CyclinD1基因过表达慢病毒载体的构建和对神经干细胞增殖的影响

Construction of CyclinD1 Gene Overexpression Lentivirus Vector and Its Effect on the Proliferation of Neural Stem Cells

目的:针对小鼠cyclinD1基因构建质粒并进行慢病毒包装,转染小鼠神经干细胞,检测其表达水平。方法:根据cyclinD1基因信息,采用DNA重组技术将Nestin promoter-Ccnd1基因插入plenti6慢病毒表达载体,重组获得慢病毒载体plenti-D1;经测序鉴定后,转染293T细胞生产病毒液,并检测病毒滴度。设立空白对照组、阴性病毒对照组及过表达慢病毒感染组。将病毒转染小鼠胚胎神经干细胞,经实时荧光定量PCR和western blot法分析转染前后3组cyclinD1表达情况,MTT法检测不同MOI值对神经干细胞增殖的影响。结果:测序结果证实cyclinD1基因正确插入载体中,成功构建小鼠cyclinD1基因过表达载体。实时荧光定量PCR结果显示过表达慢病毒感染组cyclinD1 mRNA较其他2组明显升高;Western Blot鉴定cyclinD1蛋白表达成功。plenti-D1的MOI值为10、20、50时均明显促进神经干细胞增殖。结论:cyclinD1基因慢病毒表达载体能感染小鼠胚胎神经干细胞,外源基因稳定表达。cyclinD1基因过表达能促进神经干细胞的增殖。

Objective：In order to study the effect of cyclinD1 on neural stem cell proliferation, the cyclinD1 gene overexpression lentiviral vector was constructed and the expression of cyclinD1 in neural stem cells in mice after transfection was tested. Methods： Nestin promoter-Ccnd1 gene was inserted into plenti6 lentiviral expression vectors by DNA recombination technique. Lentiviral vector plenti-D1 was established after recombination. Recombinant lentiviral vector was detected by DNA sequencing. The plenti-D1 was transfected into 293 T cell line. The viruses yielded by 293 T cell were transfected into mouse embryonic neural stem cells. The expression of cyclinD1 was detected by real-time PCR and Western Blot analysis after transfection. We studied the effects of different MOI values of plenti-D1 on neural stem cell proliferation by MTT assay. Results： It was confirmed by DNA sequencing that the cyclinD1 gene sequencing was correctly inserted into the vector, and that the cyclinD1 gene overexpression lentiviral vector was successfully constructed. After extracting the infected cells, the real-time PCR showed cyclinD1 m RNA overexpression was significantly higher than the control groups; Western Blot analysis was used to detect expression of cyclinD1 protein in neural stem cells. The cyclinD1 gene overexpression lentiviral vector was significantly up-regulated in m RNA level or in protein level in neural stem cells. When the MOI was 10, 20, and50, the viruses significantly promoted the proliferation of neural stem cells. Conclusion： The cyclinD1 gene overexpression lentiviral vector was successfully constructed and it efficiently up-regulated the expression of cyclinD1 in mouse embryonic neural stem cells. Cyclin D1 overexpression can promote the proliferation of neural stem cells.