携带siANGPTL4重组腺病毒的构建及其对MG63增殖的抑制作用

Construction of recombinant adenovirus with siANGPTL4 gene and its inhibitive effect on MG63 cell proliferation

目的应用AdEasy腺病毒改良载体系统构建携带siANGPTL4基因的重组腺病毒,进一步研究ANGPTL4基因对骨肉瘤细胞株MG63增殖的影响。方法将设计的siRNA靶点寡聚核苷酸克隆到pSES-HUS载体构建重组质粒pSES-siANGPTL4,在B J5183大肠杆菌内与pAdEasy1骨架质粒完成同源重组;经脂质体介导转入HEK293细胞包装、扩增并经反复感染获得携带siANGPTL4基因的重组腺病毒Ad-siANGPTL4;有限稀释法检测病毒滴度;根据加入的重组腺病毒携带的基因不同将实验细胞分为阴性对照组、RFP组(加入Ad-RFP)、ANGPTL4组(加入Ad-ANGPTL4)及siANGPTL4组(加入Ad-siANGPTL4),利用腺病毒感染人骨肉瘤细胞株MG63,RT-PCR法检测细胞内ANGPTL4基因相对表达强度。结晶紫染色及细胞计数观察ANGPTL4基因对MG63细胞增殖的影响。结果成功构建携带siANGPTL4基因的重组腺病毒Ad-siANGPTL4,病毒滴度达1.2×1011～2.6×1011efu/m l;阴性对照组、RFP组、ANGPTL4组及siANGPTL4组细胞ANGPTL4基因表达的相对强度分别为(104.87±5.21)、(110.95±4.15)、(145.24±7.26)、(72.81±3.61);siANGPTL4组相对表达强度显著降低(P<0.05)。结晶紫染色及细胞计数显示,ANGPTL4基因过度表达可促进MG63细胞增殖;该基因抑制后MG63细胞增殖明显减缓(P<0.05)。结论成功构建了Ad-siANGPTL4重组腺病毒并感染MG63使其内源性表达的ANGPTL4基因沉默;该基因的表达沉默抑制MG63细胞体外增殖。

Objective To construct recombinant adenovirus with si-angiopoietin-like 4（siANGPTL4） gene using modified AdEasy system,and to investigate the effect of ANGPTL4 gene on the proliferation of osteosarcoma cell line MG63.Methods The designed siRNA oligonucleotide fragments of ANGPTL4 gene were cloned into shuttle plasmid pSES-HUS to construct recombinant plasmid pSES-siANGPTL4,and homologous recombination was completed between pSES-siANGPTL4 and backbone plasmid pAdEasy1 in E.coli BJ5183 to construct recombinant adenoviral plasmid pAdEasy-siANGPTL4.Recombinant adenovirus Ad-siANGPTL4 was then packaged and amplified in HEK293 cells after liposome-mediated transfection.MG63 cells were divided into four groups,i.e.,negative control group,RFP group（infected with Ad-RFP）,ANGPTL4 group（infected with Ad-ANGPTL4）,and siANGPTL4 group（infected with Ad-siANGPTL4）.The viral titers were measured by limiting dilution assay,and the relative expression degrees of ANGPTL4 gene in MG63 cells were tested by RT-PCR.The effect of ANGPTL4 gene on MG63 cell proliferation was observed by Crystal Violet staining and cell counting.Results The recombinant adenovirus with siANGPTL4 gene was constructed,and the viral titers were（1.2-2.6）×1011 efu/ml.The relative expression of ANGPTL4 gene in MG63 cells of the negative control group,RFP group,ANGPTL4 group and siANGPTL4 group were 104.87±5.21,110.95±4.15,145.24±7.26 and 72.81±3.61,respectively.In the siANGPTL4 group,the relative expression degree of ANGPTL4 gene was lowered significantly（P0.05）.It was proved by Crystal Violet staining and cell counting results that MG63 cell proliferation was improved by ANGPTL4 gene overexpression and decreased significantly by gene silencing（P0.05）.Conclusion Recombinant adenovirus Ad-siANGPTL4 carrying siANGPTL4 gene with high titer can be constructed by modified AdEasy system,which can silence ANGPTL4 gene and inhibit proliferation of MG63 cells in vitro.