GAP-43在骨髓间充质干细胞向神经样细胞分化过程中表达的实验研究

Study of GAP-43 expression on differentiation of bone mesenchymal stem cells to neuron-like cells

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 growth associated protein -43（GAP-43） expression on the differentiation of bone marrow mesenchymal stem cells （BMSCs） to neuron-like ceils in vitro in order to understand it＇s effect on this process.Methods BMSCs of rat were isolated and cultivated in vitro,the purified BMSCs of the third generation were identifieated by flow cytometer.BMSCs were induced and differentiated by beta mereaptoethanol （BME） and brain-derived neurotrophie factor （BDNF）, respectively. Morphological changes of induced cells were observed,and GAP-43,nesfin,NSE and NF of induced cells in two groups were detected by immunocy-toehemistry. The positive rates of GAP-43 staining in two groups were compared by statistic method on 2,4,6,8 hours after induction respectively.Results The third generation BMSCs were identificated with CD90（ ＋ ）,CD71（ ＋ ）,CD29（ ＋ ）,and CD45（-） by flow cytometer. BMSCs can be induced to differentiate into neuron-like cells by BME and BDNF respectively. The induced cells expressed GAP-43, nestin,NSE,NF. On 2,4,6,8 hours after induction,the positive rates of GAP-43 were 77.1% ,83.4% ,87.0% ,82.5% in BME group, and 69.3 %,78.1%, 82.2%, 86.8 % in BDNF group respectively. BMSCs expressed GAP-43 persistently after induction in both two groups.There were no significant differences between two groups in positive rates of GAP-43 staining （ P 〉 0.05）. Condusion GAP-43 can express persistently in the process that BMSCs are induced to differentiate into neuron-like cells by either chemical inducer or neurotrophie factor, and play a important role in this process.