中文摘要：

目的构建携带人酪氨酸羟化酶（hTH）的荧光真核表达质粒-pEGFP-C2-hTH,转染骨髓基质细胞源神经干细胞（BMSCs-D-NSCs）,观察外源EGFP和hTH基因在BMSCs-D-NSCs中的表达情况.方法应用基因重组技术,将pWAV2-TH中的TH目的基因亚克隆到荧光真核表达载体pEGFP-C2,以酶切和测序鉴定重组质粒pEGFP-C2-hTH的正确性;pEGFP-C2-hTH经NucleofectorTM核转染仪转染培养的恒河猴BMSCs-D-NSCs,24 h后观察绿色荧光蛋白的瞬时表达情况,10 d后行TH单克隆抗体的免疫组化和TH基因的RT-PCR. 结果（1）酶切、PCR和DNA序列鉴定均证实插入片段的正确性;（2）细胞转染24 h后,荧光显微镜下可观察到绿色荧光蛋白（GFP）的表达,观察到80%的转染细胞发出绿色荧光;转染10 d后细胞的RT-PCR检测到hTH基因的表达,TH单克隆抗体免疫组化结果显示转染细胞呈阳性染色,同时在荧光显微镜下观察到绿色荧光.结论构建的hTH荧光真核表达重组质粒pEGFP-C2-hTH,经电转染方法转染至BMSCs-D-NSCs内,成功表达hTH和EGFP,为BMSCs-D-NSCs基因治疗提供了实验基础.

英文摘要：

Objective To observe the expression of hTH gene in NSCs-BMSCs by way of constructing a recombinant plasmid-pEGFP-C2-hTH which can express hTH （human tyrosine hydroxylase） gene with pEGFP-C2 in eukaryocyte and then transfecting the plasmid to bone marrow stromal cells-derived neural stem cells （BMSCs-D-NSCs）. Mehtods With the technology of gene re-arrangement, hTH gene in pWAV2-TH plasmid was subcloned into pEGFP-C2 vector to obtain pEGFP-C2-hTH plasmid, with its correctness evaluated by the means of restriction enzyme analysis and sequencing, pEGFP-C2-hTH was transfected into NSCs-BMSCsSCs with NucleofectorTM device. After 24 hours, the transient expression of GFP was observed under fluorescence microscope, and the expression of TH gene was measured by RT-PCR and immunohistochemistry of TH monoclonal antibody. Results Correct construction of pEGFP-C2-hTH was identified by methods of restriction enzyme analysis, PCR amplification and nucleotide sequence determination. 80% transfected cells emitted out green fluorescence under fluorescent microscope after 24h after transfection and the transfected cells expressed hTH gene by RT-PCR, displayed TH-positive by immunohistochemistry and emitted out green fluorescence under fluorescent microscope after 10 days after transfection. Conclusion The constructed fluorescent eukaryotic expression plasmid- pEGFP-C2-hTH can successfully express hTH and EGFP in NSCs-BMSCs of Rhesus monkey, which may provide an experimental basis for the application of NSCs-BMSCs to cell transplantation in the treatment of Parkinson＇ s disease.