大鼠胰腺导管上皮细胞体外诱导的研究

目的:观察大鼠胰腺导管上皮经四步法诱导分化为胰岛样细胞团后移植治疗糖尿病的效果?方法:采用Ⅴ型胶原酶胆管内灌注消化法,经Ficoll不连续密度梯度离心后分别获得导管上皮细胞和新鲜胰岛?导管上皮细胞经原代培养及传代后取2~6代细胞开始进行四步18天的诱导,分化为胰岛样细胞团,行免疫荧光鉴定?将链脲佐菌素造模成功的18只SD大鼠采用完全随机法均分成3组,空白对照组?新鲜胰岛组?胰岛样细胞团组进行左肾包膜下移植?分别给予RMPI1640?新鲜胰岛和胰岛样细胞团,移植后隔日固定时间监测血糖?结果:经免疫荧光鉴定,分离纯化的胰腺导管细胞具有干细胞特性,诱导后的胰岛样细胞团胰岛素?胰高血糖素染色均为阳性?移植后空白对照组血糖维持在高血糖范围?新鲜胰岛组移植后血糖逐步下降至正常水平,并在2周之内基本稳定在正常范围内但略有上升?胰岛样细胞团组移植后前2天血糖不降反升,之后有所下降但始终未能降至正常范围,然后又逐渐上升?结论:胰腺导管上皮细胞经该方案诱导后分化为胰岛样细胞团,使糖尿病大鼠血糖有一定程度的下降,但下降幅度及维持时间不能令人满意,可能与移植量不够及诱导的胰岛样细胞团功能不良有关?

Differentiation of rat pancreatic duct stem cells into islet-like cell clusters

Objective:To observe the effects of islet-liking cell clusters inducing from pancreatic duct stem cells in treatment of diabetic rats by transplantation. Methods:After intraductal collagenase digestion,purified by Ficoll density gradient centrifugation,fresh islets and pancreatic duct stem cells were obtained. Then the cells were cultured and expanded in vitro. The 2nd to 6th generation of duct cells were induced in four stages to differentiate into islet-like cell clusters. Islet-like cell clusters were detected by immunofluorescence staining before transplantation. The rat diabetic model was constructed by intraperitoneal injection with streptozotocin(STZ). Eighteen diabetes rats were randomly divided into three groups:diabetic control group,islet transplantation group,islet-liking cell clusters transplantation group. The implants were transplanted into the subcapsular in the left kidneys. The levels of non-fasting blood glucose of rats in 3 groups were measured every other day. Results:The pancreatic duct cells isolated and purified had the characteristic of stem cells by immunofluorescence staining. The results of immunofluorescence staining revealed that insulining and glucagon stain of the islet-like cell clusters were positive. After transplantation,the level of blood glucose of rats in the control group was high,keeping in the range of diabetes. The blood glucose level of rats in the islet transplantation group decreased after transplantation,which was in the normal range for two weeks. The blood glucose level of rats implanted with islet-like cell clusters increased in the first two days after transplantation and decreased after then,and was higher than the normal range during two weeks. Conclusion:Islet-like cell clusters generated in vitro from pancreatic duct cells has some effects on reversing the diabetes in diabetic rats,although the blood glucose control effect is not satisfying.