摘要
目的:原位移植胎鼠胰腺干细胞,探讨其在糖尿病鼠胰腺微环境中转分化为胰岛样细胞团的可行性。方法:分离纯化SD大鼠胎鼠胰腺干细胞;荧光原位杂交(fluorescentin situhybridization,FISH)检测SRY上的性别决定区(sex determining region on Y,SRY)以鉴别雄雌;免疫细胞化学检测巢蛋白(Nestin)、胰十二指肠同源异型盒基因(PDX-1)的表达及流式细胞术测定Nestin细胞含量以鉴定胰腺干细胞;分胰腺实质内移植组、实验对照组及空白对照组,10只/组;监测各组大鼠血糖及血浆胰岛素含量,8周后取大鼠胰腺组织切片观察,FISH检测SRY,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,免疫印迹(Western blotting)检测PDX-1及胰岛素的蛋白表达水平。结果:有5只胎鼠经FISH检测为雄性。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%。胰腺实质内移植组大鼠于移植后第3周血糖开始下降,血浆胰岛素水平逐渐升高;第5周血糖及血浆胰岛素均达到正常水平并维持。取第8周大鼠胰腺组织切片HE染色可见外源性细胞团,FISH检测SRY阳性。RT-PCR示胰腺实质内移植组胰腺组织内胰岛素的mRNA表达明显高于实验对照组(P<0.05),而Nestin及PDX-1的mRNA表达量高于实验对照组及正常组(P<0.05)。Western blotting示干细胞移植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:胎鼠胰腺干细胞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,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策略。
AIM ： To observe the possibility of differentiation of fetal rat pancreatic stem cells into islet - like cell cluster by transplantation of fetal rat pancreatic stem cells into pancreatic parenchyma in diabetic SD rats. METHODS： The pancreatic stem cells （PSCs） were harvested from pancreatic rudiments of SD rat embryos onembryonic day 16. SRY DNA was examined 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 three groups including 10 pancreatic parenchymal orthotopic transplantation, 10 experimental controls and 10 normal controls. In orthotopic transplantation group, 1 × 10^6 male fetal pancreatic stem cells per rat were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume of PBS was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodically. 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 assessed by Western blotting. RESULTS： 5 of 12 fetal rats were male according to FISH. After passaged 3 generations, the PSCs expressed nestin and PDX - 1 according to immunostaining while identified by flow cytometry with 74.1% of PSCs expressed nestin. The orthotopic transplantation of PSCs led to stable reduction in hyperglycemia and increase in insulin level in serum （ 3 weeks after trans- plantation）, culminating （5 weeks post -transplantation） in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Exogenous islet - like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipient＇s pancreata 8 weeks post - transplantation. The expression levels of insulin mRNA and pro- tein in the ort.hotopic transplanted recipient＇s pancreata were higher than those in experimental control （P 〈0. 05）, and the expressions of PDX - 1 mRNA and protein were also higher than those in normal control （P〈0.05 ）. CONCLUSION： When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat differentiates into isletlike cell cluster, gains comparable function with normal islets and reverses experimental diabetes.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第8期1595-1599,共5页
Chinese Journal of Pathophysiology
基金
深圳市科技计划资助项目(No.200601015)
关键词
胎鼠
胰腺干细胞
原位移植
糖尿病
Fetal rats
Pancreatic stem cells
Orthotopic transplantation
Diabetes mellitus
