胰岛干细胞分离及诱导分化的实验研究  

Research on the methods including differentiation and isolation of pancreatic stem cells

作　　者：陈自力[1] 孙诚谊[1] 潘耀振[1] 陈玲[1] 肖宏枝 

机构地区：[1]贵阳医学院附属医院肝胆外科,贵州贵阳550001

出　　处：《中国现代医学杂志》2014年第8期40-45,共6页China Journal of Modern Medicine

基　　金：贵州省科技攻关课题(No:黔科合201031492010)

摘　　要：目的①比较两种不同的分离方法在胰岛干细胞分离过程中的效果差异。②探讨维甲酸(Retinoic Acid)联合活化素A(Activin A)在胰岛干细胞诱导分化过程中的价值。方法随机分组比较麻醉状态下消化酶原位灌注法和离体机械消化法在胰岛干细胞分离过程中的效果差异;将分离出的胰岛干细胞进行贴壁培养,培养过程中加入维甲酸及活化素A做为实验组,取不加维甲酸﹑活化素A及加入高糖的培养液作为实验对照组,通过观察细胞生长状态及绘制细胞生长曲线来分析该处理因素对胰岛干细胞的影响,从而了解其在胰岛干细胞分离及分化过程中的价值。结果①麻醉状态下原位灌注消化法消化酶用量小,消化彻底,巢蛋白(Nestin)阳性细胞率高。②维甲酸联合活化素A明显地促进Nestin阳性细胞呈簇状生长,并形成类胰岛样细胞团;通过免疫组织化学染色鉴定发现胰岛样细胞团中心部位胰岛素抗体阳性细胞及周围区域胰高血糖素抗体阳性细胞数明显增多,通过记录类胰岛细胞团形成时间,维甲酸及活化素A组明显短于高糖组及空白组,差异具有统计学意义(P<0.01)。结论胰岛干细胞分离过程中尽量减少对细胞的机械破坏,分化过程中联合应用维甲酸及活化素A明显地促进Nestin阳性细胞呈簇状生长,形成类胰岛样细胞团,为体外获得大量的胰岛细胞从而解决移植细胞来源的难题提供了可能。[Objective] To compare the difference between the methods of isolation in pancreatic stem cells and investigates the value of Activin A and retinoic acid in the process of differentiation in pancreatic stem cells. [Methods] Firstly, we grouped the SD rat at random, then compared the difference between the methods of isolation involving digestive ferment perfusion in original position narcotically and mechanical digestion in vitro. We cultured the stem cells by using the methods above, and mixed the revulsant into the culture fluid. We divided the Activin A and retinoic acid as the experimental group, the high glucose and nothing as the control group. We observed the state of affairs and drew the growth curvature of the cells, analyzed the effect of these factor of influence. In the end, we investigated the value of estimating the ability of differentiation. [Results] The methods of digestive ferment perfusion in original position digesting completely, which cost lower quantum of digestive ferment. Activin A and retinoic acid promoted the growth of the pancreatic stem cells obviously and formed the similar structure of pancreatic island. In the resuks of immunohistochemistry assays, we found that the expression of insulin and Glucagon increased obviously in the core. The time of formation the similar structure of pancreatic island, compared to the control group, has significantly differences （P 〈0.01）. [Conclusion] The process of isolating in pancereatic stem cells should avoid cell mechanical damage and mix Activin A, retinoic acid, which may promote the growth of pancereatic stem cells obviously and the ability of forming the pancreatic island. It is possible to solve the problem of obtaining and transplanting.

关 键 词：胰岛干细胞 糖尿病 诱导分化 分离培养 

分 类 号：R-332[医药卫生] R329.28