sox9和LMP1基因慢病毒载体共转染兔骨髓间充质干细胞后的表达

Cotransfection of sox9 and LMP1 genes in rabbit bone mesenchymal stem cells through lentivirus vectors in vitro

背景:与传统细胞因子诱导液诱导的方法相比,将基因通过慢病毒转染入宿主细胞,可以得到长期高效的持续表达,采取双/多基因共修饰治疗的策略,较采用单一的因子来诱导骨髓间充质干细胞向类髓核细胞方向转化可获得更好的效果。目的:构建sox9基因慢病毒载体以及LMP1基因慢病毒载体,并共转染兔骨髓间充质干细胞,观察SOX9和LMP1基因的表达情况。方法:双酶切从Gene Art提供的含sox9基因序列的质粒13ABIV6C_1366933并克隆到p Lenti6.3_MCS_IRES2-EGFP载体,获得含sox9基因的质粒p LMIG-13GS0345-1。通过单链oligo设计及合成LMP1基因序列,并克隆到p Lenti6.3_MCS_IRES2-Ds Red-Monomer载体,获得含LMP1基因的质粒13GS0318-1。再通过293T细胞包装后获得绿色荧光蛋白标记的sox9基因慢病毒载体(Lenti-SOX9-GFP),以及红色荧光蛋白的LMP1基因慢病毒载体(Lenti-LMP1-RFP)。将Lenti-SOX9-GFP和Lenti-LMP1-RFP共转染兔骨髓间充质干细胞,观察sox9基因及LMP1基因的表达情况。结果与结论:测序结果显示质粒p LMIG-13GS0345-1和13GS0318-1中的插入序列与基因库中sox9(NM_000346)及LMP1(AF345904.1)基因序列完全一致,成功构建Lenti-SOX9-GFP和Lenti-LMP1-RFP慢病毒载体。Lenti-SOX9-GFP和Lenti-LMP1-RFP共转染兔骨髓间充质干细胞后同一视野下可观察到绿色和红色荧光蛋白的共表达,RT-PCR与Western Blot检测结果证明Lenti-SOX9-GFP和Lenti-LMP1-RFP可成功高效共转染目的细胞并可在m RNA及蛋白水平同时表达SOX9及LMP1。结果说明实验成功构建了绿色荧光蛋白标记的携带sox9基因的Lenti-SOX9-GFP慢病毒载体,以及红色荧光蛋白的携带LMP1基因的Lenti-LMP1-RFP慢病毒载体,为sox9和LMP1基因共修饰组织工程髓核的研究提供前期实验基础。

BACKGROUND： Compared with the traditional method of cytokines, gene transfection into a host cell by lentivirus can achieve a sustained long-term efficient expression. Double/multiple gene co-modification therapy is superior to single gene therapy to induce the differentiation of bone marrow mesenchymal stem cells into nucleus pulposus-like cells. OBJECTIVE： To build two recombinant lentivirus vectors separately expressing sox9 and LMP1 genes, and then co-transfect rabbit bone marrow mesenchymal stem cells for observing the expression of sox9 and LMP1 genes.METHODS： Double enzyme digestion was performed for plasmid 13abiv6c_1366933 containing sex9 gene sequences provided by GeneArt, and the sox9 gene was cloned into pLenti6.3_MCS IRES2-EGFP carrier to harvest the plasmid pLMIG-13GS0345-1 including sox9 gene. Through single oligo design and synthesis of LMP1 gene sequences that were cloned into pLenti6.3_MCS_IRES2 DsRed-Monomer carrier, the plasmid gs0318 13-1 including LMP1 gene was obtained. The plasmids were packed by 293T ceils to obtain the recombinant lentivirus vector carrying sox9 gene marked by green fluorescent protein markers （Lenti-SOX9-GFP） and the recombinant lentivirus vector carrying LMP1 gene marked by red fluorescent protein markers （Lenti-LMP1-RFP）. The Lenti-soxg-GFP and Lenti-LMP1-RFP were co-transfected into rabbit bone marrow mesenchymal stem cells to observe the expression of sox9 and LMP1 genes. RESULTS AND CONCLUSION： Sequencing results showed that the insert sequences of plasmids pLMIG-13GS0345-1 and 13GS0318-1 were completely consistent with the sox9 gene （NM_000346） and LMP1 gene （AF345904.1） sequences in the Genbank respectively, indicating that the Lenti-SOX9-GFP and Lenti-LMP1-RFP were successfully constructed. The expression of green fluorescent protein and red fluorescent protein could be observed in the same view after co-transfection of rabbit bone marrow mesenchymal stem cells by Lenti-SOX9-GFP and Lenti-LMP1-RFP. The RT-PCR and western blot assay results also proved that the Lenti-SOX9-GFP and Lenti-LMP1-RFP could successfully end efficiently co-transfect the bone marrow mesenchymal stem cells, and mRNA and protein expressions of SOX9 and LMP1 gene could be successfully observed. The recombinant lentivirus vectors Lenti-SOX9-GFP and Lenti-LMP1-RFP were successfully constructed and successfully co-expressed in the rabbit bone marrow mesenchymal stem cells, which provided preliminary experimental basis for further research in nucleus pulposus tissue engineering.