体外共培养诱导骨髓间充质干细胞向胰岛样细胞分化

目的 观察成熟胰岛细胞对小鼠骨髓间充质干细胞(bone marrow derived mesenchymalstem cells,mMSCs)的诱导分化作用,为胰岛细胞移植治疗糖尿病提供移植细胞来源.方法 采用贴壁法分离培养小鼠mMSCs,并传代扩增.应用共聚焦显微镜观察细胞形态,流式细胞仪分析其表面分子.经胆总管注入Ⅳ型胶原酶消化胰腺,行密度梯度离心获得胰岛细胞.运用Transwell共培养体系,取第3代mMSCs与胰岛细胞共培养,倒置显微镜观察细胞的生长及形态变化,细胞免疫化学法检测mMSCs胰岛素的表达,胰岛素释放试验检测胰岛素分泌.以单独培养mMSCs作为对照组.结果 从小鼠骨髓中获得的细胞培养48 h后呈长梭形,体积较大,1周后呈集落式生长,可传代培养.细胞表面分子Sca-1、CD29、CD44、CD105呈阳性,且表达水平较高,而CD34、CD45阴性,证实为mMSCs.与小鼠胰岛细胞共培养7 d后,部分mMSCs细胞胰岛素免疫组化染色呈阳性,胰岛素分泌量为(16.83±0.15)μIU/ml.结论 从小鼠骨髓中分离培养的mMSCs与胰岛细胞共培养后可以被诱导分化为胰岛样细胞,为在胰岛细胞移植治疗糖尿病中存在的供体缺乏和免疫排斥问题提供了新的解决方法 .

In vitro co-culture induced mesenchymal stem cells differentiate to islet cell

Objective To observe the effects of differentiation of mature islet cells of mice on marrowmesenchymal stem cells (mMSCs). To provide transplant source for islet cell transplantation in the treatment of diabetes. Methods The culture, isolation and passage of mMSCs was performed by using patch wall, cell shape was observed by confocal microscope, and flow cytometry analysis was used to determine their biological characteristics. The type Ⅳ collagenase was injected into common bile duct to digest the pancreas, and then gradient centrifugation was used to isolate islet cells. The transwell co-culture system was used for third generation of mMSCs and isolated islet cells, then inversion microscope was used to observe the cell growth and morphological changes, immunochemistry methods was applied to detect the expression of insulin in mMSCs, and insulin release test was performed to determine the secretion of insulin. The control group consisted of cultured mMSCs alone. Results The cells from mouse bone marrow were found to be in long spindle shape with large volume after 48 hours in culture. One week later the cells grew in the form of colony with serial subcultivation. The cell surface molecules including Sca-l, CD29, CD44, CD105 were positive with high level of expression;while the cell surface molecules including CD34, CD45 were negative, all of these results confirmed that the ceils were mMSCs. After 7 days of coculture with mice islet cells, part of mMSCs cells were positively stained by insulin immunohistochemisty, the insulin secretion was (16.83±0. 15)μIU/ml.Conclusions After cocultured with islet cells, mMSCs isolated from mouse bone marrow could differentiate into islet like cells. These cells may be used in the islet cells transplantation in the treatment of diabetes, which provided a solution to the problems of donor-shortage and immunologic rejection.