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目的观察生长相关蛋白-43(GAP-43)在体外培养的骨髓间充质干细胞(BMSCs)向神经样细胞分化中的表达情况以探讨其在骨髓间充质干细胞定向分化过程中的作用。方法体外培养大鼠骨髓间充质干细胞,选取第3代细胞,经流式细胞仪检测细胞表面标志物进行鉴定,并分别应用化学诱导剂β巯基乙醇(BME)和脑源性神经生长因子(BDNF)诱导BMSCs分化,观察诱导后细胞的形态学变化,并应用免疫细胞化学方法对诱导后的细胞进行鉴定、分析生长相关蛋白43、神经巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)的表达情况。应用统计学方法比较两组细胞经诱导后2、4、6、8 h GAP-43免疫细胞化学染色阳性率。结果第三代BMSCs经流式细胞仪检测CD90(+)、CD71(+)、CD29(+),CD45(-),经两种方法诱导BMSCs均可分化为神经样细胞,分化后细胞均表达GAP-43、nestin、NSE、NF。诱导后2,4,6,8 h GAP-43表达阳性率BME组为77.1%,83.4%,87.0%,82.5%,BDNF组:69.3%,78.1%,82.2%,86.8%。GAP-43随诱导分化时间增加而持续表达,两组间表达阳性率无统计学差异。结论GAP-43在化学诱导剂及生长因子诱导BMSCs分化为神经样细胞过程中持续表达,对于骨髓间充质干细胞向神经样细胞的分化具有重要意义。
Objective To observe the expression of growth-associated protein-43 (GAP-43) in differentiation of neuroblastoma cells from bone marrow-derived mesenchymal stem cells (BMSCs) in vitro to explore its role in the directional differentiation of bone marrow mesenchymal stem cells. Methods Rat bone marrow mesenchymal stem cells were cultured in vitro and the third generation of cells were selected. The cell surface markers were identified by flow cytometry. The chemical inducer β-mercaptoethanol (BME) and brain-derived nerve growth factor BDNF) to induce the differentiation of BMSCs. The morphological changes of the induced cells were observed. The induced cells were identified by immunocytochemistry. The expressions of growth-associated protein 43, nestin, neuron-specific enolase NSE), neurofilament (NF) expression. The positive rate of GAP-43 immunocytochemical staining was compared between the two groups after 2, 4, 6 and 8 h of induction by statistical methods. Results The third generation of BMSCs could detect CD90 (+), CD71 (+), CD29 (+) and CD45 (-) by flow cytometry. BMSCs could differentiate into neuron- GAP-43, nestin, NSE, NF. The positive rate of GAP-43 expression at 2, 4, 6 and 8 h after induction was 77.1%, 83.4%, 87.0% and 82.5% in BME group and 69.3%, 78.1%, 82.2% and 86.8% in BDNF group. The expression of GAP-43 persisted with the induction of differentiation, and there was no significant difference between the two groups. CONCLUSION: GAP-43 is expressed continuously in the process of differentiation of BMSCs into neuron-like cells induced by chemoattractant and growth factor, which is of great significance for the differentiation of bone marrow mesenchymal stem cells into neuron-like cells.