慢病毒重组同源盒基因HOXA4表达载体的构建及其感染人脐带间充质干细胞的实验性研究

Construction of Recombinant Lentivirus Containing Human HoxA4 Gene and Their Transfection Efficiency in Human Umbilicalcord Mesenchymal Stem Cells

目的 构建携带同源基因HOXA4的慢病毒表达载体,并测定其对人脐带间充质干细胞的感染效率.方法 使用酶切及PCR技术从含有HOXA4基因的质粒克隆模版HOXA4-MSCV逆转录载体中获取目的 基因HOXA4,并将HOXA4基因重组到慢病毒载体表达质粒上Lenti-GFP-CTB,通过酶切、测序验证HOXA4基因后,将Lenti-GFP-HOXA4质粒、和辅助包装质粒pRsv-REV、pMDlg-pRRE、PMD2G共同转染人胚胎肾上皮细胞系293T细胞,获得携带HOXA4基因的重组慢病毒Lentiviral-HOXA4;然后感染人脐带间充质干细胞,通过荧光显微镜及流式细胞术检测其感染效率.结果 成功构建携带HOXA4基因的慢病毒表达载体Lentiviral-HOXA4,并获得高纯度的慢病毒浓缩液.经检测病毒滴度达2.11×108 TU/ml.成功转染HOXA4基因的脐带间充质干细胞表达绿色荧光蛋白,当病毒感染复数（MOI）值为60时转染效率最高,达（95.4±4.3）%.结论 成功构建携带人HOXA4基因的慢病毒,并可以在体外有效转染人脐带间充质干细胞.

Objective To construct recombinant lentivirus vector containing human HOXA4 gene and test its transfection in human umbilicalcord mesenehymal stem cells （hUCMSCs） . Methods HOXA4 gene was amplified from plasmid HOXA4 -MSCV by PCR technique and subcloned into the expression plasmid of lentiviral-GFP-CTB vector. The lentiviral-GFP-HOXA4 expression vector was constructed and confirmed by endoenzyme digestion and sequencing. Recombinant Lentiviral-GFP-HOXA4 was produced by 293T cells following the co-transfeetion of lentiviral-GFP- HOXA4 and packaging plasmids pRsv-REV pMDlg-pRRE and PMD2G. The recombinant lentiviral- GFP-HOXA4 was then used to infect hUCMSCs. Results The recombinant lentiviral-GFP-HOXA4 was produced successfully. High-purified virus supernatant was obtained, with virus titer of 2. 11 x lOs TU/ml and efficient infection in hUCMSCs expressing green fluorescence （95.37%） 60 h after transfection. Conclusion The lentiviruse vector with human HOXA4 cDNA was established suc- cessfully, with effective transfeetion in hUCMSCs.