【作者】 张悦；

【导师】 王春友； 余小舫；

【作者基本信息】 华中科技大学 ， 普通外科， 2008， 博士

【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。更多还原

【Abstract】 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 vivoOBJECTIVE: 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 mellitusOBJECTIVE: 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.更多还原