体外诱导大鼠骨髓间充质干细胞分化为胰岛素分泌细胞

In vitro differentiation of rat bone marrow mesenchymal stem cells into insulin-producing cells

目的：国内外对骨髓间充质干细胞体外成功诱导分化为胰岛素分泌细胞的报道屡见不鲜,但这些诱导方法都比较繁琐,而且大多都应用了细胞因子。实验拟验证体外非细胞因子方法诱导骨髓间充质干细胞分化为胰岛素分泌细胞的可能性。方法：实验于2005-11/2007-01在大连医科大学诊断学实验中心,校一级实验室完成。①实验材料：4-6周龄Wistar大鼠由大连医科大学实验动物中心提供,实验过程中对动物处置符合动物伦理学标准。②实验方法：无菌条件下取大鼠的骨髓细胞,并通过贴壁时间差异性去除单核细胞从而获得骨髓干细胞。应用二甲基亚砜及高糖环境依次对大鼠骨髓间充质干细胞进行体外诱导,以直接加入含体积分数为0.10胎牛血清的L-DMEM培养基培养的为对照。③实验评估：通过形态、双硫腙染色、免疫组织化学及RT-PCR方法对诱导后的细胞进行检测,采用ELISA方法对诱导后细胞分泌的胰岛素进行检测。结果：①培养10d时,诱导组细胞呈细胞团样改变,内层的细胞为圆形,外层的细胞呈长梭形并包绕着内层细胞。对照组细胞也呈团样改变,但形态不规则,呈发散状,细胞呈梭形类似骨髓间充质细胞。②诱导组的细胞团双硫腙染色后呈明显猩红色,对照组形成的细胞团双硫腙染色不显色。③诱导组成团及单个存在的圆形和梭形细胞中有胰岛素存在,对照组细胞团及未成团细胞中无胰岛素存在。④在内参蛋白GAPDH表达量大致相等的条件下,诱导组诱导的细胞表达Ins1和Ins2,对照组培养的细胞不表达Ins1和Ins2。⑤ELISA法证实诱导后细胞向胞外分泌胰岛素,与对照组比较差异显著（P〈0.05）。结论：大鼠骨髓间充质干细胞体外经过二甲基亚砜和高糖环境诱导可分化为胰岛素分泌细胞。

AIM： A variety of methods by which bone marrow mesenchymal stem cells （BMSCs） are induced into insulin-producing cells have been reported, and most are very complicated and cytokines are used as the induction factor limiting its clinical application. In this experiment, we tried to explore the possibility of BMSCs in vitro differentiating into insulin-producing cell without cytokine. METHODS： Experiments were performed at the Experimental Center of Diagnostics, Dalian Medical University from November 2005 to January 2007. （1）4-6 weeks Wistar rat were bought from Experimental Animal Center of Dalian Medical University. All procedure was accordant with experiment guideline of animal ethical standards. （2）BMSCs were harvested from bone marrow cells of rats by taking out monocytes by difference in adherence. In vitro rat BMSCs were induced by Dimethyl Sulfoxide （DMSO） and high-glucose culture in turns. Cells cultured in L-DMEM medium containing volume fraction of 0.10 fetal bovine serum （FBS） were served as control. （3）Differentiated cells were detected by morphological changes, dithizone staining, immunocytochemistry and reverse transcription-polymerase chain reaction （RT-PCR）. Insulin excreted from differentiated cells was tested with enzyme-labeled immunosorbernt assay （ELISA）. RESULTS： （1）At day 10, there were cells in clusters in the induction group, and inter-layer cells were round, outer-layer cells were long fusiform. There were irregular cell clusters in the control group, and the cells were divergent fusiform. （2）Cell masses were scarlet in the induction group after dithizone staining, and non-stained in the control group. （3）Insulin was found in the single round and fusiform cells in the induction group, but no insulin was found in the control group. （4）With the same expression of glyceraldehydes phosphate dehydrogenase （GAPDH）, Insl and Ins2 expression were found in the induction group, but no Insl and Ins2 were seen in the control group. （5）ELISA demonstrated that insulin was excreted from differentiated cells and there were significant differences between both groups （P 〈 0.05）. CONCLUSION： Rat BMSCs can differentiate into insulin-producing cells in vitro by DMSO and high-glucose culture.