NGF基因重组慢病毒载体的构建及其在人脐带血间充质干细胞的表达

目的:构建神经生长因子(NGF)的慢病毒表达载体,并观察其转染人脐带间充质干细胞后的表达情况。方法:采用实时定量PCR(RT-PCR)方法获取NGF基因编码片段,并将构建的慢病毒载体质粒与包装质粒和包膜质粒共转染293T细胞,包装生产慢病毒。应用相同滴度的慢病毒转导等量间充质干细胞(MSCs),观察转染后细胞的生长形态及生长曲线,再采用RT-PCR、Western Blot方法检测NGF m RNA、蛋白质的表达水平。结果:经PCR、酶切和测序结果证明成功构建NGF基因重组慢病毒载体。同时NGF基因重组慢病毒载体能够成功转染人脐带间充质干细胞,转染率达95.35%,转染后干细胞在NGF m RNA及蛋白质的表达方面较对照组明显升高,同时经倒置显微镜观察及生长曲线实验证实转染后干细胞的生长与对照组相比无明显差异。结论:重组NGF的慢病毒表达载体能够高效的转染人脐带间充质干细胞,基因转染后干细胞的增殖分化能力与未转染细胞差异无统计学意义,可作为一种高效的干细胞转染方法。

Construction of Lentiviral Expression Vector of NGF and Its Expression in Human Umbilical Cord Mesenchymal Stem Cell

ABSTRACT Objective: To investigate the effects of nerve growth factor gene transfection via a recombinant lentiviral virus vector on the proliferation and cell cycle of human umbilical cord mesenchymal stem cells cultured in vitro. Methods: The NGF gene was cloned into lentiviral shuttle plasmid, and identified by the enzyme cut assay, PCR and gene sequencing. It was then transfected into 293T cells to construct the recombinant lentiviral vector plasmid. Cellular proliferation was determined by cell growth curve and Cell Counting Kit-8 assay. The infection efficiency of lentiviral virus transfection was detected by fluorescence microscope and the protein expression of NGF was tested by Western blot. Results: NGF gene recombinant lentiviral vector was successfully transfected into human umbilical cord mesenchymal stem cells, and the transfection rate reached 95.35%. The expression of NGF mRNA and protein in stem cells was sig- nificantly higher than that in the control group after transfection. It was confirmed by inverted microscope observation and growth curve experiment that the growth of stem cells after transfection was not significantly different from that of the control group. Conclusion: These findings suggest that the recombinant lentiviral virus-mediated nerve growth factor gene can transfect the umbilical cord mes- enchymal stem cells. There was no significant difference in the proliferation and differentiation ability of stem cells after transfection with untransfected cells.