性胎儿,分离出股骨和胫骨,取约1cm左右胎骨植入BALB/c裸鼠皮下,建立BALB/c-nu-hu鼠-人嵌合体;胎骨植入三周后,确认胎骨存活,将IL-6依赖的人MM细胞株XG-7悬液注入胎骨骨髓腔内建立人浆细胞瘤BALB/c-nu-hu模型;将两种细胞混合注入裸鼠皮下;单独皮下注入XG-7细胞。观察三种模型的肿瘤生长情况。40天后,取胎骨、肿瘤组织分别行H&E染色及抗CD34、CD59、CD138及VEGF免疫组织化学染色;X线平片检查裸鼠骨密度及胎骨骨密度前后变化。
【结果】
XG-7细胞与MSCs的共培养后其抗无血清诱导的凋亡及自噬能力增强,处于S期的细胞较多,增殖活性强。两种细胞间存在GJIC的物质交流,且阻断GJIC后细胞增殖的速度减慢。人胎骨组织能够在BALB/c裸鼠体内生长存活。动物实验中皮下混合注入两种细胞及在BALB/c-nu-hu鼠-人嵌合体基础上接种XG-7细胞组肿瘤生长速度快,而单独注入组未见肉眼可见肿瘤长出。肿瘤具有多种与浆细胞瘤相似的病理学特征,免疫组化CD138、CD59阳性显示了其仍具有浆细胞表面标记,且血管CD34阳性表明了其人源性;模型裸鼠出现恶病质,X线显示胎骨出现骨破坏。
【结论】
MSCs增强XG-7细胞抗凋亡及自噬的能力,并可促进XG-7细胞的生长,对其生存增殖起到庇护作用。MSCs与XG-7间存在GJIC,这种GJIC可能参与了MSCs促进XG-7生长的作用。成功建立崭新的BALB/c-nu-hu鼠-人嵌合性小生境,可作为一种建立肿瘤模型可靠的基础动物载体。利用BALB/c-nu-hu鼠-人嵌合体或MSCs及XG-7细胞混合输注均可建立的浆细胞瘤模型,且模型肿瘤生长速度快,实验周期短,重复性好,可为进一步研究人多发性骨髓瘤的治疗奠定良好的基础。
Multiple myeloma (MM) is a malignant plasma cell disorder. It is characterized by heterogeneous anemia, bone disease, renal impairment, hypercalcaemia and infections due to abnormal accumulation of malignant plasma cells in the marrow microenvironment. Multiple myeloma is still incurable and accounts for approximately 10%-15% of human hematopoietic malignancies.Many researches have shown that marrow microenvironment play an important role in the proliferation of human myeloma cells and drug tolerance.
【Objective】
To research the effects of the mesenchyma stem cells (MSCs) on the proliferation of multiple myeloma cells.Establish plasmacytoma model to determine the effects of the mesenchymal stem cells on the proliferation of multiple myeloma cells in vivo.
【Methods】
MSCs from fetal bone marrow were isolated and cultured. Direct immunofluorescence assay was employed to analyze the phenotype of those cells by using flow cytometry . Interleukin-6 (IL-6)–dependent human MM cell line XG-7 were cocultured with the MSCs.Before and after coculture,the apoptosis,autophagy and cell cycle of XG-7 were detected by means of annexin V / PI double staining, MDC staining , PI staining. To confirm the presence of gap junctions intercellular communication (GJIC) between cocultured MSCs and XG-7, we use both calcein AM and DiI to labele XG-7 cells. Transwell inserts were used to separate the two kinds of cells in adjacent and remote cocultures, The proliferation of XG-7 cell line was observed in the two different coculture ways. Then we establish plasmacytoma model in there ways to determine the effects of the MSCs on the growth of multiple myeloma cells in vivo: Subcutaneously transplantation of 1cm long segment of 16-18 weeks old human fetal thigh or tibia bone into BALB/c-nu mice to develope a novel model of BALB/c-nu-hu chimera, and three weeks later XG-7 cells were inoculated into the human fetal bone to establish plasmacytoma; the mixture of MSCs and XG-7 cells was injected directerly into subcutaneous of BALB/c-nu mice in the second group; In the third group we injected sigle XG-7 cells into subcutaeous BALB/c-nu mice. 40 days later,human fetal bone implanted as well as the tumor were taken out respectively, stained by hematoxylin and eosin (H&E) and monoclonal mouse anti-human CD34、CD59、CD138 and VEGF followed by morphological examination. Take the X-ray film of mouse model at the start and the end of the experiment.
【Results】
After cocultured with MSCs, XG-7 cells enhanced the resistance of apoptosis and autophagy, at the same time its proliferation was increased. From fluorescence microscope we observed GJIC between cocultured MSCs and XG-7. Inhibit the GJIC by remote cocultures could step down the growth of XG-7; Fetal bone can survive in the subcutaneous site of BALB/c nude mice;After injection the mixture of the two cells, or injection XG-7 cell into human fetal bone in BALB/c-nu-hu chimera, tumor grown fast and characterized by typical plasmacytoma.And the resorption of the human bones can slso be observed in BALB/c-nu-hu model.But no evidence shows that the tumor was growing in BALB/c-nu mice with single XG-7 cells injection.
【Conclusion】
MSCs could protect XG-7 cells from apoptosis and autophagy, and obviosely promote the proliferation of XG-7 cells. GJIC dose existence between cocultured MSCs and XG-7, and it may participate the promotion of growth .The BALB/c-nu-hu chimeric model is a novel vector for researching hominine hematopoiesis and bone based on animal. The MM cell can growth successfully based on the BALB/c-nu-hu chimera or subcutaneous injection of mixture cells.
引文
1. Barlogie B, Shaughnessy J, Munshi N, Epstein J. Plasma cell myeloma. In: Beutler E, Lichtman M, Coller B, Kipps T, Seligsohn U, eds. Williams Hematology (ed 6). New York:McGraw-Hill;2001:1279-1304.
2. Mitsuyoshi Urashima, Benjamin P. Chen, Shirley Chen, et al. The Development of a Model for the Homing of Multiple Myeloma Cells to Human Bone Marrow. Blood, 1997 90: 754-765
3. Bataille R, Harousseau JL. Multiple myeloma. N Engl J Med[J]. 1997. 336:1657-1664
4. Chu MS,Chang CF,Yang CC,et a1.Signalling pathway in the in duction of neurite outgrowth in human mesenchymal stem cells[J].Cell Signal,2006,18(4):519-530
5. Yaccoby S, Pearse RN, Johnson CL, Barlogie B, Choi Y, Epstein J. Myeloma interacts with the bone marrow microenvironment to induce osteoclastogenesis and is dependent on osteoclast activity. Br J Haematol. 2002;116:278-290.
6. Podar K,Chauhan D,Anderson KC.Bone marrow mocroenvironment and the idification of new targets for myeloma therapy.Leukemia.2009;23:10-24.
7. Peacok CD,Wang Q,Gesell GS,et al.Hedgehog sigaling maintains a tumor stem cell compartment in multiple myelama.PNAS2007;104:4048-53.
8. Basak GW,srivastava AS and Malhotra R,et al.Multiple myeloma bone marrow niche.Curr Pharm Biot 2009;10:335-346
9. Silvertris F, Ciavarella S and matteo MD. Bone-resorbing cells in multiple myeloma: Osteoclasts, Myeloma Cell Polykaryons,or both? The Oncologist 2009;14:264-275.
10. Giuliani N,Mangoni M and Rizzoli V.Osteogenic differentiation of mesenchymal stem cells in multiple myeloma:Identification of potential therapeutic targets.Exp Hemat2009;37:879-886.
11. Wong RC, Pebay A,nguyen L T, et al.Presence of functional gap junctions in human embryonic stem cells[J]. Stem Cells. 2004,22(6):883-889.
12. Henry J. Donahue, Zhongyong Li, Zhiyi Zhou,et al. Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication. Am. J. Physiol. Cell Physiol.278: C315–C322, 2000.
13. Ciovacco WA, Goldberg CG, Taylor AF, Lemieux JM, Horowitz MC, Donahue HJ, Kacena MA. The role of gap junctions in megakaryocyte-mediated osteoblast proliferation and differentiation. Bone. 2009;44:80-86
14. Elizabeth A Hanna, Sandra Umhauer, Stacie L Roshong, et al. Gap junctional intercellular communication and connexin43 expression in human ovarian surface epithelial cells and ovarian carcinomas in vivo and in vitro. Carcinogenesis, Jul 1999; 20: 1369 - 1373.
15. Nichel BM, DeFranci BH and Gay VL,et al.Clathrin and Cx43 gap junction plaque endocytosis.Biochemical and Biophy Research Communications.2008;374:679-682.
16. Daniel-Wojcik A,Misztal K,Bechyne I and Ian M.Cell motility affects the intensity of gap junctional coupling in prostate carcinoma and melanoma cell populations.Intl J Oncol 2008;33;309-315
17. Li Z;Zhou,Z;Donahue HJ.Alterations in Cx43 and OB-cadherin affect breast cancer cell metastatic potential.Clin&Exp Metastasis,2008;25:265-272
18. Park SY, Lee HE, Li H, Shipitsin M, Gelman R, Polyak K. Heterogeneity for stem cell-related markers according to tumor subtype and histologic stage in breast cancer. Clin Cancer Res. 2010 ;16:876-87.
19. Tekpli X, Rivedal E, Gorria M, Landvik NE, Rissel M, Dimanche-Boitrel MT, Baffet G, Holme JA, Lagadic-Gossmann D. The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis. Toxicol Appl Pharmacol. 2010;242:231-40.
20. Levine B,Kroemer G .Autophagy in the pathogenesis of disease.Cell,2008,132:27-42
21. Kuma A,Hatano M,Matsui M,el a1.The role of autophagy during the early neonatal starvation period[J].Nature,2004,432:1032一1036.
22. Hara T,Nakamura K,Matsui M,el a1.Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice[J].Nature,2006,441:885—889
23. Klara G, Santiago S, Katalin P. Mouse plasmacytoma: an experimental model of human multiple myeloma. Haematologica, 2001; 86:227-236
24. Seishi Kyoizumi, Charles M. Baum, Hideto Kaneshima,et al. Implantation and Maintenance of Functional Human Bone Marrow in SCID-hu Mice. Blood[J]. 1992 April. Vol79(7): 1704-1711
25. Kyoizumi S, Murray LJ, Namikawa R Preclinical analysis of cytokine therapy in the SCID-hu mouse. Blood, 81:1479, 1993
26. R Namikawa, R Ueda, and S Kyoizumi. Growth of human myeloid leukemias in the human marrow environment of SCID-hu mice. Blood, Oct 1993; 82: 2526 - 2536.
27. Shmuel Yaccoby, Bart Barlogie, Joshua Epstein. Primary Myeloma Cells Growing in SCID-hu Mice: A Model for Studying the Biology and Treatment of Myeloma and Its Manifestations. The American Society of Hematology. 1998. 92:2908-2913
1、Niemann A, Takatsuki A, Elsasser H-P.The lysosomotropic agent monodansylcadaverine alsoacts as a solvent polarity probe. J.Histocbem. Cytocbem. 48(2),251-258.
2、Henry J. Donahue, Zhongyong Li, Zhiyi Zhou,et al. Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication. Am. J. Physiol. Cell Physiol.278: C315–C322, 2000.
3、Wendy A,Carolyn G, Amanda F,et al.The role of gap junctions in megakaryocyte mediated osteoblast proliferation and differentiation.Bone.2009.44:80-86.
4、A. F. Taylor, M. M. Saunders, D. L. Shingle,et al. Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions. Am J Physiol Cell Physiol 292:545-552, 2007.
5、Mitsuyoshi Urashima, Benjamin P. Chen, Shirley Chen, et al. The Development of a Model for the Homing of Multiple Myeloma Cells to Human Bone Marrow. Blood, 1997 90: 754-765
6、Preston S L,Alison M R,Forbes S J,et a1.The new stem cell biology:something for everyone [J]. J Clin Pathol:Mol pathol,2003,56:86-96.
7、Bossolasco P,Cova L,Calzarossa C,et al.Neuro-glial differentiation of human bone marrow stem cells in vitro.Exp Neurol.2005:193(2 ):312 -325.
8、Majumdar M K,Thieda M A,Mosea J D, et a1. Phenotypic and functional comparison of cultures of marrow—derived mesenchymal stem cells(Msc)and stromal cells[J].J Cell Physiol,1998,176(1):186-192
9、Terstappen LW ,Johnson S,Segres-Nolton IM ,et a1.Identification and characterization of plasma cells in normal human bone marrow by high—resolution flow cyrometry. Blood,1990,76 ,1739- l 747
10、Wallace S R,OkenM M,Lunetta K L,et a1. Abnormalities of bone marrow mesenchymal cells in multiple myeloma patients[J].Cancer.2002,91(7):1219-1230.
11、Wong RC, Pebay A,nguyen L T, et al.Presence of functional gap junctions in human embryonic stem cells[J]. Stem Cells. 2004,22(6):883-889.
1. Seishi Kyoizumi, Charles M. Baum, Hideto Kaneshima,et al. Implantation and Maintenance of Functional Human Bone Marrow in SCID-hu Mice. Blood[J]. 1992 April. Vol79(7): 1704-1711
2.中华人民共和国科学技术部.关于善待实验动物的指导性意见.2006-09-30.
3. State Council of the People's Republic of China. Administrative Regulations on Medical Institution. 1994-09-01.中华人民共和国国务院.医疗机构管理条例. 1994-09-01.
4. Reiko Namikawa, Katherine N. Weilbaecher, Hideto Kaneshima, et al. Long Term Human Hematopoiesis in the SCID-hu Mouse. J . Exp. Med[J].1990. 172:1055-1063.
5. Mc Cune JM, Namikawa R, Kaneshima H, et al. The SCID-hu mouse: Murine model for the analysis of human hematolymphoid differentiation and function[J]. Science,1988. 241:1632
6. Kyoizumi S, Murray LJ, Namikawa R Preclinical analysis of cytokine therapy in the SCID-hu mouse. Blood, 81:1479, 1993
7. R Namikawa, R Ueda, and S Kyoizumi. Growth of human myeloid leukemias in the human marrow environment of SCID-hu mice. Blood, Oct 1993; 82: 2526 - 2536.
8. Shmuel Yaccoby , Joshua Epstein. The Proliferative Potential of Myeloma Plasma Cells Manifest in the SCID-hu Host.Blood, 1999;94: 3576-3582
9. Yaccoby S, Wezeman MJ, Henderson A, et al. Cancer and the microenvironment: myeloma-osteoclast interactions as a model. Cancer Res. 2004;64:2016-2023.
10. Flanagan. SP. "Nude", a new hairless gene with pleiotropic effects in the mouse. Genet[J]. 1966. Research 8: 295-309
11. Pantelouris EM. Absence of thymus in a mouse mutant. Nature[J]. 1968. 217: 370-371
12. Shang-Yi Huang, Hwei-Fang Tien, Fang-Hsein Su, et al. Nonirradiated NOD/SCID-Human Chimeric Animal Model for Primary Human Multiple Myeloma. American Journal of Pathology[J]. 2004. 164, 2:747-756
1. K. R. Mcintimif, R. M. Asofsky, M. Potter, et al. Macroglobulin-Producing Plasma-Cell Tumor in Mice: Identification of a New Light Chain. Science, Oct 1965; 150: 361-363
2. Mitsuyoshi Urashima, Benjamin P. Chen, Shirley Chen, et al. The Development of a Model for the Homing of Multiple Myeloma Cells to Human Bone Marrow. Blood, 1997 90: 754-765
3. Bataille R, Harousseau JL. Multiple myeloma. N Engl J Med[J]. 1997. 336:1657-1664
4. Giuliani N, Colla S, and Rizzoli V. New insight in the mechanism of osteoclast activation and formation in multiple myeloma: Focus on the receptor activator of NF-κB ligand (RANKL). Exp Hematol[J]. 2004. 32: 685-691
5. Pierfrancesco Tassone, Paola Neri, Daniel R, et al. Aclinically relevant SCID-hu in vivo model of human multiple myeloma. Blood.2005;106:713-716)
6. Shmuel Yaccoby, Bart Barlogie, Joshua Epstein. Primary Myeloma Cells Growing in SCID-hu Mice: A Model for Studying the Biology and Treatment of Myeloma and Its Manifestations. The American Society of Hematology. 1998;29:08-2913
7. Chu MS,Chang CF,Yang CC,et a1.Signalling pathway in the in duction of neurite outgrowth in human mesenchymal stem cells[J].Cell Signal,2006,18(4):519-530
8. Shmuel Yaccoby , Joshua Epstein. The Proliferative Potential of Myeloma Plasma Cells Manifest in the SCID-hu Host.Blood, 1999;94: 3576-3582
9. Yaccoby S, Wezeman MJ, Henderson A, et al. Cancer and the microenvironment: myeloma-osteoclast interactions as a model. Cancer Res. 2004;64:2016-2023.
1. Guid T.Multiple myeloma and other plasma disorders [M]//Hoffman R.Hematology- basic principles and practice.4th ed.Elsevier,2005:1501-1505.
2.徐秀芹,刘春霞.多发性骨髓瘤40例血清免疫学特征.河北医药2008; 30:1523-1524.
3. Solly S. Remarks on the pathology of mollities ossium with cases. Med Chir Trans Lond. 1844. 27: 435-461
4. Robert A, Kyle and S, Vincent Rajkumar. Multiple myeloma. Blood. 2008; 111: 2962-2972
5. Bence Jones H. On the new substance occurring, 2969 in the urine of a patient with mollities ossium. Philos Trans R Soc Lond. 1848.138:55-62
6. Fleischer R XXIV. Ueber das Vorkommen des sogenannten Bence Jones’schen Eiweisskorpers im normalen Knochenmark. Arch Pathol Anatom Physiol Klin Med. 1880;80:842-849.
7. Kyle RA. Multiple myeloma: an odyssey of discovery. Br J Haematol[J]. 2000. 111:1035-1044
8. Robert A. Kyle, Morie A. Gertz, Thomas E,et al. Review of 1027 Patients With Newly Diagnosed Multiple Myeloma. Mayo Clin. Proc., Jan 2003; 78: 21 - 33.
9. Wright JH. A case of multiple myeloma. Trans Assoc Am Phys. 1900. 15:137-147
10. Arinkin MI. Die intravitale Untersuchungsmethodikdes Knochenmarks. Folia Haematol. 1929; 38: 233- 340
11. Perlzweig WA, Delrue G, Geschicter C. Hyperproteinemia associated with multiple myelomas: report of an unusual case. JAMA. 1928. 90:755-757
12. Longsworth LG, Shedlovsky T, MacInnes DA. Electrophoretic patterns of normal and pathological human blood, serum, and plasma. J Exp Med[J].1939. 70:399-413
13. Waldenstro¨m J. Studies on conditions associated with disturbed gamma globulin formation (gammopathies).Harvey Lect. 1961;56:211-231.
14. Fonseca R,Barlogie B,Bataille R,et a1.Genetics and cytogenetics of m ultiple myeloma:a workshop report.Cancer Ras,2004,64(4):1546—1558.
15.王良哲.多发性骨髓瘤发病机制研究新进展.国际输血及血液学杂志.2007; 30(4):319-322
16. Bergsagel PL,Kuehl WM.Critical roles for immunoglobulin translocations and cyclin D dysregulation in multiple myeloma[J].Immunol Rev,2003,194:96-104.
17. Greco C ,D’Agnano I,V itelli G,et a1.c-MYC deregulation is involved in melphalan resistance of multiple myeloma:role of PDGF-BB.Int J Immunopathol Pharmacol,2006,l9(1):67-79.
18.李勇华,侯健.多发性骨髓瘤的发病机制.中国全科医学.2007;10:1492-1495
19.侯健,傅卫军.努力提高多发性骨髓瘤的基础和临床研究水平.国际输血及血液学杂志.2007;30:289-290
20. Zojer, N., H. Ludwig, et al. Patterns of somatic mutations in VH genes reveal pathways of clonal transformation from MGUS to multiple myeloma. Blood [J]. 2003. 101(10): 4137-9
21. Preston S L,Alison M R,Forbes S J,et a1.The new stem cell biology:something for everyone [J]. J Clin Pathol:Mol pathol,2003,56:86-96.
22. Majumdar MK ,Thiede MA,Havnesworth SE ,et al.Human marrow-derived mesenchymal stem cells (MSCs) express hematopietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages. J Hematother stem Cell Res.2000 :9 (6 ):841-848
23. Teoh G,Anderson KC. Interaction of tumor and host cells with adhesion and extracellular matrix molecules in the development of multiple myeloma. Hematol Oncol Clin North Am.1997;11[1]:27-42
24. Urashima M,Chauhan D,Uchiyama H,et a1. CD40 ligand triggered interleukin-6 secretion in multiple myeloma.Blood.1995;85:1903
25. Bernard Klein, Xue-Guang Zhang, Zhao-Yang Lu. Interleukin-6 in Human Multiple Myeloma. Blood, 1995; 85: 863 - 872.
26. AdnanA,David H.Multiple mydoma:an old disease with new hope for the future [J].Cancer J Chin.2002,51:273-285
27. Dharminder C, Surender K, Atsushi O,et al. Interleukin-6 inhibits Fas-induced apoptosis and stress-activated protein kinase activation in multiple myeloma cells. Blood, Jan 1997; 89: 227– 234
28. A Lichtenstein, Y Tu, C Fady, R Vescio,et al. Interleukin-6 inhibits apoptosis of malignant plasma cells. Cell Immunol, May 1995; 162(2): 248-55
29. Kumar S,Witzig TE,Timm M,et a1.Bone marrow angiogenic ability and expression ofangiogenic cytokines in myeloma:Evidence favoring loss of marrow angiogenesis inhibitory activity with disease progression.Blood,2004,104(4):1159-1165.
30. Vacca A,Ribatti D,Roncali L,et a1. Bone marrow angiogenesis and progression in multiple myeloma[J].Br J Haematol,1994,87(3):503-508
31. Dankbar B,Padro T,Leo R,et a1.Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma [J]. Blood, 2000, 95(8): 2630-2636
32. Podar K,Tai Y T,Davies F E,et a1.Vascular endothelial growth factor triggers signaling cascades mediating multiple myeloma cell growth and migration [J].Blood,2001,98(2): 428-435.
33. Lin B,Podar K,Gupta D,et a1.The vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584 inhibits growt h and migration of multiple myeloma cells in the bone marrow microenvironment [J].Cancer Res, 2002, 62(17):5019-5026
34. Podar K ,Catley L P,Tai Y T,et a1.GW 654652,the pan-inhibitor of VEGF receptors,blocks the growth and migration of multiple myeloma cells in the bo ne marrow microenvironment [J].Blood,2004,103(9):3474-3479.
35. Uchiyama H,Anderson K C.Cellular adhesion molecules[J].Transf Med Rev. 1994; 8:84-95.
36. Hata H,et al Autocrine growth by two cytokine IL-6 and TNF, in the myeloma cell line KHMIA. Acra Hematol,1990;83:-93
37. Asosingh K,Gunthert U,Bakkus MH,et a1.In vivo induction of insulinlike growth factor-1 receptor and CD44v6 confers homingand adhesion to murine multiple myeloma cells.Cancer Res,2000,60(11):3096-3104.
38. Qiang YW.Yao L,Tosato G,et aI.Insulin—like growth factor-1 induces migration and invasion of human multiple myeloma cells.Blood,2004,l03(1):30l-308.
39. Qiang Y,Kopantzev E,Rudikoff S.Insulinllke growth factor-1 signaling in multiple myeloma:downstream elements,functionalcorrelates,and pathway cross-talk.Blood,2002,99(11): 4138-4146.
40. Mitsiades N,Mitsiade C S,poulalki V,et a1.Apoptotie signaling induced by immunomodulatory thalidomide analogs in human multiple myeloma cells :therapeuticimplications.[J]Blood,2002,99(12):4525-4530.
41. Owen M E,Cave J,Joyner C J.Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F [J].J Cell Science,1987,87:731-738.
42. Boiret N,Rapatel V,Veyrat-Masson R,et a1.Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow. Exp Hematol 2005;33(2):219-225
43. Phinney D. G., Prockop D. J. Mesenchymal stem/multi-potent stromal cells (MSCs): the state of transdifferentiation and modes of tissue repair -current views. Stem Cells[J]. 2007. 25:2896-2902
44. Wang X ,Hisha H,Taketanis S,et al.Characterization of mesenchymal stem cells isolated from mouse fetal bone marrow .Stem Cells.2006:24 (3 ):482– 493
45. Wislet-Gendebien S,Hans G,Leprince P, et al. Plasticity of cultured mesenchymal stem cells:switch from nestin-positive to excitable neuron-like phenotype. Stem Cell. 2005:23 (3 ):392 -402
46. Bossolasco P,Cova L,Calzarossa C,et al.Neuro-glial differentiation of human bone marrow stem cells in vitro.Exp Neurol.2005:193(2 ):312 -325.
47. Wallace S R,Oken M M.Lunetta K L,et a1.Abnormalities of bone marrow mesenchymal cells in multiple myeloma patients[J].Cancer,2002,91(7):1219-1230
48. Fermand JP, Ravaud P, Chevret S, et al. Highdose therapy and autologous peripheral blood stem cell transplantation in multiple myeloma: up-front or rescue treatment? Results of a multicenter sequential randomized clinical trial. Blood[J]. 1998. 92:3131-3136.