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目的构建PLCε基因的shRNA表达载体及其功能鉴定。方法设计并合成针对PLCε基因mRNA的寡核苷酸序列,插入绿色荧光蛋白质粒真核表达载体pGenesil中构建重组载体pGenesil-PLCε。重组载体经酶切、测序鉴定后转染膀胱癌细胞,RT-PCR及Western blot检测对膀胱癌细胞株PLCε的抑制作用,流式细胞术(FCM)及MTT检测其对膀胱癌细胞株增殖活力影响。结果成功构建了针对2个PLCε基因的shRNA表达载体。RT-PCR、Western blot检测结果显示:2个重组质粒载体转染T24细胞株后对PLCεmRNA及对PLCε蛋白表达均有明显抑制,流式细胞术结果表明细胞阻滞于G0/G1期,MTT检测表明重组的载体质粒明显抑制T24细胞增殖活力。结论针对PLCε基因的shRNA能有效抑制膀胱癌T24细胞中PLCε基因的表达及膀胱癌细胞增殖。
Objective To construct the shRNA expression vector of PLCε gene and its function identification. Methods The oligonucleotide sequence targeting PLCε gene was designed and synthesized. The recombinant plasmid pGenesil-PLCε was constructed by inserting the green fluorescent protein plasmid pGenesil into pGenesil. The recombinant vector was transfected into bladder cancer cells by enzyme digestion and sequencing. The inhibitory effect of PLCε on bladder cancer cell line PLCε was detected by RT-PCR and Western blot. The proliferation and proliferation of bladder cancer cell lines were detected by flow cytometry (FCM) and MTT assay influences. Results The shRNA expression vector targeting two PLCε genes was successfully constructed. The results of RT-PCR and Western blot showed that the expression of PLCεmRNA and PLCε protein in T24 cells transfected by two recombinant plasmids were significantly inhibited. The results of flow cytometry showed that cells were arrested in G0 / G1 phase and MTT assay The results showed that the recombinant vector plasmid significantly inhibited T24 cell proliferation activity. Conclusion shRNA targeting PLCε gene can effectively inhibit the expression of PLCε gene in bladder cancer T24 cells and the proliferation of bladder cancer cells.