论文部分内容阅读
目的探讨联合应用碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和纹状体条件培养液定向诱导大鼠骨髓间充质干细胞(BMMSCs)分化为多巴胺能神经元的可能性。方法1.BMMSCs的分离培养:分离纯化健康成年Wistar大鼠BMMSCs进行传代培养。2.纹状体条件培养液的制备:出生24h内的新生Wistar大鼠,分离纹状体组织用于制备纹状体条件培养液。3.BMMSCs的诱导分化:取体外培养的第5代间充质干细胞,用含bFGF和EGF的预诱导液进行预诱导,24h后去除预诱导液,换用纹状体条件培养液进行诱导。4.倒置显微镜下观察细胞形态变化,并用细胞免疫化学技术检测细胞内神经元特异烯醇化酶(NSE)和酪氨酸羟化酶(TH)的表达。结果大鼠BMMSCs经bFGF、EGF和纹状体条件培养液诱导后细胞胞体逐渐回缩成团,形成梭形,部分细胞可见突起伸出,类似神经元。细胞免疫化学检测,联合诱导后NSE阳性表达为(72.70±14.81)%,TH阳性表达为(34.50±15.93)%。结论bFGF、EGF联合纹状体条件培养液体外可定向诱导大鼠BMMSCs分化为多巴胺能神经元。
OBJECTIVE: To investigate the possibility of directional differentiation of rat bone marrow mesenchymal stem cells (BMMSCs) into dopaminergic neurons by combining application of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and striatal conditioned medium. Isolation and Culture of BMMSCs: BMMSCs of healthy adult Wistar rats were isolated and purified for subculturing. 2. Preparation of Striatal Conditioned Culture Medium: Newborn Wistar rats born within 24h were isolated for preparation of striatal conditioned medium. Induction and differentiation of BMSCs: The passage 5 mesenchymal stem cells cultured in vitro were pre-induced with pre-induction solution containing bFGF and EGF. After 24 hours, the pre-induction solution was removed and replaced with striatal conditioned medium. The morphological changes of cells were observed under an inverted microscope, and the expression of neuron specific enolase (NSE) and tyrosine hydroxylase (TH) was detected by immunocytochemistry. Results After BMMSCs were induced by bFGF, EGF and striatum, the cell bodies of rats were gradually retracted into clusters and formed fusiform shape. Some neurites were seen protruding from some cells. Immunocytochemistry showed that the positive expression of NSE was (72.70 ± 14.81)% and the positive expression of TH was (34.50 ± 15.93)% after combined induction. Conclusion bMSCs can differentiate into dopaminergic neurons in bFGF and EGF combined with conditioned medium of striatum.