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目的构建BAG-1、Bcl-2双靶区反义RNA重组载体并初步探讨其对SGC-7901细胞增殖活性的影响。方法从SGC-7901细胞总RNA中逆转录扩增包括全部编码序列的BAG-1和Bcl-2 cDNA,利用生物工程技术,反向插入真核细胞双表达载体pVITRO2的多克隆位点中,转染SGC-7901细胞,MTT法检测对细胞增殖的影响,RT-PCR法观察对细胞BAG-1和Bcl-2 mRNA表达水平的变化,流式细胞术检测对细胞周期的影响。结果限制性内切酶和测序分析表明,双表达质粒pVITRO2-AsBAG-1-Bcl-2构建成功。MTT法检测显示pVITRO2-AsBAG-1-Bcl-2载体抑制SGC-7901细胞增殖,并呈时间依赖性,其中以72 h转染组抑制作用最显著(P<0.01);与对照组比较,pVITRO2-AsBAG-1-Bcl-2组BAG-1和Bcl-2mRNA表达水平下降(P<0.05),细胞凋亡比例升高(P<0.01)。结论成功构建反义双靶区重组载体pVITRO2-AsBAG-1-Bcl-2,并发现其能抑制SGC-7901细胞增殖并引起细胞凋亡。
Objective To construct antisense RNA recombinant vector of BAG-1 and Bcl-2 double targets and to investigate the effect on the proliferation of SGC-7901 cells. Methods The BAG-1 and Bcl-2 cDNAs were amplified by RT-PCR from total RNA of SGC-7901 cells. The recombinant plasmids were inserted into the multiple cloning site of eukaryotic cell double expression vector pVITRO2 by bioengineering, The cell proliferation was detected by MTT assay. The expression of BAG-1 and Bcl-2 mRNA was detected by RT-PCR and the cell cycle was analyzed by flow cytometry. Results Restriction endonuclease and sequencing analysis showed that the double expression plasmid pVITRO2-AsBAG-1-Bcl-2 was successfully constructed. The results of MTT assay showed that pVITRO2-AsBAG-1-Bcl-2 could inhibit the proliferation of SGC-7901 cells in a time-dependent manner, of which the inhibition was most significant at 72 h (P <0.01). Compared with the control group, pVITRO2 -AsBAG-1-Bcl-2 decreased the expression of BAG-1 and Bcl-2 mRNA (P <0.05), and the percentage of apoptosis increased (P <0.01). Conclusion The recombinant antisense vector pVITRO2-AsBAG-1-Bcl-2 was successfully constructed and found to inhibit the proliferation and induce the apoptosis of SGC-7901 cells.