不同血清浓度对bFGF联合EGF诱导BMSCs向神经细胞分化的影响

发布时间：2017-12-31 19:30

本文关键词：不同血清浓度对bFGF联合EGF诱导BMSCs向神经细胞分化的影响　出处：《神经解剖学杂志》2013年01期 　论文类型：期刊论文

【摘要】：目的:探讨不同血清浓度条件下碱性成纤维生长因子(basic fibroblast growth factor,bFGF)联合表皮生长因子(epidermal growth factor,EGF)诱导骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向神经细胞分化方向的影响。方法:全骨髓贴壁法体外分离培养BMSCs,流式细胞法检测BMSCs表面骨髓基质标志CD90和造血细胞标志CD45,MTT法绘制第3(P3)代BMSCs生长曲线。取P4代细胞分为3组给予不同诱导条件,分别给予含20 ng/ml的bFGF和20 ng/ml EGF的无胎牛血清(A组)、10 ml/L胎牛血清(B组)及100 ml/L胎牛血清(C组)的DMEM/F-12培养液进行诱导,倒置显微镜观察细胞的形态变化,诱导6 d时免疫组织化学法检测细胞内神经元特异性烯醇化酶(NSE)及胶质纤维酸性蛋白(GFAP)的表达,台盼蓝染色检测各组细胞的活力。结果:BM-SCs经3-4次传代培养后形态大致呈单一的长梭形或扁平形,流式细胞鉴定显示CD90阳性,CD45阴性;细胞生长曲线近似呈"S"形;诱导后可见细胞胞体收缩,呈双极、多极并伸出突起;诱导6 d时3组活细胞率分别为:86.57%、95.29%、97.32%;免疫组化各组均见NSE、GFAP表达,3组NSE阳性率高于GFAP,A组NSE阳性率高于B、C组,差异具有统计学意义(P0.01),C组NSE阳性率高于B组,差异具有统计学意义(P0.01),A组GFAP阳性率低于B、C组,差异具有统计学意义(P0.01),C组GFAP阳性率高于B组,差异具有统计学意义(P0.01)。结论:bFGF联合EGF可诱导BMSCs向神经细胞分化,在无血清条件下趋向于向神经元分化,在高浓度血清条件下趋向胶质细胞分化。
[Abstract]:Objective: to study basic fibroblast growth factor of basic fibroblast growth factor (BFGF) under different serum concentrations. BFGF combined with epidermal growth factor. EGF-induced bone mesenchymal stem cells. Methods: whole bone marrow adherent method was used to isolate and culture BMSCs in vitro. Bone marrow stromal marker (CD90) and hematopoietic marker (CD45) on the surface of BMSCs were detected by flow cytometry. MTT method was used to draw the BMSCs growth curve of the 3rd generation. The P4 passage cells were divided into 3 groups under different induction conditions. Group A was given fetal bovine serum containing 20 ng/ml bFGF and 20 ng/ml EGF, respectively. 10 ml/L fetal bovine serum group B) and 100 ml/L fetal bovine serum group C) were induced by DMEM/F-12 culture medium, and the morphological changes of cells were observed by inverted microscope. The expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry at 6 d after induction. Trypan blue staining was used to detect the viability of the cells in each group. Results the morphology of BM-SCs was approximately a single spindle or flattened shape after 3-4 passages. Flow cytometry showed that CD90 was positive. CD45 negative; The cell growth curve was approximately "S" shape. After induction, the cell bodies contracted, showing bipolar, multipolar and protruding processes. On the 6th day after induction, the living cell rates of the three groups were respectively: 86.57 and 95.299.32; The positive rate of NSE in three groups was higher than that in group A (P 0.01). The positive rate of NSE in group C was higher than that in group B, and the difference was statistically significant. The positive rate of GFAP in group A was lower than that in group B (P 0.01). The positive rate of GFAP in group C was higher than that in group B. the difference was statistically significant (P 0.01). Conclusion: GFAP combined with EGF can induce the differentiation of BMSCs into neural cells. It tends to differentiate into neurons in serum-free condition and glial cell in high serum condition.
【作者单位】：南昌大学第二附属医院骨一科;南昌大学研究生院医学部;九江学院分析与检测实验室;
【基金】：江西省科技支撑计划项目(20111BBG70022-2)
【分类号】：R329
【正文快照】： 骨髓间充质干细胞(bone mesenchymal stemcells,BMSCs)是来源于中胚层的一类未分化间质干细胞,具有很强的自我复制能力及多向分化潜能[1],在特定条件下可向外胚层的神经元样细胞、星形胶质样细胞和少突胶质样细胞分化[2]。在体外培养中,目前主要采用化学药物和生物制剂诱导干

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