骨髓间充质干细胞向早期神经元样细胞诱导分化的研究

背景骨髓间充质干细胞(mesenchymal stem cells,MSCs)不仅能分化成间质细胞,而且能向实质细胞(如心肌细胞)转化.在体外定向诱导MSCs分化成神经细胞,是目前国内外研究热点,具有重大理论意义.目的探讨大鼠MSCs体外诱导分化成神经元的能力.设计随机空白对照实验的研究.地点、材料和干预实验在吉林大学公共卫生学院毒理教研室完成.实验动物采用Wistar大鼠(吉林大学实验动物中心),6周龄,雌雄不限.将2～4代的rMSCs接种在铺有盖玻片的24孔板中.分为3个实验组,一个对照组.实验组分别加入终浓度为0.25,0.50和1.00 mmol/L异丁基甲基黄嘌呤(isobutylmethlxanthine,IBMX)诱导传代的rMSCs,待细胞分化后进行形态学观察,对照组中不加诱导剂,并用免疫细胞化学方法进行细胞鉴定.主要观察指标培养MSCs的生长情况,诱导后细胞的形态学变化及nestin(小鼠抗大鼠,单克隆),神经元特异性烯醇化酶(neuron-specific enolase,NSE)抗体(兔抗鼠,多抗)、微管相关蛋白-2a,bmicrotubule-associated protein-2a,b,MAP-2a,b)的免疫细胞化学检测.结果加入IBMX后,0.25mmol/L组分化成神经元样细胞较少.1.0mmol/L组细胞加入IBMX第2天开始可见细胞陆续死亡.0.5 mmol/L组2 d即可见有神经元样细胞出现,细胞具有典型的神经元形态特征,0.5 mmol/LIBMX诱导细胞效果最好,2 d即可见有神经元样细胞出现,6 d神经元样细胞占细胞总数的(23.2±2.3)%,NSE染色阳性.对照组中未诱导细胞表达nestin,诱导后3 d阳性细胞增多,6 d减少.实验组和对照组均不表达MAP-2a,b.结论体外rMSC能扩增、传代,并被诱导分化成早期神经元样细胞.

Research of the differentiation of mesenchymal stem cells into early neuron-like cells

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate not only into interstitial cells but also into parenchyma cell( e. g. myocardial cells) .It is a hotspot of national and international researches to directionally induce MSCs to differentiate into neurocyte in vitro, which has an important theoretical significance.OBJECTIVE: To investigate the ability of MSCs to differentiate into neuron in vitro in rat.DESIGN: A randomly blank-control study.SETTING, MATERIALS AND INTERVENTIONS: Research was completed at Toxicology Department of Public Health School of Jilin University.Six-week old Wistar rats of either gender obtained from experimental animal center of Jilln University were employed. The 2nd to 4th generation of rMSCs were inoculated on 24-well plates covered by microscopic coverglass. There were 3 experiment groups and one control group. Isobutylmethylxanthine (IBMX) with a concentration of 0.25, 0.50 and 1.00 mmol/L was added respectively into 3 experiment group to induce rMSCs generation. Morphologic observation was operated after cellular differentiation. There was no inductive agent added into control group. Cellular identification was operated by immunocytochemistry.MAIN OUTCOME MEASURES: The growth of cultured MSCs; cellular morphologic changes after induction; the immunocytochemical assay of nestin (mouse antirat, monoclone), neuron-specific enolase(NSE) antibody(rabbit anti mouse, mutil-antibodies), and microtubule-associated protein-2a, b (MAP-2a, b).RESULTS: After being added with IBMX, there was little neuron-like cells differentiation in 0.25 mmol/L group; cells in 0. 1 mmol/L group gradually died since the 2nd day; neuron-like cells could be seen on the 2nd day in 0.5mmol/L group with typical morphological characteristics. 0.5 mmol/L IBMX had a best inductive effectiveness. Neuron-like cells could be seen on the 2nd day and on the 6th day, neuron-like cells were with(23.2 ± 2.3)% of the total cells, NSE dye reaction was positive. Non-differentiating cellular expression was nestin. With 3-day induction, positive cells increased, and decreased on the 6th day.CONCLUSION: rMSC can be amplified, generated in vitro and to be induced into early phase neuron-like cells.