胰腺干细胞诱导分化后同种移植治疗糖尿病的实验研究
Pancreatic stem cells differentiation and allograft for the treatment of diabetes
摘要 目的 探讨胰腺干细胞体外定向分化潜能,评价诱导分化后细胞同种移植对糖尿病的治疗效果.方法 体外分离、纯化成体Wistar大鼠胰腺干细胞,运用荧光免疫染色法对胰腺干细胞表面进行鉴定,然后在肝细胞生长因子、尼克酰胺等共刺激下诱导细胞定向分化.双硫腙(DTZ)染色对诱导后细胞进行胰岛细胞鉴定,ELISA染色光电酶标记法检测其胰岛素分泌功能.采用链脲佐菌素(STZ)腹腔注射建立Wistar大鼠糖尿病模型,将40只造模成功的大鼠随机分为胰岛细胞移植组(移植组)和安慰剂注射组(对照组),分别于移植前1 d及移植后1、2、3、4周经尾静脉采血检测血清胰岛素和血糖水平.结果 本实验成功获取成体大鼠胰腺干细胞,细胞表面CK19、Pdx-1和Nestin表达均为阳性.诱导分化后的细胞DTZ染色呈棕红色,在高糖刺激下表达、分泌胰岛素.移植后4周内移植组血清胰岛素水平平均为(11.41±1.52)mU/L,血糖水平平均为(8.22±2.7)mmol/L,对照组分别为(9.30±1.56)mU/L和(12.23±3.8)mmol/L,两组相差显著(P<0.05).结论 体外分离成体胰腺干细胞可被定向诱导分化为具有胰岛素分泌功能的胰岛样细胞,同种移植后对糖尿病具有治疗作用.
Abstract:
Objective To investigate the potential of pancreatic stem cells (PSCs) directed differentiation in vitro, and to evaluate the effects of differentiated PSCs allograft on the treatment of diabetes.Methods The PSCs of adult Wistar rats were separated and purified in vitro. The surface of PSCs was determined by immunofluorescence staining, and then it was stimulated by hepatocyte growth factor (HGF) and nicotinamide to induce directed differentiation. Dithizone dyeing was used to determine the islet-like cells after induction, and ELISA staining method was used to detect the insulin levels. Streptozotocin peritoneal injection was used to induce the diabetic rat mode. 40 rats were randomly allocated into pancreatic islet cells allograft group (experiment group) and placebo group. The serum insulin and glucose levels 1 d before transplantation and 1, 2, 3, 4 week after transplantation were measured. Results PSCs of adult Wistar rats were successfully obtained, and the expression of CK19, Pdx-1 and Nestin on cell surface was positive. Dithizone dyeing for directed differentiation cells showed brownish red color. The cells could express and secrete insulin after hyperglycaemia stimulation. The serum insulin and glucose levels 4 week after transplantation were (11.41 ±1.52) mU/L and (8.22 ± 2.7) mmol/L, which were (9.30 ± 1.56) mU/L and (12.23 ± 3.8) mmol/L in the placebo group, and difference was statistically significant (P<0.05). Conclusions PSCs can be induced and directed differentiated in vitro into islet-like clusters with insulin secretion function. And its allograft has the potential for the treatment of diabetes.
Author: SHI Guang-jun ZHANG Lei XU Ping LUAN Shao-Hai YU Jiang CHEN Hao-qiang
作者单位: 青岛市立医院肝胆胰外科,山东青岛,266071 济宁医学院附属医院急诊外科
刊 名 中华胰腺病杂志 ISTIC
年,卷(期): 2011, 11(1)
分类号: R73
机标分类号: R73 R58
在线出版日期: 2011年5月16日
基金项目: 青岛市科技局基金项目计划