尼克酰胺联合Exendin-4对STZ大鼠骨髓干细胞的体内诱导作用
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摘要
目的
近年来,糖尿病发病率呈上升趋势,而且更趋年轻化。预计截止到2025年,世界范围内将有3.34亿人患有糖尿病。胰岛移植是一种有效治疗糖尿病的方法,不仅可以避免胰岛素注射引起的低血糖和胰岛素抵抗,更重要的是能减少和改善糖尿病并发症。然而胰岛移植由于胰腺供体严重缺乏而受到限制,并且移植后出现的免疫排斥问题也无法解决。干细胞可定向分化为胰岛素分泌细胞(Insulin-producing cells,IPCs),成为糖尿病治疗的一个新的研究方向。骨髓干细胞(Bone marrow stem cells,BMSCs)是成体干细胞的一种,具有多向分化潜能和较强的可塑性。最近的研究表明,在体外适当的条件下,BMSCs可以转分化为IPCs,将提供无限的可供移植的IPCs,避免了由于供体缺乏及免疫排斥反应带来的问题。但是离体干细胞移植存在易污染、易变异等问题。因此,本研究探讨尼克酰胺(Nicotinamide,N)联合长效胰高血糖素样肽-1类似物(Exendin-4,E-4)在链脲佐菌素(STZ)依赖糖尿病大鼠模型体内能否诱导BMSCs转分化IPCs,对大鼠糖尿病是否有治疗作用。
材料和方法
一、实验动物分组及处理
50只雄性Wistar大鼠按随机数字表随机分为两组:正常组(10只)和造模组(40只)。造模组大鼠一次性腹腔注射1%STZ 55mg/kg,建立STZ大鼠模型,将造模成功大鼠随机分为糖尿病未治疗组、单纯动员组、单纯用药组和动员并诱导组,每组10只。动员并诱导组大鼠分别皮下注射10mg/kg.d重组人粒细胞集落刺激因子(rhG-CSF,商品名特尔津),连续5天,在注射rhG-CSF第2天始,分别腹腔注射100 mg/kg.d N,连续7天,然后再给予腹腔注射3ug/kg.d的E-4,连续7天;单纯用药组大鼠分别皮下注射10mg/kg.d生理盐水,连续5天,在注射生理盐水第2天始,腹腔注射100 mg/kg.d N,连续7天,然后再给予腹腔注射3ug/kg.d的E-4,连续7天;单纯动员组大鼠分别皮下注射10mg/kg.d rhG-CSF,连续5天,然后再给予腹腔注射同体积生理盐水;正常组及糖尿病未治疗组大鼠分别腹腔注射同体积的生理盐水。
二、生化指标的检测及方法
整个实验过程中,各组大鼠每周测一次体重,每2周测一次血糖。治疗前和实验结束时,各取血测血清胰岛素(ELISA法)。实验终止时,用10%水合氯醛3ml/kg腹腔注射麻醉,取胰腺、肝脏和脾脏,用免疫组织化学染色(SABC法)检测其中胰岛素染色阳性细胞的表达。图像采集分析系统对各组大鼠胰腺组织中胰岛素染色阳性细胞占整个胰岛面积百分比进行分析。
结果
一、生化检测结果
治疗前,糖尿病未治疗组、单纯动员组、单纯用药组及动员并诱导组大鼠血糖、血清胰岛素无显著性差异。治疗2周时,动员并诱导组及单纯动员组大鼠的血糖低于糖尿病未治疗组及单纯用药组(P＜0.05),但仍高于正常组(P＜0.05);治疗6周时,动员并诱导组大鼠的血糖明显低于糖尿病未治疗组、单纯动员组、单纯用药组,但高于正常组(P＜0.05);血清胰岛素水平高于糖尿病未治疗组、单纯动员组、单纯用药组(P＜0.05),与正常组无显著性差异。单纯动员组、单纯用药组大鼠的血糖、血清胰岛素与糖尿病未治疗组无显著性差异(P＞0.05)。整个实验中,正常组大鼠的体重始终高于其它各组(P＜0.05)。
二、免疫组织化学及图像分析结果
实验终止时,免疫组织化学检测各组大鼠胰腺中胰岛素阳性细胞的百分比发现:正常组明显高于其他各组(P＜0.01);动员并诱导组低于正常组,但是明显高于糖尿病未治疗组(P＜0.01)、单纯动员组(P＜0.01)及单纯用药组(P＜0.01);单纯动员组、单纯用药组与糖尿病未治疗组无显著性差异(P＞0.05)。动员并诱导组大鼠的肝脏和脾脏中均发现胰岛素染色阳性细胞的表达,其他各组大鼠的肝脏和脾脏中均未发现胰岛素染色阳性细胞的表达。
结论
1、N联合E-4可以在体内将BMSCs转分化为IPCs。
2、由BMSCs分化而来的IPCs可以改善STZ大鼠的高血糖症,提高胰岛素分泌量,也有一定的胰岛修复作用,但对糖尿病的长期治疗效果尚待进一步研究。
Objective
In recent years,the incidence of diabetes on the rise,more and younger.The closing is expected by the year 2025,worldwide there will be 334 million people with diabetes.Islet transplantation is an effective way to treat diabetes,insulin injections can be avoided not only caused by low blood sugar or insulin resistance and,more importantly,can reduce the complications of diabetes and improve.However,due to pancreatic islet transplant donor and a serious lack of restrictions,and the emergence of post-transplant immune rejection problems to solve.Stem cells can be directed to differentiate into insulin-secreting cells to become a new treatment for diabetes research.Bone marrow stem cells are adult stem cells as a has more potential to divide and clear plastic.Recent studies have shown that in extracorporeal right conditions,can be bone marrow stem cells to differentiate into insulin-secreting cells,will provide unlimited islet cells for transplantation to avoid because of a lack Donor and immune rejection problems brought about.However,in vitro stem cell transplantation existence of e-pollution and easy variation,and other issues.As a result,this study was to explore Nicotinamide and Exendin-4 in the(STZ)-dependent diabetic model in the body can induce bone marrow stem cells IPCs,on whether the treatment of diabetic rats role.
Materials and methods
50 male Wistar rats were randomly divided into two groups randomly:normal group(10 wistar rats in each group),model group(40 wistar rats in each group).The model group were treated with single intraperitoneal injection of 1%streptozotocin (STZ,55 mg/kg body wt) to establish diabetes model.STZ diabetic rats were randomly allocated into four groups:diabetic group,mobilization group,treatment group,induct- ion group(10 wistar rats in each group).In induction group the rats were treated for 5 days with once-daily subcutaneously injection of recombination human Granulocytecolony stimulating factor(rhG-CSF,10 mg/kg body wt),in addition,on the 2nd day after mobilization injection,treat the rats with intraperitoneal injection of nicotinamide(100 mg/kg.d),for 7 days;and then give them intraperitoneal injection of Exendin-4(3ug/kg.d),for 7 days;The treatment group rat treated for 5 days with once-daily subcutaneously injection of normal saline(10 mg/kg body wt),in addition, on the 2nd day after mobilization injection,treat the rats with intraperitoneal injection of nicotinamiamide(100 mg/kg.d),for 7 days;and then give them intraperitoneal injection of Exendin-4(3ug/kg.d),for 7 days;The mobilization of rats were injected rhG-CSF(10mg/kg.d),for 5 days,then given the same volume of normal saline injection,14 days in a row;Normal and diabetic rats were injected with the volume of normal saline.
In the process of experiment,the weight were measured every week,measured the blood glucose every two weeks,before treatment and the end of the study measured the fasting serum insulin concentration.At the end of the study,treat the rats with intraperitoneal injection of 10%Chloral Hydrate(3 ml/kg body wt) to anesthetize them. Remove the liver,spleen,pancreas,and then measure the expression of insulin positive cell by immunohistochemistry,the area of insulin positive cell in pancreatic islet were measured by using image analysis software.
Results
Before treatment,the blood glucose and fasting serum insulin concentration level has no significant difference among diabetes group,mobilization group,treatment group,induction group.At 2 weeks of treatment,the blood glucose level of mobilization group and induction group is higher than the normal group but lower that the diabetes group and treatment group(p＜0.05).At the end of study,the blood glucose level of induction group is significant lower than diabetes group,(p＜0.05),but still higher than normal group,the fasting serum insulin concentration of induction group is significant higher than diabetes group,mobilization group,treatment group,(p＜0.05), compared with normal group,the fasting serum insulin concentration level has no significant difference.Compared with diabetes group,the blood glucose and fasting serum insulin concentration of mobilization group and treatment group have no significant difference(P＞0.05).In the whole process,the weight of normal group is always higher than other groups.
At the end of our experiment we found that the ratio of insulin positive cell in normal group which have been measured by immunohisto chemistry was conspicuous higher than other each group,the ratio of induction group is lower than normal group, but higher than diabetes group and mobilization group,and there has no significant difference between mobilization and diabetes group(P＞0.05).We found insulin positive cell expression in liver and spleen in induction groups.We did not find any expression of insulin positive cell in liver and spleen in other groups.
Conclusion
Combination therapy with Nicotinamide and Exendin-4 could induce differention of bone marrow stem cell into insulin-producing cell,and the cells can improve high blood sugar and improve insulin secretion,but also a certain role in pancreatic islet repaired,but the long-term diabetes treatment to be studied further.
近年来,糖尿病发病率呈上升趋势,而且更趋年轻化。预计截止到2025年,世界范围内将有3.34亿人患有糖尿病。胰岛移植是一种有效治疗糖尿病的方法,不仅可以避免胰岛素注射引起的低血糖和胰岛素抵抗,更重要的是能减少和改善糖尿病并发症。然而胰岛移植由于胰腺供体严重缺乏而受到限制,并且移植后出现的免疫排斥问题也无法解决。干细胞可定向分化为胰岛素分泌细胞(Insulin-producing cells,IPCs),成为糖尿病治疗的一个新的研究方向。骨髓干细胞(Bone marrow stem cells,BMSCs)是成体干细胞的一种,具有多向分化潜能和较强的可塑性。最近的研究表明,在体外适当的条件下,BMSCs可以转分化为IPCs,将提供无限的可供移植的IPCs,避免了由于供体缺乏及免疫排斥反应带来的问题。但是离体干细胞移植存在易污染、易变异等问题。因此,本研究探讨尼克酰胺(Nicotinamide,N)联合长效胰高血糖素样肽-1类似物(Exendin-4,E-4)在链脲佐菌素(STZ)依赖糖尿病大鼠模型体内能否诱导BMSCs转分化IPCs,对大鼠糖尿病是否有治疗作用。
材料和方法
一、实验动物分组及处理
50只雄性Wistar大鼠按随机数字表随机分为两组:正常组(10只)和造模组(40只)。造模组大鼠一次性腹腔注射1%STZ 55mg/kg,建立STZ大鼠模型,将造模成功大鼠随机分为糖尿病未治疗组、单纯动员组、单纯用药组和动员并诱导组,每组10只。动员并诱导组大鼠分别皮下注射10mg/kg.d重组人粒细胞集落刺激因子(rhG-CSF,商品名特尔津),连续5天,在注射rhG-CSF第2天始,分别腹腔注射100 mg/kg.d N,连续7天,然后再给予腹腔注射3ug/kg.d的E-4,连续7天;单纯用药组大鼠分别皮下注射10mg/kg.d生理盐水,连续5天,在注射生理盐水第2天始,腹腔注射100 mg/kg.d N,连续7天,然后再给予腹腔注射3ug/kg.d的E-4,连续7天;单纯动员组大鼠分别皮下注射10mg/kg.d rhG-CSF,连续5天,然后再给予腹腔注射同体积生理盐水;正常组及糖尿病未治疗组大鼠分别腹腔注射同体积的生理盐水。
二、生化指标的检测及方法
整个实验过程中,各组大鼠每周测一次体重,每2周测一次血糖。治疗前和实验结束时,各取血测血清胰岛素(ELISA法)。实验终止时,用10%水合氯醛3ml/kg腹腔注射麻醉,取胰腺、肝脏和脾脏,用免疫组织化学染色(SABC法)检测其中胰岛素染色阳性细胞的表达。图像采集分析系统对各组大鼠胰腺组织中胰岛素染色阳性细胞占整个胰岛面积百分比进行分析。
结果
一、生化检测结果
治疗前,糖尿病未治疗组、单纯动员组、单纯用药组及动员并诱导组大鼠血糖、血清胰岛素无显著性差异。治疗2周时,动员并诱导组及单纯动员组大鼠的血糖低于糖尿病未治疗组及单纯用药组(P＜0.05),但仍高于正常组(P＜0.05);治疗6周时,动员并诱导组大鼠的血糖明显低于糖尿病未治疗组、单纯动员组、单纯用药组,但高于正常组(P＜0.05);血清胰岛素水平高于糖尿病未治疗组、单纯动员组、单纯用药组(P＜0.05),与正常组无显著性差异。单纯动员组、单纯用药组大鼠的血糖、血清胰岛素与糖尿病未治疗组无显著性差异(P＞0.05)。整个实验中,正常组大鼠的体重始终高于其它各组(P＜0.05)。
二、免疫组织化学及图像分析结果
实验终止时,免疫组织化学检测各组大鼠胰腺中胰岛素阳性细胞的百分比发现:正常组明显高于其他各组(P＜0.01);动员并诱导组低于正常组,但是明显高于糖尿病未治疗组(P＜0.01)、单纯动员组(P＜0.01)及单纯用药组(P＜0.01);单纯动员组、单纯用药组与糖尿病未治疗组无显著性差异(P＞0.05)。动员并诱导组大鼠的肝脏和脾脏中均发现胰岛素染色阳性细胞的表达,其他各组大鼠的肝脏和脾脏中均未发现胰岛素染色阳性细胞的表达。
结论
1、N联合E-4可以在体内将BMSCs转分化为IPCs。
2、由BMSCs分化而来的IPCs可以改善STZ大鼠的高血糖症,提高胰岛素分泌量,也有一定的胰岛修复作用,但对糖尿病的长期治疗效果尚待进一步研究。
Objective
In recent years,the incidence of diabetes on the rise,more and younger.The closing is expected by the year 2025,worldwide there will be 334 million people with diabetes.Islet transplantation is an effective way to treat diabetes,insulin injections can be avoided not only caused by low blood sugar or insulin resistance and,more importantly,can reduce the complications of diabetes and improve.However,due to pancreatic islet transplant donor and a serious lack of restrictions,and the emergence of post-transplant immune rejection problems to solve.Stem cells can be directed to differentiate into insulin-secreting cells to become a new treatment for diabetes research.Bone marrow stem cells are adult stem cells as a has more potential to divide and clear plastic.Recent studies have shown that in extracorporeal right conditions,can be bone marrow stem cells to differentiate into insulin-secreting cells,will provide unlimited islet cells for transplantation to avoid because of a lack Donor and immune rejection problems brought about.However,in vitro stem cell transplantation existence of e-pollution and easy variation,and other issues.As a result,this study was to explore Nicotinamide and Exendin-4 in the(STZ)-dependent diabetic model in the body can induce bone marrow stem cells IPCs,on whether the treatment of diabetic rats role.
Materials and methods
50 male Wistar rats were randomly divided into two groups randomly:normal group(10 wistar rats in each group),model group(40 wistar rats in each group).The model group were treated with single intraperitoneal injection of 1%streptozotocin (STZ,55 mg/kg body wt) to establish diabetes model.STZ diabetic rats were randomly allocated into four groups:diabetic group,mobilization group,treatment group,induct- ion group(10 wistar rats in each group).In induction group the rats were treated for 5 days with once-daily subcutaneously injection of recombination human Granulocytecolony stimulating factor(rhG-CSF,10 mg/kg body wt),in addition,on the 2nd day after mobilization injection,treat the rats with intraperitoneal injection of nicotinamide(100 mg/kg.d),for 7 days;and then give them intraperitoneal injection of Exendin-4(3ug/kg.d),for 7 days;The treatment group rat treated for 5 days with once-daily subcutaneously injection of normal saline(10 mg/kg body wt),in addition, on the 2nd day after mobilization injection,treat the rats with intraperitoneal injection of nicotinamiamide(100 mg/kg.d),for 7 days;and then give them intraperitoneal injection of Exendin-4(3ug/kg.d),for 7 days;The mobilization of rats were injected rhG-CSF(10mg/kg.d),for 5 days,then given the same volume of normal saline injection,14 days in a row;Normal and diabetic rats were injected with the volume of normal saline.
In the process of experiment,the weight were measured every week,measured the blood glucose every two weeks,before treatment and the end of the study measured the fasting serum insulin concentration.At the end of the study,treat the rats with intraperitoneal injection of 10%Chloral Hydrate(3 ml/kg body wt) to anesthetize them. Remove the liver,spleen,pancreas,and then measure the expression of insulin positive cell by immunohistochemistry,the area of insulin positive cell in pancreatic islet were measured by using image analysis software.
Results
Before treatment,the blood glucose and fasting serum insulin concentration level has no significant difference among diabetes group,mobilization group,treatment group,induction group.At 2 weeks of treatment,the blood glucose level of mobilization group and induction group is higher than the normal group but lower that the diabetes group and treatment group(p＜0.05).At the end of study,the blood glucose level of induction group is significant lower than diabetes group,(p＜0.05),but still higher than normal group,the fasting serum insulin concentration of induction group is significant higher than diabetes group,mobilization group,treatment group,(p＜0.05), compared with normal group,the fasting serum insulin concentration level has no significant difference.Compared with diabetes group,the blood glucose and fasting serum insulin concentration of mobilization group and treatment group have no significant difference(P＞0.05).In the whole process,the weight of normal group is always higher than other groups.
At the end of our experiment we found that the ratio of insulin positive cell in normal group which have been measured by immunohisto chemistry was conspicuous higher than other each group,the ratio of induction group is lower than normal group, but higher than diabetes group and mobilization group,and there has no significant difference between mobilization and diabetes group(P＞0.05).We found insulin positive cell expression in liver and spleen in induction groups.We did not find any expression of insulin positive cell in liver and spleen in other groups.
Conclusion
Combination therapy with Nicotinamide and Exendin-4 could induce differention of bone marrow stem cell into insulin-producing cell,and the cells can improve high blood sugar and improve insulin secretion,but also a certain role in pancreatic islet repaired,but the long-term diabetes treatment to be studied further.
引文
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2 Hori Y, Rulifson I C, Tsai B C, et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells [J]. Proc Natl Acad Sci USA, 2002, 99(25): 16105-16110.
3 Shim JH, Kim SE, Woo DH, et al. Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. [J]. Diabetologia. 2007, 50(6): 1228-1238.
4 Jiang J, Au M, Lu K, et al. Generation of insulin-producing islet-like clusters from human embryonic stem cells. [J]. Stem Cells. 2007, 25(8): 1940-1953.
5 Pittengre MF,Mackay AM, Beck SC, et al. Multilineage potential of adult mesenchymal stem cells[J]. Science, 1999,284(5411): 143-147.
6 Kucia M, Ratajczta J, Reca R, et al. Tissue specific muscle, neural and liver stem/progenitor cells reside in the bone marrow, respond to an SDF-1 gradient and are mobilized into peripheral blood during stress and tissue injury [J]. Blood Cells Mol Dis, 2004, 32(1): 52-57.
7 Bruder SP, Kutyh AA, Shea M, et al. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells [J]. J Ortho Res, 1998, 16:155-162.
8 Ferrari G, Coletta M, Paolucci E, et al. Muscle regeneration by bone marrow-derived myogenic progenitors [J]. Science, 1998, 279(5356):1528-1530.
9 Brazerlton TR, Rossi FM V, Keshet GI, et al. From marrow to brain expression of neuronal phenotypers in adult mice [J]. Science, 2000, 290(5497): 1775-1779.
10 Ereira RF, Halford KW,0 Hara MD, et al. Cultured adlherent cells from marrow can long-lasting precursor cells for bone, cartilage, and lung in irradiated mice [J]. Proc Natl Aci USA, 1995, 92(11): 4857-4861.
11 Rockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues [J]. Science, 1997, 276(5 309): 71-74.
12 Sanchez-Ramos J, Song S. Cardozo-pelaze F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro [J]. Exp Neurol, 2000, 164(2): 247-252.
13 Nuttall M E, Patton AJ, Olivera DL, et al. Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype :implications for osteogenic disorders[J]. J Bone Miner Res, 1998. 13(3): 371-376.
14 Mashima H, Ohnishi H, Wakabayashi K, et al. Betacellulin and active A coordinately convert anmylase-secreting pancreatic AR42J cells into insulin-secreting cells [J]. J Clin Invest, 1996, 97(7): 1647-1654.
15 Tang QQ,Jiang MS,Lane MD.Repressive effect of Spl on the C/EBP alpha gene promoter:Role in adipocyte differentiation[J].Mol Cell Biol,1999,19(7):4855-4865
16 Klemm DJ,Roesler WJ,Boras T,et al.Insulin stimulates cAMP-response element binding protein activity in Hep G2 and 3T3-L1 cell line[J].J Biol Chem,1998,273(2):917-923.
17 郭丽,尹飞,王晓丽,等.大鼠骨髓间充质干细胞向神经元的诱导分化[J].吉林大学学报:医学版,2003,29(1):51-54.
18 Li G,Fei Y,Hong QM.et al.Dfferentation of mesenchymal stem cells into dopaminergic neuron-like cells in vitro[J].Biomed Environ Sci,2005,18:36-42.
19 Le Douarin NM.On the origin of pancreatic endocrine cells[J].Cell,1988,53(2):169-171.
20 Jmaes E.Origin and differentiation of human and murine stroma[J].Stem Cell,2002,20:205-214
21 Docherty K.Growth and development of the islets of langerhans:implication for the treatment of diabetes mellitus[J].Curr Opin Pharmacol,2001,1(6):641-650.
22 Hunziker E,Stein M.Nestin-experssing cells in the pancreatic islets of langerhans[J].Biochem Biophys Res Commun,2000,271(1):116-119.
23 Mitsui K,Tokuzawa Y,Itoh H,et al.The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.[J]Cell,2003,113:631-642.
24 Ianus A,Holz GG,Theise ND,et al.In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion[J].J Clin Invest,2003,111:843-850.
25 Kojima H,Fujimiya M,Matsumura K,et al.Extrapancreatic insulin-producing cells in multiple organs in diabetes[J].Proc Natl Acad Sci USA.2004;101(8):2458-2463.
26 Lechner A,Yang YG,Blacken RA,et al.No evidence for significant transdifferentiation of bone marrow into pancreatic beta-cells in vivo.[J].Diabetes.2004;53(3):616-623.
27 Hess D,Li L,Martin M,et al.Bone marrow-derived stem cells initiate pancreatic regeneration [J].Nat Blotechnol.2003,21(7):763-770.
28 Choi JB,Uchino H,Azuma K,et al.Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells[J].Diabetologia,2003,46(10):1366-1374.
29 Tang DQ,Cao LZ,Burkhardt BR,et al.In vivo and in vitro charancterization of insulin-producing cells obtained from murine bone marrow[J].Diabetes,2004,53(7):1721-1732.
30 Oh SH,Muzzonigro TM,Bae SH,et al.Adult bone marrow-derived cells transdifferentiating into insulin-producing cells for the treatment of type Ⅰ diabetes[J].Lab Invest,2004,84(5):607-617.
31 MathewsV,Hanson P,Ford E,et al.Recruitment of bone marrow-derived endotheial cells to sites of pancreatic beta-cell injury[J].Diabetes,2004,53(1):91-98.
32 Chen LB, Jiang XB, Yang L. Differentiation of rat marrow mesenchymal stem cells into pancreatic islet beta-cells [J].World J Gastroenterol, 2004, 10(20): 3016-3020.
33 Blyszczuk P, Czyz J, Kania G, et al. Exp ression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells [J]. Proc Natl Acad Sci USA, 2003, 100:998-1003.
34 Soria B, Roche E, Leon Quinto T, et al. Insulin secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin induced diabetic mice [J]. Diabetes, 2000, 49: 157-162.
35 Kim D, Gu Y, IshiiM, et al. In vivo functioning and transp lantable mature pancreatic islet-like cell clusters differentiated from embryonic stem cell[J]. Pancreas, 2003,27: E34-E41.
36 Lumelsky N, Blondel O, Laeng P, et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets [J]. Science, 2001, 292: 1389-1394.
37 Huang HX, Tang XM. Phenotyp ic determination and characterization of nestin-positive precursors derived from human fetal pancreas [J]. Lab Invest, 2003, 83: 539-547.
38 Ramiya VK, MaraistM, Arfors KE, et al. Reversal of insulin dependent diabetes using islets generated in vitro from pancreatic stem cells[J]. NatMed, 2000, 6: 278 -282.
39 Li Y, Zhang R, Qiao H, et al. Generation of insulin-producing cells from PDX-1 genemodified human mesenchymal stem cells [J]. J Cell Phvsiol. 2007,211(1): 36-44.
40 Wu XH, Liu CP, Xu KF, et al. Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells [J]. Word J Gastroentrol. 2007 Jun 28; 13(24): 3342-9.
2 Bodnar CA,Sen A,Kallos MS,et al.Characterization of human islet-like structures generated from pancreatic precursor cells in culture.Biotechnol Bioeng.2006 Apr 5;93(5):980-8.
3 Ibii T,Shimada H,Miura S,et al.Possibility of insulin-producing cells derived from mouse embryonic stem cells for diabetes treatment.J Biosci Bioeng.2007 Feb;103(2):140-6.
4 Vaca P,Berna G,Araujo,R,et al.Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells.Exp Cell Res.2008 Mar 10;314(5):969-74.
5 Tayaramma T,Ma B,Rohde M,et al.Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells.Stem Cells.2006 Dec;24(12):2858-67.
6 Zhao M,Amiel SA,Ajami S,et al.Amelioration of streptozotocin-induced diabetes in mice with cells derived from human marrow stromal cells.Plos ONE.2008 Jul 16;3(7):e2666.
7 Hisanaga E,Park KY,Yamada S,et al.A simple method to induce differentiation of murine bone marrow mesenchymal cells to insulin-producing cells using conophylline and betacellulin-delta4.Endocr J.2008 Jul;55(3):535-43.
8 Dong-Qi Tang,Li-Zhen Cao,Brant R,et al.In Vivo and In Vitro Characterization of Insulin-Producing Cells Obtained From Murine Bone Marrow.Diabetes.2004 Jul;53(7):1721-32.
9 武晓泓,刘翠萍,茅晓东等.大鼠骨髓间充质干细胞体外诱导分化为胰岛素阳性细胞的潜能[J].中国临床康复,2005,9(34):1-4.
10 Abraham,E.J.,Leech,et al.Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells.Endocrinology.2002,143:3152-3161.
11 Xu,G.,Stoffers,D.A,Habener,J,et al.Exendin-4 stimulates both beta-cell replication and neogenesis,resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats.Diabetes,1999 Dec;48,2270-2276.
12 Movassat,J.,Beattie,G.M.,Lopez,A.D.,et al.Exendin 4 up-regulates expression of PDX 1and hastens differentiation and maturation of human fetal pancreatic cells.J.Clin.Endocrinol.Metab.2002 Oct;87,4775-4781.
13 Suen PM,Li K,Chan JC,et al.In vivo treatment with glucagon-like peptide 1 promotes the graft function of fetal islet-like cell clusters in transplanted mice.Int J Biochem Cell Biol.2006;38(5-6):951-60.
14 Sun Y, Chen L, Hou XG, et al. Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitro.Chin Med J (Engl). 2007 May 5; 120(9): 771-6.
15 15.D. Eberhard, D. Tosh and J. M. W. Slack. Origin of pancreatic endocrine cells from biliary duct epithelium. Cell Mol Life Sci. 2008 Sep 22.
16 Cho YM, Lim JM, Yoo DH, et al. Betacellulin and nicotinamide sustain PDXl expression and induce pancreatic beta-cell differentiation in human embryonic stem cells. Biochem Biophys Res Commun.2008 Febl; 366(1):129-34.
17 Kojima H, Fujimiya M, Matsumura K, et al. Extrapancreatic insulin-producing cells in multiple organs in diabetes. Proc Natl Acad Sci USA.2004;101:2458-2463.
18 Lavazais E, Pogu S, Sa(?) P, et al. Cytokine mobilization of bone marrow cells and pancreatic lesion do not improve streptozotocin-induced diabetes in mice by transdifferentiation of bone marrow cells into insulin-producing cells. Diabetes Metab, 2007; 33(1): 68-78.
1 Lumelsky N, Blondel O, Laeng P, et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets [J]. Science, 2001, 292: 1389-1394.
2 Hori Y, Rulifson I C, Tsai B C, et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells [J]. Proc Natl Acad Sci USA, 2002, 99(25): 16105-16110.
3 Shim JH, Kim SE, Woo DH, et al. Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. [J]. Diabetologia. 2007, 50(6): 1228-1238.
4 Jiang J, Au M, Lu K, et al. Generation of insulin-producing islet-like clusters from human embryonic stem cells. [J]. Stem Cells. 2007, 25(8): 1940-1953.
5 Pittengre MF,Mackay AM, Beck SC, et al. Multilineage potential of adult mesenchymal stem cells[J]. Science, 1999,284(5411): 143-147.
6 Kucia M, Ratajczta J, Reca R, et al. Tissue specific muscle, neural and liver stem/progenitor cells reside in the bone marrow, respond to an SDF-1 gradient and are mobilized into peripheral blood during stress and tissue injury [J]. Blood Cells Mol Dis, 2004, 32(1): 52-57.
7 Bruder SP, Kutyh AA, Shea M, et al. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells [J]. J Ortho Res, 1998, 16:155-162.
8 Ferrari G, Coletta M, Paolucci E, et al. Muscle regeneration by bone marrow-derived myogenic progenitors [J]. Science, 1998, 279(5356):1528-1530.
9 Brazerlton TR, Rossi FM V, Keshet GI, et al. From marrow to brain expression of neuronal phenotypers in adult mice [J]. Science, 2000, 290(5497): 1775-1779.
10 Ereira RF, Halford KW,0 Hara MD, et al. Cultured adlherent cells from marrow can long-lasting precursor cells for bone, cartilage, and lung in irradiated mice [J]. Proc Natl Aci USA, 1995, 92(11): 4857-4861.
11 Rockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues [J]. Science, 1997, 276(5 309): 71-74.
12 Sanchez-Ramos J, Song S. Cardozo-pelaze F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro [J]. Exp Neurol, 2000, 164(2): 247-252.
13 Nuttall M E, Patton AJ, Olivera DL, et al. Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype :implications for osteogenic disorders[J]. J Bone Miner Res, 1998. 13(3): 371-376.
14 Mashima H, Ohnishi H, Wakabayashi K, et al. Betacellulin and active A coordinately convert anmylase-secreting pancreatic AR42J cells into insulin-secreting cells [J]. J Clin Invest, 1996, 97(7): 1647-1654.
15 Tang QQ,Jiang MS,Lane MD.Repressive effect of Spl on the C/EBP alpha gene promoter:Role in adipocyte differentiation[J].Mol Cell Biol,1999,19(7):4855-4865
16 Klemm DJ,Roesler WJ,Boras T,et al.Insulin stimulates cAMP-response element binding protein activity in Hep G2 and 3T3-L1 cell line[J].J Biol Chem,1998,273(2):917-923.
17 郭丽,尹飞,王晓丽,等.大鼠骨髓间充质干细胞向神经元的诱导分化[J].吉林大学学报:医学版,2003,29(1):51-54.
18 Li G,Fei Y,Hong QM.et al.Dfferentation of mesenchymal stem cells into dopaminergic neuron-like cells in vitro[J].Biomed Environ Sci,2005,18:36-42.
19 Le Douarin NM.On the origin of pancreatic endocrine cells[J].Cell,1988,53(2):169-171.
20 Jmaes E.Origin and differentiation of human and murine stroma[J].Stem Cell,2002,20:205-214
21 Docherty K.Growth and development of the islets of langerhans:implication for the treatment of diabetes mellitus[J].Curr Opin Pharmacol,2001,1(6):641-650.
22 Hunziker E,Stein M.Nestin-experssing cells in the pancreatic islets of langerhans[J].Biochem Biophys Res Commun,2000,271(1):116-119.
23 Mitsui K,Tokuzawa Y,Itoh H,et al.The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.[J]Cell,2003,113:631-642.
24 Ianus A,Holz GG,Theise ND,et al.In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion[J].J Clin Invest,2003,111:843-850.
25 Kojima H,Fujimiya M,Matsumura K,et al.Extrapancreatic insulin-producing cells in multiple organs in diabetes[J].Proc Natl Acad Sci USA.2004;101(8):2458-2463.
26 Lechner A,Yang YG,Blacken RA,et al.No evidence for significant transdifferentiation of bone marrow into pancreatic beta-cells in vivo.[J].Diabetes.2004;53(3):616-623.
27 Hess D,Li L,Martin M,et al.Bone marrow-derived stem cells initiate pancreatic regeneration [J].Nat Blotechnol.2003,21(7):763-770.
28 Choi JB,Uchino H,Azuma K,et al.Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells[J].Diabetologia,2003,46(10):1366-1374.
29 Tang DQ,Cao LZ,Burkhardt BR,et al.In vivo and in vitro charancterization of insulin-producing cells obtained from murine bone marrow[J].Diabetes,2004,53(7):1721-1732.
30 Oh SH,Muzzonigro TM,Bae SH,et al.Adult bone marrow-derived cells transdifferentiating into insulin-producing cells for the treatment of type Ⅰ diabetes[J].Lab Invest,2004,84(5):607-617.
31 MathewsV,Hanson P,Ford E,et al.Recruitment of bone marrow-derived endotheial cells to sites of pancreatic beta-cell injury[J].Diabetes,2004,53(1):91-98.
32 Chen LB, Jiang XB, Yang L. Differentiation of rat marrow mesenchymal stem cells into pancreatic islet beta-cells [J].World J Gastroenterol, 2004, 10(20): 3016-3020.
33 Blyszczuk P, Czyz J, Kania G, et al. Exp ression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells [J]. Proc Natl Acad Sci USA, 2003, 100:998-1003.
34 Soria B, Roche E, Leon Quinto T, et al. Insulin secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin induced diabetic mice [J]. Diabetes, 2000, 49: 157-162.
35 Kim D, Gu Y, IshiiM, et al. In vivo functioning and transp lantable mature pancreatic islet-like cell clusters differentiated from embryonic stem cell[J]. Pancreas, 2003,27: E34-E41.
36 Lumelsky N, Blondel O, Laeng P, et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets [J]. Science, 2001, 292: 1389-1394.
37 Huang HX, Tang XM. Phenotyp ic determination and characterization of nestin-positive precursors derived from human fetal pancreas [J]. Lab Invest, 2003, 83: 539-547.
38 Ramiya VK, MaraistM, Arfors KE, et al. Reversal of insulin dependent diabetes using islets generated in vitro from pancreatic stem cells[J]. NatMed, 2000, 6: 278 -282.
39 Li Y, Zhang R, Qiao H, et al. Generation of insulin-producing cells from PDX-1 genemodified human mesenchymal stem cells [J]. J Cell Phvsiol. 2007,211(1): 36-44.
40 Wu XH, Liu CP, Xu KF, et al. Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells [J]. Word J Gastroentrol. 2007 Jun 28; 13(24): 3342-9.