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Construction and expression of recombinant plasmid-pEGFP-C2-hTH in the neural stem cells derived from bone marrow stromal cells
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF NEUROMEDICINE
- Issue:
- 5
- DOI:
- 10.3760/cma.j.issn.1671-8925.2006.05.003
- Key Word:
- 酪氨酸羟化酶;绿色荧光蛋白;神经干细胞;基因转染;恒河猴
Abstract： 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.
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