SOX9基因慢病毒载体的构建及其在骨髓间充质干细胞中的表达

Construction of ientiviral vector containing SOX9 gene and its expression in bone marrow mesenchymal stem cells

目的 构建SOX9基因的慢病毒载体并使其在兔骨髓间充质干细胞（MSCs）中表达.方法 通过RT-PCR获得人SOX9基因编码区（CDS）片段,将该片段克隆入质粒Pwpxl-MOD2中产生Pwpxl-MOD2/SOX9,将Pwpxl-MOD2/SOX9、pRsv-REV、pMDlg-pRRE、pMD2G共转染293T细胞,获得携带目的基因SOX9基因的重组病毒.同时共转染Pwpxl-MOD2,pRsv-REV,pMDlg-pRRE,pMD2G进另一组293T细胞包装产生空载体慢病毒作为阴性对照.包装好的慢病毒体外感染MSCs,逆转录-聚合酶链反应（RT-PCR）法及免疫印迹检测SOX9的表达;四甲基噻唑蓝（MTT）法测定慢病毒对细胞增殖能力的影响.结果 酶切、PCR及测序鉴定证实慢病毒载体质粒Pwpxl-MOD中插入片段为基因SOX9.病毒感染MSCs后,经RT-PCR法和免疫印迹证实SOX9基因在MSCs中表达.MTT法检测示慢病毒感染对骨髓间克质干细胞的生长未产生显著影响.结论 成功构建SOX9基因的慢病毒载体并感染骨髓间充质干细胞后能够稳定表达SOX9.为研究椎间盘退变的基因及生物学治疗提供了实验依据.

Objective To construct the lentiviral vector containing SOX9 gene and to detect its expression in MSCs derived from rabbit bone marrow. Methods Human sox-9 gene coding region fragment was obtained by RT-PCR （reverse transcription-polymerase chain reaction） and then cloned into the plasmid of Pwpxl-MOD2 to form Pwpxl-MOD2/SOX9. Pwpxl-MOD2/SOX9, pRsv-REV, pMDlg-Prre and pMD2G were co-transfected into 293T cells to obtain recombinant virus containing SOX9 gene. Meanwhile, Pwpxl-MOD2, pRsv-REV, pMDlg-pRRE and pMD2G were transfected into another group of 293T cells as a control group packing into blank lentiviral vector. Then the packed lentiviral vector was transfected into MSCs which derived from rabbit bone marrow. The expression of SOX9 was detected by both RT-PCR and Western blot. Identification and proliferation of MSCs was determined by MTT after transfection. Results The sequencing and restriction analysis showed that SOX9 gene fragment was correctly connected and cloned into the plasmid Pwpxl-MOD in lentiviral vectors. After transfection, the expression of SOX9 gene in MSCs was confirmed by RT-PCR and Western blot. MTT showed the growth of MSCs had no significant effect after transfection with lentiviral vector. Conclusion Lentiviral vector carrying SOX9 gene has been successfully constructed. There is a stable expression in transfected MSCs. Thus it will facilitate the exploratory development of gene and biological therapy for intervertebral disc degeneration.