胎鼠胰腺干细胞体外对胰岛的保护作用观察
Protective effect of pancreatic stem cells in vitro on the islets of langerhans
摘要 目的:观察胎鼠胰腺干细胞体外对胰岛功能的保护作用。方法将孕16 d的SD大鼠胎鼠胰腺干细胞分离、纯化、培养传代,免疫细胞化学法及流式细胞术鉴定;分离纯化SD大鼠胰岛,双硫腙鉴定后分为A组和B组,分别行单纯胰岛培养及胰岛与胰腺干细胞联合培养14 d,期间观察胰岛形态变化、检测胰岛存活率及细胞凋亡率, ELISA法检测胰岛素分泌量、计算刺激指数。取两组培养7 d后悬浮生长的胰岛移植入糖尿病大鼠左肾包膜下,术后每天尾静脉采血以快速血糖测试仪检测血糖。结果胎鼠胰腺干细胞培养传代3代后免疫细胞化学可见巢蛋白(Nestin)阳性细胞,流式细胞术测定其含量占74.1%;培养第7、14天时B组胰岛存活率显著高于A组(P均<0.01),培养第7天时B组胰岛细胞凋亡率显著低于A组(P<0.05);培养第7、14天时B组高糖刺激后胰岛素分泌量及刺激指数均显著高于A组(P均<0.01);B组移植后大鼠血糖第5天降至正常。结论胎鼠胰腺干细胞与胰岛联合培养可明显延长胰岛体外存活时间并使其保持良好的活性。
Abstract:
Objective To investigate the protective effect of pancreatic stem cells ( PSCs) of fetal rats in vitro on the islets of langerhans.Methods The PSCs of SD rat embryos on embryonic 16th day were isolated, purified, cultivated and passaged , then were identified by immuocytochemistry and flow cytometry .The rat's islets were isolated from the pancreas . After being identified by dithizone , the islets were divided into two groups:group A which underwent islet alone culturing and group B which underwent co-culturing of islet and PSCs for 14 days.The morphological changes , survival rate and ap-optosis rate of islets were observed under inverted microscope , fluorescence microscope and flow cytometry ( FCM) in both groups.The amount of insulin secretion was measured by ELISA;and insulin stimulation index was calculated with the in-sulin release test.After being cultured for 7 days, the islets in the two groups were transplanted to the peplos of the left kid-ney in SD rats with diabetes , respectively .The blood glucose of the SD rats was tested after transplantation .Results The immuocytochemistry showed the PSCs were nestin-positive after being cultivated and passaged for the 3rd generation.Flow cytometry showed its content accounted for 74.1%;on the 7th, 14th day, the survival rate of islets in group B was signifi-cantly higher than that in group A (all P<0.01), while on the 7th day, the apoptosis rate of islets in group B was signifi-cantly lower than that in group A (P<0.05).On the 7th , 14th day, under the high glucose stimulation , the levels of in-sulin secretion and insulin stimulation index in group B were significantly higher than those in group A (all P<0.01).Af-ter transplantation in group B , the blood glucose of rats dropped to normal on the 5th day.Conclusion Co-cultivation of islet and PSCs can significantly prolong the survival time of islets and preserve viability of islets .
Author: ZHANG Yu-sen CHANG Cai-hong ZHANG Yue LI Fu-rong BAO Shi-yun
作者单位: 深圳市人民医院,暨南大学第二临床医学院,广东深圳518020 海南医学院
刊 名 山东医药 ISTICPKU
年,卷(期): 2014, (15)
分类号: R458.5
机标分类号: R71 TP2
在线出版日期: 2014年6月7日
基金项目: 国家自然科学基金资助项目(81260125/Ho704)。