胎鼠胰腺干细胞体内移植治疗1型糖尿病的实验研究
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摘要
临床胰岛移植最近几年的突破性进展,为糖尿病的治愈带来了希望,但是供体缺乏制约了该项技术的广泛应用。干细胞具有高度分化和自我更新的的能力,是解决胰岛细胞来源的热点途径之一。本研究采用干细胞体内移植的方法,观察干细胞在体内分化的效果,为糖尿病的治疗提供一种新的思路。
第一部分不同移植部位对胎鼠胰腺干细胞分化为胰岛样细胞团的影响
【摘要】目的:利用胎鼠胰腺干细胞移植入1型糖尿病模型大鼠,探讨胎鼠胰腺干细胞在不同移植部位体内分化为胰岛细胞团的可能性,为1型糖尿病的治疗寻找一种新的可行的方法。方法:分离纯化孕16日SD大鼠胎鼠胰腺干细胞培养传代;荧光原位杂交(FISH)检测Y染色体;取第3代雄性胎鼠胰腺干细胞行Nestin、PDX-1免疫组化及流式细胞术鉴定。每只雌性糖尿病模型移植雄性胎鼠1×106个胰腺干细胞,分为胰腺原位移植组(10只)、肝内移植组(10只)、肾包膜下移植组(10只)、实验对照组(10只)及空白对照组(10只),定期监测各组大鼠血糖及血浆胰岛素含量,8周后处死大鼠,取相应组织观察胎鼠胰腺干细胞体内转分化情况并行FISH检测Y染色体鉴定。结果:12只胎鼠中有5只经FISH检测为雄性。雄性胎鼠胰腺干细胞培养传代3代后细胞爬片免疫组化示存在Nestin阳性细胞,流式细胞术测定nestin阳性细胞含量占74.1%。胰腺实质内移植组在第3周血糖开始下降,血浆胰岛素水平逐渐升高;第5周血糖降至正常水平并维持,血浆胰岛素达到正常水平。病理学观察在胰腺内可见外源性胰岛样细胞团,胰岛素免疫组化阳性,Nestin免疫组化强阳性,FISH检测Y染色体阳性。而肝内移植组及肾包膜下移植组各大鼠仍维持高血糖状态,相应组织均未见外源性细胞团形成。结论:分离纯化的胎鼠胰腺干细胞在胰腺内原位移植后在体内可转分化为胰岛样细胞团,并使血糖降至正常,胎鼠胰腺干细胞原位移植可能成为1型糖尿病治疗的一种新的可行方法。
第二部分胎鼠胰腺干细胞原位移植治疗1型糖尿病的实验研究
【摘要】目的:原位移植胎鼠胰腺干细胞,探讨不同数量级的胎胰干细胞在糖尿病鼠胰腺微环境中转分化为胰岛样细胞团、治疗1型糖尿病的可行性。方法:分离纯化孕16日SD大鼠胎鼠胰腺干细胞培养传代;荧光原位杂交(FISH)检测Y染色体鉴定雄雌;取第3代雄性胎鼠胰腺细胞行Nestin、PDX-1免疫组化及流式细胞术鉴定;取第5代雄性胎鼠胰腺干细胞,分为三个数量级,分别为1×105个/只(A组,10只)、1×106个/只(B组,10只)、1×107个/只(C组,10只)各组行糖尿病大鼠胰腺内原位移植,PBS溶液胰腺实质内注射作为实验对照组(D组,10只),正常大鼠作为空白对照组(E组,10只)。定期监测各组大鼠血糖情况及留取血浆ELISA测胰岛素含量,8周后取大鼠胰腺组织切片观察,FISH检测Y染色体,逆转录-聚合酶链反应(RT-PCR)观察各组大鼠胰腺内nestin、PDX-1及胰岛素等mRNA的表达情况,Western blot检测PDX-1及胰岛素的蛋白表达水平。结果:12只胎鼠中有5只经FISH检测为雄性。雄性胎鼠胰腺干细胞培养传代3代后细胞爬片免疫组化示存在Nestin和PDX-1阳性细胞,流式细胞术测定nestin阳性细胞含量占74.1%。A组大鼠于第3周开始血糖逐渐下降,血浆胰岛素水平逐渐升高但均未能达到正常水平,B组及C组大鼠于移植后第3周血糖开始下降,血浆胰岛素水平逐渐升高;第5周血糖降至正常水平并维持,血浆胰岛素达到正常水平。取第8周大鼠胰腺组织切片HE染色A、B、C组均可见外源性细胞团,FISH检测Y染色体阳性。RT-PCR检测示B组其内胰岛素的mRNA表达明显高于D组(P<0.05),与正常大鼠比较无显著性差别(P>0.05),而nestin及PDX-1的mRNA表达量高于D组及E组(P<0.05)。Western blot示B组胰腺组织内胰岛素含量接近正常大鼠(P>0.05),而PDX-1的含量高于正常大鼠(P<0.05)。结论:糖尿病大鼠每只予1×106个胎鼠胰腺干细胞进行原位移植后可在体内转分化为胰岛样细胞团且具有良好的功能,移植后可使血糖降至正常,胎鼠胰腺干细胞原位移植可能为1型糖尿病的治疗提供了一个新的可行方法。
Clinical pancreatic islet transplantation has developed rapidly these several years. This development makes the cure of diabetes mellitus hopeful, but the low availability of donor limits the number of islet transplants that can be performed. stem cells have great potentialities of the continuous proliferation and differentiation,and thus,it is thought to be a valuable source for generation of islet cells. Our reserch sought to observe the differentiation of stem cell in vivo by cell transplantation, and attempt to offer compelling opportunities for the development of new, innovative approaches for treating Diabetes.
Part 1 Effects of transplanted site on fetal rat pancreatic stem cells transdifferented into islet-like cell cluster in vivo
OBJECTIVE: To observe the possibility of fetal rat pancreatic stem cell transdifferentiating into islet-like cell cluster by transplanting fetal rat pancreatic stem cells into type 1 diabetic SD rat.
METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16 and examined SRY DNA by Fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry. Adult female SD rats were divided into five groups including 10 pancreatic parenchymal orthotopic transplantation, 10 intrahepatic transplantation,10 renal subcapsular place transplantation, 10 experimental control and 10 normal control.1×106 fetal pancreatic stem cells per rat were injected into diabetic rat’s pancreatic parenchyma, portal vein and renal subcapsular space respectively. Glucose and insulin level were monitored in serum periodic. Related tissues were excised for histological and morphometric analysis to assess the transdifferentiation and SRY DNA were examined by FISH.
RESULTS:After passaged 3 generations, the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry 74.1% of PSCs expressed nestin. The PSCs’orthotopic transplantation led to stable reduction in hyperglycemia and increasing insulin level in serum (3 weeks after transplantation) and culminated (5 weeks post- transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Neither intrahepatic transplantion nor renal subcapsular transplantation showed decrease of blood glucose or increase of insulin content in serum. No exogenous islet-like cell clusters immunostained by anti-insulin mAb and anti-nestin mAb were found except the recipient’s pancreata 8 weeks post-transplantation. The exogenous islet-like clusters expressed SRY DNA according to FISH.
CONCLUSION:After passaging 3 generation we can get pure pancreatic stem cells.When orthotopic transplant into pancreatic parenchyma PSCs can differenciate into islet-like cell cluster and reverse experimental diabetes.
Part 2 Study on orthotopic transplantion 0f fetal rat pancreatic stem cell to treat type 1 diabetes mellitus
OBJECTIVE: To observe the possibility of fetal rat pancreatic stem cell differentiating into islet-like cell cluster by transplanting fetal rat pancreatic stem cells into diabetic SD rat pancreatic parenchyma.
METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16 and examined SRY DNA to discriminate gender by Fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry.Adult SD rats were divided into five groups including 1×105 PSCs per rat pancreatic parenchymal orthotopic transplantation(group A, 10 rats), 1×106 PSCs per rat pancreatic parenchymal orthotopic transplantation(group B, 10 rats), 1×107 PSCs per rat pancreatic parenchymal orthotopic transplantation(group C, 10 rats),10 experimental control(group D, 10 rats) and 10 normal control(group E, 10 rats). In orthotopic transplantatation group male fetal pancreatic stem cells were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume PBS solution was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodic.8 weeks after transplantation pancreata were excised for histological and morphometric analysis. SRY DNA was detected by FISH. Nestin, PDX-1 and insulin mRNA expression in pancreata were detected by RT-PCR, insulin and PDX-1 protein contents were assess by western blot.
RESULTS:After passaged 3 generations,the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry 74.1% of PSCs expressed nestin. When orthotopic transplanted,both 1×106 and 1×107 PSCs could lead to stable reduction in hyperglycemia and increasing insulin level in serum (3wees after transplantation),culminating (5weeks post-transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse while 1×105 PSCs could also lead to reduction in hyperglycemia and increasing insulin level in serum without culminating in restoration of normoglycemia. Exogenous islet-like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipient’s pancreata 8 weeks post-transplantation. The expression level of insulin mRNA and protein in the recipient pancreata of group B were higher than experimental control(P<0.05),and the expression of nestin and PDX-1 mRNA and protein were also higher than normal control(P<0.05).
CONCLUSION:When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat can differenciate into islet-like cell cluster, gain comparable function with normal islets and reverse experimental diabetes.
第一部分不同移植部位对胎鼠胰腺干细胞分化为胰岛样细胞团的影响
【摘要】目的:利用胎鼠胰腺干细胞移植入1型糖尿病模型大鼠,探讨胎鼠胰腺干细胞在不同移植部位体内分化为胰岛细胞团的可能性,为1型糖尿病的治疗寻找一种新的可行的方法。方法:分离纯化孕16日SD大鼠胎鼠胰腺干细胞培养传代;荧光原位杂交(FISH)检测Y染色体;取第3代雄性胎鼠胰腺干细胞行Nestin、PDX-1免疫组化及流式细胞术鉴定。每只雌性糖尿病模型移植雄性胎鼠1×106个胰腺干细胞,分为胰腺原位移植组(10只)、肝内移植组(10只)、肾包膜下移植组(10只)、实验对照组(10只)及空白对照组(10只),定期监测各组大鼠血糖及血浆胰岛素含量,8周后处死大鼠,取相应组织观察胎鼠胰腺干细胞体内转分化情况并行FISH检测Y染色体鉴定。结果:12只胎鼠中有5只经FISH检测为雄性。雄性胎鼠胰腺干细胞培养传代3代后细胞爬片免疫组化示存在Nestin阳性细胞,流式细胞术测定nestin阳性细胞含量占74.1%。胰腺实质内移植组在第3周血糖开始下降,血浆胰岛素水平逐渐升高;第5周血糖降至正常水平并维持,血浆胰岛素达到正常水平。病理学观察在胰腺内可见外源性胰岛样细胞团,胰岛素免疫组化阳性,Nestin免疫组化强阳性,FISH检测Y染色体阳性。而肝内移植组及肾包膜下移植组各大鼠仍维持高血糖状态,相应组织均未见外源性细胞团形成。结论:分离纯化的胎鼠胰腺干细胞在胰腺内原位移植后在体内可转分化为胰岛样细胞团,并使血糖降至正常,胎鼠胰腺干细胞原位移植可能成为1型糖尿病治疗的一种新的可行方法。
第二部分胎鼠胰腺干细胞原位移植治疗1型糖尿病的实验研究
【摘要】目的:原位移植胎鼠胰腺干细胞,探讨不同数量级的胎胰干细胞在糖尿病鼠胰腺微环境中转分化为胰岛样细胞团、治疗1型糖尿病的可行性。方法:分离纯化孕16日SD大鼠胎鼠胰腺干细胞培养传代;荧光原位杂交(FISH)检测Y染色体鉴定雄雌;取第3代雄性胎鼠胰腺细胞行Nestin、PDX-1免疫组化及流式细胞术鉴定;取第5代雄性胎鼠胰腺干细胞,分为三个数量级,分别为1×105个/只(A组,10只)、1×106个/只(B组,10只)、1×107个/只(C组,10只)各组行糖尿病大鼠胰腺内原位移植,PBS溶液胰腺实质内注射作为实验对照组(D组,10只),正常大鼠作为空白对照组(E组,10只)。定期监测各组大鼠血糖情况及留取血浆ELISA测胰岛素含量,8周后取大鼠胰腺组织切片观察,FISH检测Y染色体,逆转录-聚合酶链反应(RT-PCR)观察各组大鼠胰腺内nestin、PDX-1及胰岛素等mRNA的表达情况,Western blot检测PDX-1及胰岛素的蛋白表达水平。结果:12只胎鼠中有5只经FISH检测为雄性。雄性胎鼠胰腺干细胞培养传代3代后细胞爬片免疫组化示存在Nestin和PDX-1阳性细胞,流式细胞术测定nestin阳性细胞含量占74.1%。A组大鼠于第3周开始血糖逐渐下降,血浆胰岛素水平逐渐升高但均未能达到正常水平,B组及C组大鼠于移植后第3周血糖开始下降,血浆胰岛素水平逐渐升高;第5周血糖降至正常水平并维持,血浆胰岛素达到正常水平。取第8周大鼠胰腺组织切片HE染色A、B、C组均可见外源性细胞团,FISH检测Y染色体阳性。RT-PCR检测示B组其内胰岛素的mRNA表达明显高于D组(P<0.05),与正常大鼠比较无显著性差别(P>0.05),而nestin及PDX-1的mRNA表达量高于D组及E组(P<0.05)。Western blot示B组胰腺组织内胰岛素含量接近正常大鼠(P>0.05),而PDX-1的含量高于正常大鼠(P<0.05)。结论:糖尿病大鼠每只予1×106个胎鼠胰腺干细胞进行原位移植后可在体内转分化为胰岛样细胞团且具有良好的功能,移植后可使血糖降至正常,胎鼠胰腺干细胞原位移植可能为1型糖尿病的治疗提供了一个新的可行方法。
Clinical pancreatic islet transplantation has developed rapidly these several years. This development makes the cure of diabetes mellitus hopeful, but the low availability of donor limits the number of islet transplants that can be performed. stem cells have great potentialities of the continuous proliferation and differentiation,and thus,it is thought to be a valuable source for generation of islet cells. Our reserch sought to observe the differentiation of stem cell in vivo by cell transplantation, and attempt to offer compelling opportunities for the development of new, innovative approaches for treating Diabetes.
Part 1 Effects of transplanted site on fetal rat pancreatic stem cells transdifferented into islet-like cell cluster in vivo
OBJECTIVE: To observe the possibility of fetal rat pancreatic stem cell transdifferentiating into islet-like cell cluster by transplanting fetal rat pancreatic stem cells into type 1 diabetic SD rat.
METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16 and examined SRY DNA by Fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry. Adult female SD rats were divided into five groups including 10 pancreatic parenchymal orthotopic transplantation, 10 intrahepatic transplantation,10 renal subcapsular place transplantation, 10 experimental control and 10 normal control.1×106 fetal pancreatic stem cells per rat were injected into diabetic rat’s pancreatic parenchyma, portal vein and renal subcapsular space respectively. Glucose and insulin level were monitored in serum periodic. Related tissues were excised for histological and morphometric analysis to assess the transdifferentiation and SRY DNA were examined by FISH.
RESULTS:After passaged 3 generations, the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry 74.1% of PSCs expressed nestin. The PSCs’orthotopic transplantation led to stable reduction in hyperglycemia and increasing insulin level in serum (3 weeks after transplantation) and culminated (5 weeks post- transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Neither intrahepatic transplantion nor renal subcapsular transplantation showed decrease of blood glucose or increase of insulin content in serum. No exogenous islet-like cell clusters immunostained by anti-insulin mAb and anti-nestin mAb were found except the recipient’s pancreata 8 weeks post-transplantation. The exogenous islet-like clusters expressed SRY DNA according to FISH.
CONCLUSION:After passaging 3 generation we can get pure pancreatic stem cells.When orthotopic transplant into pancreatic parenchyma PSCs can differenciate into islet-like cell cluster and reverse experimental diabetes.
Part 2 Study on orthotopic transplantion 0f fetal rat pancreatic stem cell to treat type 1 diabetes mellitus
OBJECTIVE: To observe the possibility of fetal rat pancreatic stem cell differentiating into islet-like cell cluster by transplanting fetal rat pancreatic stem cells into diabetic SD rat pancreatic parenchyma.
METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16 and examined SRY DNA to discriminate gender by Fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry.Adult SD rats were divided into five groups including 1×105 PSCs per rat pancreatic parenchymal orthotopic transplantation(group A, 10 rats), 1×106 PSCs per rat pancreatic parenchymal orthotopic transplantation(group B, 10 rats), 1×107 PSCs per rat pancreatic parenchymal orthotopic transplantation(group C, 10 rats),10 experimental control(group D, 10 rats) and 10 normal control(group E, 10 rats). In orthotopic transplantatation group male fetal pancreatic stem cells were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume PBS solution was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodic.8 weeks after transplantation pancreata were excised for histological and morphometric analysis. SRY DNA was detected by FISH. Nestin, PDX-1 and insulin mRNA expression in pancreata were detected by RT-PCR, insulin and PDX-1 protein contents were assess by western blot.
RESULTS:After passaged 3 generations,the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry 74.1% of PSCs expressed nestin. When orthotopic transplanted,both 1×106 and 1×107 PSCs could lead to stable reduction in hyperglycemia and increasing insulin level in serum (3wees after transplantation),culminating (5weeks post-transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse while 1×105 PSCs could also lead to reduction in hyperglycemia and increasing insulin level in serum without culminating in restoration of normoglycemia. Exogenous islet-like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipient’s pancreata 8 weeks post-transplantation. The expression level of insulin mRNA and protein in the recipient pancreata of group B were higher than experimental control(P<0.05),and the expression of nestin and PDX-1 mRNA and protein were also higher than normal control(P<0.05).
CONCLUSION:When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat can differenciate into islet-like cell cluster, gain comparable function with normal islets and reverse experimental diabetes.
引文
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20、Street CN, Sipione S, Helms L,et al. Stem cell-based approaches to solving the problem of tissue supply for islet transplantation in type 1 diabetes. Int. J. Biochem. Cell Biol.2004;36:667–683.
21、R Wang, J Li, N Yashpal, et al. Nestin expression and clonal analysis ofislet-derived epithelial monolayers: insight into nestin-expressing cell heterogeneity and differentiation potential. Endocrinol, 2005,184:329-339.
22、郑宗梅,陈东明,李凌松等巢蛋白和神经元素3在人胚胎胰腺中的表达和分布.中华外科杂志,2005,43:1537-1541.
23、Lendahl U ,Zimmerman LB ,Mckay RDG. CNS stem cells express a new class of intermediate filament protein. Cell ,1990 ,60 :585-595.
24、Dahlstrand J, Lardelli M, Lendahl U. Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system. Brain Res Dev Brain Res. 1995;84:109-29.
25、Kachinsky AM, Dominov JA, Miller JB. Myogenesis and the intermediate filament protein, nestin. Dev Biol. 1994;165:216-228.
26、Mokry J, Nemecek S. Angiogenesis of extra- and intraembryonic blood vessels is associated with expression of nestin in endothelial cells. Folia Biol (Praha). 1998;44:155-61.
27、Mokry J, Nemecek S. Cerebral angiogenesis shows nestin expression in endothelial cells. Gen Physiol Biophys. 1999;18 Suppl 1:25-29.
28、Shih CC, Weng Y, Mamelak A, Identification of a candidate human neurohematopoietic stem-cell population. Blood. 2001;98:2412-22.
29、Rietze RL, Valcanis H, Brooker GF, et al. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature. 2001;412:736-739.
30、Hunziker E, Stein M. Nestin-expressing cells in the pancreatic islets of Langerhans. Biochem Biophys Res Commun. 2000;271:116-119.
31、Street CN, Lakey JR, Seeberger K, et al.Heterogenous expression of nestin in human pancreatic tissue precludes its use as an islet precursor marker. J Endocrinol. 2004;180:213-225.
32、Abraham EJ, Leech CA, Lin JC,et al. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells intoinsulin-producing cells. Endocrinology. 2002;143:3152-3161.
33、Huang H, Tang X. Phenotypic determination and characterization of nestin-positive precursors derived from human fetal pancreas. Lab Invest. 2003;83:539-547.
34、Lechner A, Leech CA, Abraham EJ,et al. Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter. Biochem Biophys Res Commun. 2002;293:670-674.
35、Palm K, Salin-Nordstr?m T, Levesque MF, et al. Fetal and adult human CNS stem cells have similar molecular characteristics and developmental potential. Brain Res Mol Brain Res. 2000;78:192-195.
36、Vaittinen S ,Lukka R ,Sahlgren C , et al . Specific and innervation2regu2 lated expression of the intermediate filament proteinnestin at neuromus2cular and myotendinous junctions in skeletalmuscle. Am J Pathol ,1999 ,154:591-600.
37、Joanette EA, Reusens B, Arany E, et al. Low2p rotein diet during early life causes a reduction in the frequency of cells immunopositive for nestin and CD34 in both pancreatic ducts and islets in the rat. Endocrinology, 2004, 145:3004-3013.
38、Peters K, Panienka R, L i J, et al. Exp ression of stem cell markers and transcrip tion factors during the remodeling of the rat pancreas after duct ligation. VirchowsArch, 2005, 446:56-63.
39、Wang R, L i J , Yashpal N, et al. Nestin exp ression and clonal analysis of islet-derived epithelial monolayers: insight into nestin-expressing cell heterogeneity and differentiation potential. Endocrinol, 2005, 184: 329-339.
40、Bernardo AS, Barrow J , Hay CW, et al. Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin /EGFP mouse. Mol Cell Endocrinol, 2006, 253:14-21.
41、Treutelaar MK, Skidmore JM, Dias-Leme CL, et al. Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet. Diabetes. 2003; 52:2503-2512.
42、Moore KA, Lemischka IR. Stem cells and their niches. Science, 2006, 311: 1880～1885.
43、Zhang W, Ge W, Li C,et al. Effects of Mesenchymal Stem Cells on Differentiation, Maturation, and Function of Human Monocyte-Derived Dendritic Cells. Stem Cells Dev. 2004 ,13:263-271.
44、Linheng Li, Ting Xie. Stem cell Niche Structure and function. Biol. 2005,21:605-631.
45、Lin H.The stem-cell niche theory: lessons from flies.Nat Rev Genet. 2002,3: 931-940.
46、Moore KA. Lemischka IR. Stem cells and their niches. Science. 2006,311:1880-1885.
47、Li L, Xie T. Stem cell Niche: structure and function. Annu Rev Cell Dev Biol. 2005,21:605-631.
1、Bonner - Weir S. Perspective : postnatal pancreatic beta cell growth. Endocrinology , 2000 , 141 : 1926-1929.
2、Bonner-Weir S. Life and death of the pancreaticβcells. Trends Endocrinol Metab , 2000 , 11 : 375-378.
3、Bonner-Weir S.β-cell turnover : its assessment and implications. Diabetes, 2001 , 50 ;S20-S24.
4、Dor Y, Brown J , Martinez OI , et al. Adult pancreatic beta cells are formed by self2duplication rather than stem cell differentiation. Nature , 2004 , 429: 41-46.
5、Teta M , Long SY, Wartschow LM , et al. Very slow turnover ofβ-cells in aged adult mice. Diabetes, 2005 , 54: 2557-2567.
6、Trucco M. Regeneration of the pancreaticβcell. Clin Invest, 2005 , 115 : 5-12.
7、Guz Y, Nasir I, Teitelman G. Regeneration of pancreatic beta cells from intra-islet precurs or cells in an experimental model of diabetes. Endocrinology, 2001,142:4956- 4968.
8、Eberhardt M, Salmon P, Mach MA, et al. Multipotential nestin and Isl-1 positive mesenchymal stem cells isolated from human pancreatic islets. Biochem Biophys Res Commun, 2006, 345:1167- 1176.
9、Gray PC, Harrison CA, Vale W, et al. Cripto forms a complex with activin and type II activin receptors and can block activin signaling. Proc Natl Acad Sci USA, 2003,100:5193- 5198.
10、List JF, Habener JF. Glucagon-like peptide 1 agonists and the development and growth of pancreatic beta-cells. Am J Physiol Endocrinol Metab, 2004,286:875- 881.
11、Stoffers DA, Kieffer TJ, Hussain MA, et al. Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes, 2000, 49:741- 748.
12、Li L, Seno M, Yamada H, et al. Promotion of beta-cell regeneration by beta cellulin in ninety percent- pancreatectomized rats. Endocrinology, 2001,142:5379- 5385.
13、Tourrel C,Bailbe D,Lacorne M,et al. Persistent improvement of type 2 diabetes in the Goto-Kakizaki rat model by expansion of the beta-cell mass during theprediabetic period with glucagonlike peptide-1 or exendin-4. Diabetes, 2002,51:1443-1452.
14、Harmon EB, Apelqvist AA, Smart NG , et al. GDF11 modulates NGN3+ islet progenitor cell number and promotes beta-cell differentiation in pancreas development. Development, 2004,131:6163-6174.
15、Jorns A, Tiedge M, Lenzen S. Thyroxine induces pancreatic beta cell apoptosis in rats. Diabetologia,2002,45:851- 855.
16、GrapirrBotton A. Majithia AR. Melton DA. Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes Dev, 2001,15:444-454.
17、Riess P, Zhang C, Saatman KE, et al. Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury. Neurosurgery. 2002,51:1043-1052.
18、Lee HJ, Kim KS, Kim EJ, et al. Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model. Stem Cells. 2007,25:1204-1212.
19、Min JY, Yang Y, Sullivan MF, et al. Long-term improvement of cardiac function in rats after infarction by transplantation of embryonic stem cells. J Thorac Cardiovasc Surg. 2003,125:361-369.
20、Prockop, DJ, Gregory, CA, Spees, JL. One strategy for cell and gene therapy: Harnessing the power of adult stem cells to repair tissues.Proceedings of the National Academy of Sciences of the United States of America, 2003,100: 11917–11923.
21、Munoz, JR, Stoutenger, BR, Robinson, AP, et al. Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice.Proceedings of the National Academy of Sciences of the United States of America, 2005,102:18171–18176.
22、Spees, JL, Olson, SD, Ylostalo, J, et al. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma.Proceedings of the National Academy of Sciences of the United States of America,2003,100: 2397–2402.
23、Spees, J L, Olson, S D, Whitney, M J, et al.Mitochondrial transfer between cells can rescue aerobic respiration.Proceedings of the National Academy of Sciences of the United States of America, 2006,103:1283–1288.
24、Ryang Hwa Lee, Min Jeong Seo, Roxanne L. Reger.et al. Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice. Proceedings of the National Academy of Sciences of the United States of America,2006,103:17438-17443.
25、Scuteri A, Cassetti A, Tredici G. Adult mesenchymal stem cells rescue dorsal root ganglia neurons from dying. Brain Research, 2006,1116:75-81.
26、Baffour R, Pakala R, Hellinga D.et al. Bone marrow-derived stem cell interactions with adult cardiomyocytes and skeletal myoblasts in vitro. Cardiovascular revascularization medicine: including molecular interventions, 2006,7:222-230.
27、Chang Liu Z, Chang TM.et al.Coencapsulation of hepatocytes and bone marrow cells: In vitro and in vivo studies.Biotechnology Annual Review.2006,12:137-151.
28、夏亚一,冯万文,孙正义等.自体骨髓间充质干细胞和软骨细胞共培养复合同种异体完全脱蛋白骨修复关节软骨全层缺损的研究.中国矫形外科杂志,2006,14:1245-1248。
29、Fraidenraich D, Stillwell E, Romero E, Rescue of cardiac defects in id knockout embryos by injection of embryonic stem cells. Science. 2004,306: 247-252.
30、Jeronia Llado,Christine Haenggeli, Nicholas J. Maragakis .et al. Neural stemcells protect against glutamate-induced excitotoxicity and promote survival of injured motor neurons. Molecular and Cellular Neuroscience.2004, 27:322-331.
1、Shapiro AM, Lakey JR, Ryan EA, et al Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid free immunosuppressive regimen. N Engl J Med 2000; 343:230-238.
2、Lumelsy N, Blondel O, Laeng P, et al Differentiation of embryonic stem cells to insulin-sercreting structures similar to pancreatic islets. Science, 2001; 292:1389-1394.
3、Hori Y, Rulifson IC, Tsai BC, et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci USA 2002; 99: 16105-16110.
4、Assady S, Moaor G, Amit M,et al. Insulin production by human embryonic stem cells. Diabetes, 2001; 50:1691-1697.
5、Suheir A, Gila M, Michal A, et al. Insulin production by human embryonic sten cells. Diabetes, 2001; 50(8):1691.
6、Rajagopal J, Anderson WJ, Kume S, et al.Insulin staining of ES cell progeny from insulin uptake. Science 2003; 299: 363.
7、Alison MR, Poulsom R, Jeffery R, et al. Hepatocytes from non-hepatic adult stem cells. Nature, 2000; 406(6793):257.
8、Ramiya VK, Maraist M, Arfors KE, et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med, 2000; 6:278-282.
9、Bonner-Weir S, Taneja M, Wair GC, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA, 2000; 97:7999-8004.
10、Hunziker E, Stein M. Nestin-expressing cells in the pancreatic islets of Langerhans. Biochem Biophys Res Commun. 2000; 271:116-119.
11、Zulewski H, Abraham EJ, Gerlach MJ, et al. Multipotential nestin-positivestem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes. 2001; 50:521-533.
12、Dor Y, Ta M, Atouf F, et al. Adult pancreas generates multipotent stem cells and pancreatic and nonpancreatic progeny. Stem Cells. 2004; 22:1070-1084.
13、Seaberg RM, Smukler SR, Kieffer TJ, et al. Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages. Nat Biotechnol. 2004; 22:1115-1124.
14、Suzuki A, Nakauchi H, Taniguchi H. Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting. Diabetes.2004; 53:2143-2152.
15、张玲,洪天配,胡江,等从胎儿胰腺组织建立单克隆胰腺前体细胞的研究中华糖尿病杂志2004; 12:442-445.
16、Piper K, Ball SG, Turnpenny LW, et al. Beta-cell differentition during human development does not rely on nestin-positive precursors: implication for stem cell-derived replacement therapy. Diabetologia. 2002; 45:1045-1047.
17、Humphrey RK, Bucay N, Beattie GM, et al. Characterization and isolation of promoter-defined nestin-positive cells from the human fetal pancreas. Diabetes. 2003; 52:2519-2525.
18、Treutelaar MK, Skidmore JM, Dias-Leme CL, et al. Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet. Diabetes. 2003; 52:2503-2512.
19、Mckinnon CM, Docherty K, Pancreatic duodenal homeobox-1, PDX-1, a major regulator beta cell identity and function. Diabetologia. 2001; 44:1203-1214.
20、Ferber S, Halkin A, Cohen H, et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and amelioratesstreptozotocin-induced hyperglycemia. Nat Med. 2000; 6:568-572.
21、Kojima H, Nakamura T, Fujita Y, et al. Combined expression of pancreatic duodenal homeobox 1 and islet factor1 induces immature enterocytes to produce insulin. Diabetes. 2002; 51:1398-1408.
22、Le BK, Tannik C, Rosendahl K, et al. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol. 2003; 31:890-896.
23、Woodlbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. Journal of Neuroscience Research. 2000; 61:364-370.
24、李艳华,白慈贤,谢超,等成人骨髓间充质干细胞体外定向诱导分化为胰岛样细胞团的研究自然科学进展2003;13:593-597.
25、贾延劼，钟乐，宋建辉，等体外诱导大鼠间充质干细胞分化为胰岛素分泌细胞中国当代儿科杂志2003;5:393-397.
26、Chen LB, Jiang XB, Yang L. Differentiation of rat marrow mesenchymal stem calls into pancreatic islet beta-cells. World J Gastroenterol. 2004; 10:3016-3020.
27、Oh SH, Muzzonigro TM, Bae SH, et al. Adult bone marrow-drived cells transdifferentiating into insulin-producing cells for the treatment of typeⅠdiabetes. Laboratory Investigation. 2004; 84:607-617.
28、Tang DQ, Cao LZ, Burkhardt BR, et al. In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes. 2004;53:1721-1732.
29、Tondreau T, Lagneaux L, Dejeneffe M, et al. Bone marrow-drived mesenchymal stem cells already express specific neural proteins before any differentiation. Differentiation. 2004; 72:319-326.
30、Ianus A, Holz GG, Theise ND, et al. In vivo derivation of glucosecompetentpancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest. 2003; 111:843-850.
31、Chiol JB, Uchinol H, Azumal K, et al. Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells. Diabetologia. 2003; 46:1366-1374.
32、Feng RQ, Du LY, Guo ZQ. In vitro cultivation and differentiation of fetal liver stem cells from mice. Cell Res. 2005; 15:401-405.
33、Suzuki A, Zheng YW, Kaneko S, et al. Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver. Cell Bio.2002;156:173-184.
34、Zalzmen M, Gupta S, Giri RK, et al. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci USA. 2003; 100:7253-7258.
35、Yang L, Li S, Taneja M, et al. In vitro transdifferention of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci USA. 2002; 99:8078-8083.
36、姚忠祥,李巍,李成仁,等.体外培养神经干细胞分化为类胰岛素组织的研究.第三军医大学学报. 2003; 25:26-28.
37、Kodama S, Davis M, Faustman DL. Diabetes and stem cells researchers turn to the lowly spleen. Sci Aging Knowledge Environ. 2005; 3:2.
38、Trucco M. Regeneration of the pancreaticβ–cell. Clin Invest. 2005; 115:5-12.
2、许曼音,陆广华,陈明道胰岛移植治疗糖尿病。糖尿病学,2003年10月998-1002。
3、Nanji SA, Shapiro AM. Advances in pancreatic islet transplantation in humans. Diabetes Obes Metab, 2006, 8:15-25.
4、杨雪菲,李富容,周汉新同种异体胰岛细胞和干细胞来源胰岛移植的现况与未来.中华器官移植杂志,2006,27:126-128.
5、J.B.Chol, H.Uchino, K.Azuma,etal. Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells.Diabetologia,26 July 2003.
6、Tissue Engineers Build New Bone.Science,2000,289:1498-1500.
7、杨明,王春友,刘涛等应用蛋白质组学的方法筛选胰腺干细胞标记物.生物化学与生物物理进展,2006,33:540-547.
8、Ramiya VK, Maraist M, Arfors KE, et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med, 2000; 6:278-282.
9、Bonner-Weir S, Taneja M, Wair GC, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA, 2000; 97:7999-8004.
10、Hunziker E, Stein M. Nestin-expressing cells in the pancreatic islets of Langerhans. Biochem Biophys Res Commun. 2000; 271:116-119.
11、Zulewski H, Abraham EJ, Gerlach MJ, et al. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes. 2001; 50:521-533.
12、Cattaneo E, McKay R. Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor. Nature. 1990;347:762-765.
13、Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell. 1990;60:585-595.
14、Lumelsy N, Blondel O, Laeng P, et al Differentiation of embryonic stem cells to insulin-sercreting structures similar to pancreatic islets. Science. 2001; 292:1389-1394.
15、Hori Y, Rulifson IC, Tsai BC, et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci USA 2002; 99: 16105-16110.
16、Mckinnon CM, Docherty K, Pancreatic duodenal homeobox-1, PDX-1, a major regulator beta cell identity and function. Diabetologia. 2001; 44:1203-1214.
17、Ferber S, Halkin A, Cohen H, et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat Med. 2000; 6:568-572.
18、Kojima H, Nakamura T, Fujita Y, et al. Combined expression of pancreatic duodenal homeobox 1 and islet factor1 induces immature enterocytes to produce insulin. Diabetes. 2002; 51:1398-1408.
19、Noguchi H. Stem cells for the treatment of diabetes. Endocr J. 2007;54(1):7-16.
20、Street CN, Sipione S, Helms L,et al. Stem cell-based approaches to solving the problem of tissue supply for islet transplantation in type 1 diabetes. Int. J. Biochem. Cell Biol.2004;36:667–683.
21、R Wang, J Li, N Yashpal, et al. Nestin expression and clonal analysis ofislet-derived epithelial monolayers: insight into nestin-expressing cell heterogeneity and differentiation potential. Endocrinol, 2005,184:329-339.
22、郑宗梅,陈东明,李凌松等巢蛋白和神经元素3在人胚胎胰腺中的表达和分布.中华外科杂志,2005,43:1537-1541.
23、Lendahl U ,Zimmerman LB ,Mckay RDG. CNS stem cells express a new class of intermediate filament protein. Cell ,1990 ,60 :585-595.
24、Dahlstrand J, Lardelli M, Lendahl U. Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system. Brain Res Dev Brain Res. 1995;84:109-29.
25、Kachinsky AM, Dominov JA, Miller JB. Myogenesis and the intermediate filament protein, nestin. Dev Biol. 1994;165:216-228.
26、Mokry J, Nemecek S. Angiogenesis of extra- and intraembryonic blood vessels is associated with expression of nestin in endothelial cells. Folia Biol (Praha). 1998;44:155-61.
27、Mokry J, Nemecek S. Cerebral angiogenesis shows nestin expression in endothelial cells. Gen Physiol Biophys. 1999;18 Suppl 1:25-29.
28、Shih CC, Weng Y, Mamelak A, Identification of a candidate human neurohematopoietic stem-cell population. Blood. 2001;98:2412-22.
29、Rietze RL, Valcanis H, Brooker GF, et al. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature. 2001;412:736-739.
30、Hunziker E, Stein M. Nestin-expressing cells in the pancreatic islets of Langerhans. Biochem Biophys Res Commun. 2000;271:116-119.
31、Street CN, Lakey JR, Seeberger K, et al.Heterogenous expression of nestin in human pancreatic tissue precludes its use as an islet precursor marker. J Endocrinol. 2004;180:213-225.
32、Abraham EJ, Leech CA, Lin JC,et al. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells intoinsulin-producing cells. Endocrinology. 2002;143:3152-3161.
33、Huang H, Tang X. Phenotypic determination and characterization of nestin-positive precursors derived from human fetal pancreas. Lab Invest. 2003;83:539-547.
34、Lechner A, Leech CA, Abraham EJ,et al. Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter. Biochem Biophys Res Commun. 2002;293:670-674.
35、Palm K, Salin-Nordstr?m T, Levesque MF, et al. Fetal and adult human CNS stem cells have similar molecular characteristics and developmental potential. Brain Res Mol Brain Res. 2000;78:192-195.
36、Vaittinen S ,Lukka R ,Sahlgren C , et al . Specific and innervation2regu2 lated expression of the intermediate filament proteinnestin at neuromus2cular and myotendinous junctions in skeletalmuscle. Am J Pathol ,1999 ,154:591-600.
37、Joanette EA, Reusens B, Arany E, et al. Low2p rotein diet during early life causes a reduction in the frequency of cells immunopositive for nestin and CD34 in both pancreatic ducts and islets in the rat. Endocrinology, 2004, 145:3004-3013.
38、Peters K, Panienka R, L i J, et al. Exp ression of stem cell markers and transcrip tion factors during the remodeling of the rat pancreas after duct ligation. VirchowsArch, 2005, 446:56-63.
39、Wang R, L i J , Yashpal N, et al. Nestin exp ression and clonal analysis of islet-derived epithelial monolayers: insight into nestin-expressing cell heterogeneity and differentiation potential. Endocrinol, 2005, 184: 329-339.
40、Bernardo AS, Barrow J , Hay CW, et al. Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin /EGFP mouse. Mol Cell Endocrinol, 2006, 253:14-21.
41、Treutelaar MK, Skidmore JM, Dias-Leme CL, et al. Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet. Diabetes. 2003; 52:2503-2512.
42、Moore KA, Lemischka IR. Stem cells and their niches. Science, 2006, 311: 1880～1885.
43、Zhang W, Ge W, Li C,et al. Effects of Mesenchymal Stem Cells on Differentiation, Maturation, and Function of Human Monocyte-Derived Dendritic Cells. Stem Cells Dev. 2004 ,13:263-271.
44、Linheng Li, Ting Xie. Stem cell Niche Structure and function. Biol. 2005,21:605-631.
45、Lin H.The stem-cell niche theory: lessons from flies.Nat Rev Genet. 2002,3: 931-940.
46、Moore KA. Lemischka IR. Stem cells and their niches. Science. 2006,311:1880-1885.
47、Li L, Xie T. Stem cell Niche: structure and function. Annu Rev Cell Dev Biol. 2005,21:605-631.
1、Bonner - Weir S. Perspective : postnatal pancreatic beta cell growth. Endocrinology , 2000 , 141 : 1926-1929.
2、Bonner-Weir S. Life and death of the pancreaticβcells. Trends Endocrinol Metab , 2000 , 11 : 375-378.
3、Bonner-Weir S.β-cell turnover : its assessment and implications. Diabetes, 2001 , 50 ;S20-S24.
4、Dor Y, Brown J , Martinez OI , et al. Adult pancreatic beta cells are formed by self2duplication rather than stem cell differentiation. Nature , 2004 , 429: 41-46.
5、Teta M , Long SY, Wartschow LM , et al. Very slow turnover ofβ-cells in aged adult mice. Diabetes, 2005 , 54: 2557-2567.
6、Trucco M. Regeneration of the pancreaticβcell. Clin Invest, 2005 , 115 : 5-12.
7、Guz Y, Nasir I, Teitelman G. Regeneration of pancreatic beta cells from intra-islet precurs or cells in an experimental model of diabetes. Endocrinology, 2001,142:4956- 4968.
8、Eberhardt M, Salmon P, Mach MA, et al. Multipotential nestin and Isl-1 positive mesenchymal stem cells isolated from human pancreatic islets. Biochem Biophys Res Commun, 2006, 345:1167- 1176.
9、Gray PC, Harrison CA, Vale W, et al. Cripto forms a complex with activin and type II activin receptors and can block activin signaling. Proc Natl Acad Sci USA, 2003,100:5193- 5198.
10、List JF, Habener JF. Glucagon-like peptide 1 agonists and the development and growth of pancreatic beta-cells. Am J Physiol Endocrinol Metab, 2004,286:875- 881.
11、Stoffers DA, Kieffer TJ, Hussain MA, et al. Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes, 2000, 49:741- 748.
12、Li L, Seno M, Yamada H, et al. Promotion of beta-cell regeneration by beta cellulin in ninety percent- pancreatectomized rats. Endocrinology, 2001,142:5379- 5385.
13、Tourrel C,Bailbe D,Lacorne M,et al. Persistent improvement of type 2 diabetes in the Goto-Kakizaki rat model by expansion of the beta-cell mass during theprediabetic period with glucagonlike peptide-1 or exendin-4. Diabetes, 2002,51:1443-1452.
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