骨髓间充质干细胞向早期神经元样细胞诱导分化的研究(英文)

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

背景:骨髓间充质干细胞(mesenchymalstemcells,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1.00mmol/L异丁基甲基黄嘌呤(isobutylmethylxanthine,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-specificenolase,NSE)抗体(兔抗鼠,多抗)、微管相关蛋白-2a,b(microtubule-associatedprotein-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。0.5mmol/L组2d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。

BACKGROUND:Mesenchymal stem cells(MSCs) can differentiate not only into inter stitial cells but also into parenchyma cell(e.g.myocardial cells). It is a hots pot of national and international researches to directionally induce MSCs to dif ferentiate into neurocyte in vitro,which has an important theoretical significan ce. 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 Depa rtment of Public Health School of Jilin University. Six-week old Wistar rats of either gender obtained from experimental animal center of Jilin University we re employed.The 2nd to 4th generation of rMSCs were inoculated on 24-well plat es covered by microscopic coverglass.There were 3 experiment groups and one cont rol group.Isobutylmethylxanthine(IBMX) with a concentration of 0.25,0.50 and 1.0 0 mmol/L was added respectively into 3 experiment group to induce rMSCs generati on.Morphologic observation was operated after cellular differentiation.There was no inductive agent added into control group.Cellular identification was operate d by immunocytochemistry. MAIN OUTCOME MEASURES:The growth of cultured MSCs;cellular morphologic change s after induction;the immunocytochemical assay of nestin(mouse antirat, monoclon e),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 diff erentiation 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 0.5 mmol/L group with typical morphological characteristics. 0.5 mmol/L IBMX had a best inductiv e 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 reac tion 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 e arly phase neuron-like cells.