胰腺干细胞与胰岛混合培养对其转化率的影响  

Co-culture with islets to make pancreatic stem cells differentiate into insulin-producing cells

作　　者：程志强[1] 王磊[2] 胡三元[2] 王甜甜[3] 

机构地区：[1]山东大学医学院,济南250012 [2]山东大学齐鲁医院普外科,济南250012 [3]山东大学附属省立医院普外科,济南250021

出　　处：《山东大学学报（医学版）》2010年第6期61-66,共6页Journal of Shandong University:Health Sciences

基　　金：山东省博士基金资助课题(2005BS03011)

摘　　要：目的研究胰腺干细胞与胰岛混合培养对增加胎鼠胰腺干细胞向β前体细胞转化率的作用及机制。方法实验分为胰腺导管干细胞单独培养组(Ⅰ组),胰岛单独培养组(Ⅱ组)及干细胞-胰岛混合培养组(Ⅲ组)。V型胶原酶消化大鼠胰腺获得胰岛后,使用Ficoll-400梯度离心法纯化胰岛;采用胶原酶消化法获得胎鼠胰腺干细胞进行培养,通过RT-PCR及免疫组织化学染色的方法 ,检测细胞角蛋白-19(CK-19)、巢蛋白(Nestin)、胰高血糖素和胰岛素等干细胞相关标志物。干细胞诱导培养基中加入纯化的胰岛进行混合培养,通过观察干细胞表达胰岛素的阳性率评价其诱导转化率。结果 V型胶原酶逆行胰管灌注继而Ficoll-400梯度离心可获得生物活性良好的胰岛;所培养的干细胞表达CK-19、Nestin、胰高血糖素,诱导后部分表达胰岛素。干细胞-胰岛混合培养组及干细胞单独培养组诱导后,细胞胰岛素阳性率分别为38.2%和23.9%,差异有统计学意义(P<0.05);CK-19阳性率分别为89.3%和81.6%,其差异亦有统计学意义(P<0.05)。结论胰腺导管干细胞与胰岛混合培养可提高胰腺导管干细胞向β前体细胞的转化率,其作用与CK-19的表达密切相关。Objective To observe the differentiation rate of pancreatic stem cells （PSCs） when they were cocultured with islets,and then study its mechanism. Methods Isolation of rat islets was performed with collagenase-V digestion and purification by using Ficoll-400. At the same time,PSCs were harvested from pancreatic rudiments of fetal rats by using collagenase digestion and identified by immuocytochemistry and RT-PCR after culture in the second generation. Then islets and PSCs were cultured together,and insulin expression of PSCs was observed to evaluate the rate of PSCs to differentiate into insulin-producing cells. Results Islets with biological viability were obtained by using collagenase-V and Ficoll-400 in digestion and purification. Most of the PSCs expressed CK-19,nestin and glucogon,and some of the PSCs also expressed insulin after induction. The rates of insulin-positive PSCs in the islet-stem cell cocultured group and the stem cell cultured group were 38.2% and 23.9% （P0.05） respectively,and the rates of CK-19 positive PSCs in the two group were 89.3% and 81.6% respectively （P0.05）. Conclusion Cocultivation with islets can make PSCs differentiate into insulin-producing cells,which is closely related to expression of intracellular CK-19.

关 键 词：糖尿病 胰岛移植 干细胞 诱导 分化 大鼠 Wistar 

分 类 号：R458.5[医药卫生—治疗学] R-332[医药卫生—临床医学]