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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 a recombinant adenovirus carrying siANGPTL4 gene by using AdEasy adenovirus modified vector system and further investigate the effect of ANGPTL4 gene on the proliferation of osteosarcoma cell line MG63. Methods The designed siRNA target oligonucleotide was cloned into the pSES-HUS vector to construct the recombinant plasmid pSES-siANGPTL4, homologous recombination was performed with the pAdEasy1 backbone plasmid in E. coli BJ5183, and then transfected into HEK293 cells by liposome The recombinant adenovirus Ad-siANGPTL4 carrying siANGPTL4 gene was amplified by repeated infection. The virus titer was determined by limiting dilution method. The experimental cells were divided into negative control group and RFP group according to the gene of recombinant adenovirus. The expression of ANGPTL4 gene was detected by RT-PCR. The expression of ANGPTL4 was detected by RT-PCR. The expression of ANGPTL4 in Ad-ANGPTL4 cells was detected by flow cytometry. Crystal Violet Staining and Cell Counting Observed the Effect of ANGPTL4 Gene on MG63 Cell Proliferation. Results The recombinant adenovirus Ad-siANGPTL4 carrying siANGPTL4 gene was successfully constructed and the virus titer reached 1.2 × 1011 ~ 2.6 × 1011efu / ml. The relative intensity of ANGPTL4 gene expression in 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. The relative expression intensity of siANGPTL4 group was significantly decreased (P <0.05). Crystal violet staining and cell counting showed that overexpression of ANGPTL4 could promote the proliferation of MG63 cells. The proliferation of MG63 cells was inhibited by this gene significantly (P <0.05). Conclusion The recombinant adenovirus carrying Ad-siANGPTL4 was successfully constructed and MG63 was infected with MG63 to silence the endogenous expression of ANGPTL4 gene. The silencing of this gene inhibited the proliferation of MG63 cells in vitro.