NGF体外诱导hMSCs分化为神经元样细胞

发布时间：2018-08-05 20:36

【摘要】： 目的:探索人骨髓间充质干细胞(human bone marrow stromal cells,hMSCs)体外分离、培养及纯化的合适实验条件,并探讨神经生长因子体外诱导人骨髓间充质干细胞(hMSCs)分化成神经细胞的可行性。方法:①从正常成人髂骨中获取hMSCs,采用密度梯度离心法和全骨髓贴壁法相结合的方法分离人骨髓低密度单个核细胞,常规传代、培养获得纯化的hMSCs。②取第2、4、6代生长状态良好的hMSCs,以含胎牛血清的DMEM培养液培养消化后,采用MTT法,描绘细胞增殖曲线。③取第3～5代hMSCs,接近70%～80%融合后,分为实验组:用碱性成纤维生长因子(Basic fibroblast growth factor,bFGF)对hMSCs进行预诱导24h,再分别用浓度为50ng/ml、100ng/ml、150ng/ml、200ng/ml神经生长因子培养基诱导分化;对照组:仅加等量无血清培养基。通过形态学观察诱导后各组细胞形态变化。④细胞培养后,第0.5d、1d、3d、5d,采用免疫组织化学法检测神经干细胞标记神经元特异性烯醇化酶(NSE)、巢蛋白(Nestin)的表达,选细胞密度适中视野计算阳性细胞数,对比各组细胞阳性表达比例。结果:①骨髓间充质干细胞的生长曲线:培养第1～2d为潜伏期,细胞生长缓慢;此后,细胞增殖加快并迅速进入对数生长期,至第6～7d达到高峰,以后细胞增殖减慢,进入平台期。 ②hMSCs诱导分化后的形态学观察结果:12h后部分细胞形态发生改变,表现为胞体变大,有类似轴突和树突的细长突起,24h后表现更为明显,部分细胞间也可见网络状排列,细胞死亡现象不明显;对照组无明显的形态学改变,仍为长梭形细胞。 ③神经元样细胞免疫组化测定结果:不同浓度神经生长因子组NSE、Nestin表达均为阳性,表现为胞体和突起被染成棕黄色;对照组无阳性结果出现。 ④不同浓度神经生长因子作用下的神经元样细胞分化率有差别。浓度为50ng/ml、100ng/ml、150ng/ml、及200ng/ml分化率分别为(35.6±4.4)%、(61.9±3.4)%、(57.3±5.7)%和(57.2±4.8)%,不同浓度的神经生长因子诱导阳性细胞数有显著差异(F=338.71,P0.05),其中100ng/ml组的分化率最高,各实验组比较:浓度为100ng/ml、浓度为150ng/ml及浓度为200ng/ml组阳性细胞数均高于浓度为50ng/ml组(P0.01),但后三者比较,差异无显著性(P0.05)。⑤100ng/ml NGF在0.5d、1d、3d、5d的分化率分别为(27.8±5.6)%、(60.5±6.7)%、(61.9±3.4)%和(30.7±3.5)%,不同时间相同浓度下神经生长因子诱导阳性细胞数有显著差异(F=136.03,P0.01),其中第3d的分化率最高,但与第1d阳性细胞数比较,差异无显著性(P0.05),第5d的阳性细胞数减少,与第1,3d比较差异有显著性(P0.01)。结论:①本实验建立的密度梯度离心法和全骨髓贴壁法相结合,体外分离、纯化hMSCs的方法,可能是目前操作简单、不易污染及分离纯度高的一种较好的方法。 ②NGF可诱导hMSCs定向分化成神经元样细胞且存活时间长,100ng/ml浓度的NGF可以起到较好的促进hMSCs向神经元样细胞分化的作用。
[Abstract]:Objective: to explore the suitable experimental conditions for the isolation, culture and purification of human bone marrow mesenchymal stem cells (human bone marrow stromal cells) in vitro, and to explore the feasibility of nerve growth factor (NGF) inducing the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into nerve cells in vitro. Methods Human bone marrow low density mononuclear cells were isolated from normal adult iliac bone by using density gradient centrifugation and whole bone marrow adherent method. The purified hMSCs.2 was cultured and digested in the DMEM medium containing fetal bovine serum for the second and fourth generation of HMSCs. After digesting and digesting, MTT method was used to depict the cell proliferation curve. 3. The 3rd generation hMSCs were obtained, which were close to 70%, 80% of the cells fused. The experimental group was divided into two groups: the hMSCs was preinduced by basic fibroblast growth factor (Basic fibroblast growth) for 24 h, and then induced by 50ng / ml 100ng / ml NGF medium, while the control group was treated with the same amount of serum-free medium. The morphological changes of cells in each group were observed by morphological observation. 4 cells were cultured on the 1st and 3rd day after induction, and the expression of NSCs labeled neuron specific enolase (NSE),) nestin (Nestin) was detected by immunohistochemical method. The number of positive cells was calculated with moderate cell density visual field, and the proportion of positive cells in each group was compared. Results the growth curve of bone marrow mesenchymal stem cells (BMSCs): the first day of culture was the incubation period, the cell growth was slow, then the cell proliferation accelerated and rapidly entered the logarithmic growth phase, and reached the peak at the 6th day, then the cell proliferation slowed down. After 2hMSCs induced differentiation, morphological changes occurred in some cells at 12 h, showing that the cell bodies became larger, and the slender processes with axons and dendrites became more obvious 24 hours later. Some cells could also be seen network arrangement, the phenomenon of cell death was not obvious, the control group had no obvious morphological changes, The expression of NSE-nestin was positive in different concentrations of NGF, and the cell bodies and processes were stained brown. No positive results were found in the control group. 4 the differentiation rate of neuron-like cells in different concentrations of nerve growth factor was different. The differentiation rate of 200ng/ml was (35.6 卤4.4), (61.9 卤3.4), (57.3 卤5.7)% and (57.2 卤4.8), respectively. There were significant differences in the number of NGF-induced positive cells in different concentrations (F338.71P0.05). The number of positive cells in 100 ng / ml, 150ng/ml and 200ng/ml groups was higher than that in 50ng/ml group (P0.01). There was no significant difference (P0.05). 5100ng / ml NGF differentiation rate was (27.8 卤5.6), (60.5 卤6.7)%, (61.9 卤3.4)% and (30.7 卤3.5) in 0.5 day, (60.5 卤6.7), (61.9 卤3.4)% and (30.7 卤3.5), respectively. There was significant difference in the number of positive cells induced by nerve growth factor at the same concentration at different time (FF136.03 / P0.01), and the differentiation rate was the highest on the 3rd day, but compared with the number of positive cells on the first day. There was no significant difference (P0.05), but the number of positive cells decreased on the 5th day, which was significantly different from that on the 1st day (P0.01). Conclusion the density gradient centrifugation combined with the whole bone marrow adherent method is a simple method for the isolation and purification of hMSCs in vitro. 2NGF can induce hMSCs to differentiate into neuron-like cells with long survival time of 100ng / ml NGF can promote hMSCs to neuron-like cells. The role of cell differentiation.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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