表达反义单核细胞趋化蛋白-1的重组逆转录病毒对家兔动脉平滑肌单核细胞趋化蛋白-1基因表达的影响

The Effect of Antisense Monocyte Chemotactic Protein-1 Expression Mediated by Retroviral Vector on Monocyte Chemotactic Protein-1 mRNA Level in Rabbit Aortic Smooth Muscle Cells

为研究反义单核细胞趋化蛋白－１ 转基因表达对单核细胞进入动脉壁的作用，首先构建了表达反义单核细胞趋化蛋白－ １ 基因的逆转录病毒重组体，并观察它在培养的细胞中的表达。将家兔单核细胞趋化蛋白－ １ ｃＤＮＡ 反向插入到ｐＬＮＣＸ，构成ＬＮＣＸ－ａｎｔｉ－ ＭＣＰ－１ 重组病毒质粒。再将重组质粒转染φ－２ 细胞，继以φ－ ２ 细胞产生的病毒上清感染ＰＡ３１７细胞，取得Ｇ４１８ＰＡ３１７ 抗细胞克隆。上述细胞经扩增培养，收集病毒上清并感染ＮＩＨ３Ｔ３ 细胞后进行检测。结果发现，病毒的滴度为５．６×１０７ ＣＦＵＬ，感染的ＮＩＨ３Ｔ３ 细胞中有重组病毒的整合。重组病毒感染培养的家兔动脉平滑肌细胞后，用聚合酶链反应检测发现，感染的平滑肌细胞基因组ＤＮＡ中有重组病毒整合；ＲＮＡｓｌｏｔ 杂交结果显示，感染的平滑肌细胞中有反义单核细胞趋化蛋白－１ 的表达，与未感染的平滑肌细胞相比，感染的平滑肌细胞中单核细胞趋化蛋白－ １ ｍＲＮＡ 的表达明显受到抑制。结果提示，反义单核细胞趋化蛋白－ １ 逆转录病毒表达载体在培养的动脉平滑肌中能表达反义基因并抑制靶基因的表达。

Aim To investigate the inhibitory effect of antisense monocyte chemotactic protein-1 (MCP-1) expression mediated by recombinant gene transfer on the migration of monocytes into arteries in vivo, as a first step, a recombinant retroviral vector expressing antisense MCP-1 gene was constructed and its expression was observed in vitro. Methods An antisense MCP-1 recombinant vector was constructed by inserting rabbit MCP-1 cDNA into LNCX plasmid in a reversed orientation. Recombinant viral DNA was transfected into φ-2 packaging cells by lipofectamine transfecting procedure. The transiently produced viruses were used to transfer PA317 amphotropic packaging cells. G418 resistant positive PA317 clones were isolated and expanded. Virus surpernatant from G418 resistant PA317 cells was used to determine virus titer and to infect aortic smooth muscle cells. After the selection with G418 medium, the integration and expression of recombinant gene in cells were measured. Results The highest retroviruses' titer was 5.6×10 7 CFU/L. PCR result showed that recombinant viruses were integrated into genome of the infected NIH3T3 cells and cultured smooth muscle cells. RNA slot blot analysis showed that antisense MCP-1 mRNA expressing was detected in the infected smooth muscle cells. There was obvious inhibition of MCP-1 mRNA expression obtained in the infected smooth muscle cells in comparing to that the uninfected cells. Conclusions The recombinant retroviral vector containing antisense MCP-1 could inhibit target gene expression in cultured smooth muscle cells and may be used for further experimental studies in vivo.