成人骨髓间质干细胞分化为神经元样细胞前后基因表达的研究  

The Genes Change of hMSC before and after the Differentation into Neuron-like Cells

作　　者：姚晓黎[1] 张成[1] 冯善伟[1] 卢锡林[1] 刘祖国[2] 邓宇斌[3] 

机构地区：[1]中山大学附属第一医院神经科,广州510080 [2]中山大学眼科中心,广州510060 [3]中山大学病理生理教研室,广州510080

出　　处：《中药材》2005年第6期476-481,共6页Journal of Chinese Medicinal Materials

基　　金：卫生部临床学科重点项目(编号:2001321);广东省科技计划项目(项目编号:2KM05501S);广东省科技计划项目重大专项(2003A3020106);国家自然科学基金(30270732)

摘　　要：目的探讨成人骨髓间质干细胞(MSC)分化为神经元样细胞前后基因表达的变化。方法从成人骨髓分离MSC,培养扩增。用参芪液诱导hMSC分化为神经元,并用免疫组化法检测神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达来鉴定是否为神经元样细胞。采用半定量RTPCR方法检测10个基因(包括内胚层基因Ceruloplasmin;中胚层基因SM22;外胚层基因Amyloidprecursorprotein、syntaxin;生殖系基因protamine;神经元特异性基因NeuroD、NF、NSE、GFAP、Tau)在诱导分化前后的变化。结果hMSC经参芪液诱导后,可见神经元样细胞。免疫组化结果显示诱导出的神经元样细胞表达NSE、NF阳性,GFAP阴性。hMSC在分化前表达内胚层、中胚层、外胚层和生殖系基因,随着分化时间的延长,生殖系基因不表达,内胚层和中胚层基因表达减弱,外胚层基因表达基本不变。而在分化过程中外胚层基因和神经元特异性标记基因表达明显增强。结论hMSC分化为神经元样细胞的过程并不是简单的神经特异性基因的“开”和“关”,而是与神经细胞分化成熟过程中相关基因表达的量相关。Objective: To study the genes change of hMSC before and after differentiation into neuron-like cells. Methods: hMSC were separated from marrow, cultured and expanded in culture medium. After hMSC being induced to differentiate with Shenqiye, Neuron-specific enolase(MSE), neurofilament(NF), and glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry respectively. Using semi-quantitiative RT-PCR to detect ten genes change in hMSC before and after differentiation. Two genes were used to detect the gene sequence. Results: When treated with the inducer——Shenqiye, hMSC exhibited neuronal phenotype. The expressions of NSE and NF in the neuron-like cells were positive, but the glial astrocyte marker GFAP was not found. hMSC expressed germline, ectodermal, endodermal and mesodermal genes prior to neurogenasis. After differentiation, germline genes did not express, while the expressions of endodermal and mesodermal genes were weakened. Ectodermal and neuronal genes expressed stronger. Conclusion: hMSC express multipotentiality, and the neural differentiation comprises quantitative modulation of gene expression rather than simple on-off switching of neural specific genes.

关 键 词：神经元样细胞 间质干细胞分化 表达的研究 成人骨髓间质干细胞 PROTAMINE 胶质纤维酸性蛋白 神经元烯醇化酶 免疫组化法检测 protein 特异性基因 NEUROD 神经元特异性 相关基因表达 神经细胞分化 GFAP 神经丝蛋白 PCR方法 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R338.21[医药卫生—基础医学]