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

doi:10.3969/j.issn.2095-4344.2014.50.004

Abstract

Abstract:

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 sox9 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 cells 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-SOX9-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 and 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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, mesenchymal stem cells, lentivirus infections, SOX9 transcription factor

CLC Number:

R394.2

Liu Wei, Wang Jie, Xing Yong-ming, Li Chun-zhi, Chen Chang-wei, Zhao Hong, Gong De-jun. Cotransfection of sox9 and LMP1 genes in rabbit bone mesenchymal stem cells through lentivirus vectors in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(50): 8054-8060.

Figures/Tables 3

2.1 慢病毒载体构建结果测序结果显示含sox9基因的质粒pLMIG-13GS0345-1中的插入序列与SRY-related high mobility group-box gene 9，sox9(NM_000346，共 1 550 bp)完全一致，含LMP1基因的质粒13GS0318-1中的插入序列与Homo sapiens LIM mineralization protein 1 (AF345904.1，共1 374 bp)完全一致(测序图略)，未见碱基突变。序列插入位点分别位于pLenti6.3_MCS_ IRES2-EGFP载体的BamHI/AscI和pLenti6.3_MCS_ IRES2-DsRed-Monomer载体的BamHI/AscI。将构建的Lenti-SOX9-GFP和Lenti-LMP1- RFP病毒原液转染293T细胞24 h后，在荧光显微镜下可见绿色或红色荧光蛋白的表达(图1，2)。Lenti-SOX9-GFP和Lenti-LMP1-RFP慢病毒滴度测定结果分别为2.5×107 TU/mL 和2.05×108 TU/mL。 2.2 兔骨髓间充质干细胞的体外培养结果刚提取的骨髓间充质干细胞为圆形，悬浮于培养液中，可见有圆盘状血细胞混杂其中。培养7 d左右骨髓间充质干细胞已贴壁，培养14 d后骨髓间充质干细胞呈多种形态，如梭形，胞体膨大，有长短不一的胞质突起，并呈集落生长(图3A)。已传第3代的骨髓间充质干细胞，细胞纯度较高，生长速度较快，细胞密度较高，已达95%融合(图3B)。 2.3 Lenti-SOX9-GFP和Lenti-LMP1-RFP慢病毒载体转染骨髓间充质干细胞的MOI确定慢病毒转染后骨髓间充质干细胞在荧光显微镜下细胞呈多角形，胞体增大、胞质伸展相互交错，胞质内充满绿(或红)色荧光物质，DAPI标记的细胞核清晰可见，不同MOI值(50，100，150，200)条件下的转染率见表2，测定结果显示Lenti-SOX9和Lenti-LMP1的最佳MOI分别为100和150，其转染率均约为85%。 2.4 MTT法检测转染对骨髓间充质干细胞增殖的影响经MTT细胞增殖试剂盒检测Lenti-SOX9、Lenti-LMP1或空白慢病毒Lenti-NC转染(MOI 200)后5 d内骨髓间充质干细胞的生长情况，实验结果显示高转染复数的Lenti-SOX9- GFP及Lenti-LMP1-RFP慢病毒载体分别转染骨髓间充质干细胞后的细胞生长曲线与未处理的细胞基本一致，统计分析表明，各种处理细胞的生长特性差异无显著性意义(图4，P > 0.05)。表明慢病毒转染对骨髓间充质干细胞的生长增殖没有明显的影响。 2.5 Lenti-SOX9-GFP和Lenti-LMP1-RFP慢病毒载体对兔骨髓间充质干细胞的同时转染结果将Lenti-SOX9- GFP(MOI 100)和Lenti-LMP1-RFP(MOI 150)慢病毒载体同时转染第3代骨髓间充质干细胞，继续培养72 h后通过荧光显微镜观察转染荧光情况。结果见图5所示，共转染的骨髓间充质干细胞同一视野中不同激发光下既有红色荧光，也有绿色荧光蛋白表达，说明Lenti-SOX9和Lenti-LMP1慢病毒载体可以同时成功转染骨髓间充质干细胞。 2.6 Lenti-SOX9-GFP和Lenti-LMP1-RFP慢病毒转染兔骨髓间充质干细胞后sox9与LMP1 mRNA的表达情况骨髓间充质干细胞被重组慢病毒Lenti-SOX9-GFP和Lenti- LMP1-RFP共转染后48 h，抽提细胞总RNA进行RT-PCR分析。结果显示共转染组出现位置大小为320 bp及1 400 bp的条带，这与sox9(304 bp)及LMP1(1 383 bp)预期结果相符合，而空白慢病毒Lenti-NC转染组没有发现sox9或LMP1的基因表达。 sox9 mRNA表达：见图6，Lenti-SOX9与共转染处理组中sox9 mRNA(304 bp)表达稍多，条带较强，且组间比较差异无显著性意义(P > 0.05)，Lenti-LMP1组有微弱条带，与Lenti-SOX9及共转染组相比，组间比较差异有显著性意义(P < 0.05)，见表3。LMP1 mRNA表达：见图7，Lenti-LMP1与共转染处理组中LMP1 mRNA(1 385 bp)表达稍多，条带较强，且组间差异无显著性意义(P > 0.05)，Lenti-NC及Lenti-SOX9-GFP转染组未见LMP1 mRNA表达，见表3。 2.7 Lenti-SOX9-GFP和Lenti-LMP1-RFP慢病毒共转染骨髓间充质干细胞后SOX9与LMP1蛋白的表达情况：经Western Blot检测各分组处理后骨髓间充质干细胞中目的蛋白表达，可以观察到Mr 42 000处有β-Actin参比条带，而经Lenti-LMP1或Lenti-SOX9慢病毒分别转染骨髓间充质干细胞72 h后，分别在有SOX9和LMP1特征条带表达，而Lenti-NC转染组未见表达。"

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