胰腺干细胞和胰岛细胞在糖尿病 大鼠治疗中的疗效观察

Therapeutic effect of pancreatic stem cells and islet cells in the treatment of diabetic rats

摘要:

目的 探讨大鼠胰腺干细胞与胰岛细胞移植在大鼠糖尿病治疗中的疗效。方法 通过链脲菌 素复制1 型糖尿病大鼠模型并随机分为胰岛细胞组和胰腺干细胞组。用胶原酶V 分别消化新生和成年大鼠 胰腺，Percoll 梯度离心分离胰岛细胞和胰腺干细胞。采用Bonner-Weir 法诱导胰腺干细胞分化，DTZ 染色 检测移植物纯度，AO/PI 检测移植物活性。分别记录手术前后大鼠血糖水平、胰岛素水平及移植物存活时 间；高糖刺激实验和腹腔糖耐量评价移植物功能；取各组大鼠移植部位标本行HE 染色和免疫组织化学染 色，观察组织形态学变化。结果 胰岛细胞组和胰腺干细胞组血糖分别在术后3 和5 d 恢复正常（P <0.05）， 两组大鼠术后不同时间点的血糖有差异（P <0.05），胰腺干细胞组的降糖能力强于胰岛细胞组（P <0.05）。 两组大鼠移植后7 d 血清胰岛素水平均较术前升高（P <0.05）。高糖刺激实验表明，各组移植后15 和65 d 高糖刺激后的C 肽水平较刺激前升高（P <0.05），胰岛细胞组移植后65 d 高糖刺激后C 肽水平低于胰腺 干细胞组（P <0.05），胰岛素和C 肽水平监测结果表明胰腺干细胞组移植物胰岛素分泌维持时间长于胰岛 细胞组。移植后腹腔糖耐量实验结果表明移植物均功能良好。胰岛细胞组和胰腺干细胞组移植物的中位 存活时间分别为73 和88 d，Kaplan-Meier 曲线分析提示胰腺干细胞组移植物生存时间较胰岛细胞组延长 （P <0.05）。HE 染色提示肝脏门静脉移植区域可见新生血管包绕的胰岛细胞团，免疫组织化学染色证实细 胞团胰岛素阳性。结论 胰腺干细胞分化后经门静脉移植能安全有效地发挥降糖作用，疗效优于胰岛细胞 移植。

Abstract:

Objective To evaluate the efficacy of pancreatic stem cell and islet cell transplantation in the treatment of diabetes mellitus in rats. Methods The rat model of type 1 diabetes was induced by streptozotocin and randomly divided into islet cell and pancreatic stem cell groups. The pancreas of neonatal and adult rats were digested by collagenase V, and pancreatic cells and pancreatic stem cells were isolated by Percoll gradient centrifugation. The differentiation of pancreatic stem cells was induced by Bonner-Weir method. Dithizone detects graft purity and AO/PI detects graft activity. The blood glucose, insulin concentration and graft survival time of rats preoperative and postoperative were recorded; high glucose stimulation test and intraperitoneal glucose tolerance test evaluation of graft function; HE staining and insulin immunohistochemical staining were performed on the transplanted sites of rats in each group to observe the changes of histomorphology. Results Blood glucose in islet cell group and pancreatic stem cell group returned to normal 3 and 5 days after operation, respectively (P < 0.05). The blood glucose of the two groups was different at different time points after operation. The hypoglycemic ability of the pancreatic stem cell group was better than that of the islet cell group (P < 0.05). At 7 days after transplantation, the serum insulin levels of the two groups were higher than those before operation (P < 0.05), suggesting that the insulin secretion function of the graft was good. The results of high glucose stimulation test showed that the level of C peptide was significantly higher in each group after 15 and 65 days postoperatively than before (P < 0.05) and the level of C peptide was lower after 65 days postoperatively than that of pancreatic stem cells group (P < 0.05). The results of insulin and C-peptide index monitoring showed that the insulin secretion capacity of the pancreatic stem cell group was better than that of the islet cell group. The results of intraperitoneal glucose tolerance test in rats after transplantation showed that the graft function of both groups was good. The median survival time was 73 days in the islet cell group grafts and 88 days in the pancreatic stem cell group grafts. Kaplan-Meier curve analysis showed that the survival time of the pancreatic stem cells was longer than that of the islet cell group (P < 0.05). HE staining showed pancreatic islet cell mass surrounded by neovascularization in the portal tracts of the liver, and immunohistochemical staining showed positive insulin in the cell mass. Conclusions After differentiation of pancreatic stem cells, portal vein transplantation can safely and effectively reduce blood glucose and its efficacy is superior to that of islet cell transplantation.