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目的探讨RNA干扰下调survivin基因表达后腺样囊性癌细胞生物学行为变化及其与caspase-3基因表达的相关性。方法设计、合成小干扰RNA(small interfering RNA,siRNA),构建表达载体pGenesil-shRNA-survivin,将重组质粒导入人腺样囊性癌ACC-2细胞株,G418筛选阳性克隆。采用逆转录-聚合酶链反应(RT-PCR)、Western blot分别检测转染后的ACC-2细胞中survivin、caspase-3 mRNA及蛋白表达的变化,用噻唑蓝(MTT)法检测细胞的增殖,原位细胞凋亡(TUNEL)法及透射电子显微镜研究细胞凋亡。结果测序证实表达载体构建成功,在pGenesil-shRNA-survivin的ACC-2细胞株中,survivin mRNA及蛋白表达明显降低(P<0.05),caspase-3 mRNA及蛋白表达明显增加(P<0.05),survivin与caspase-3的mRNA和蛋白表达呈显著负相关(r=-0.333,P<0.05)。ACC-2细胞增殖受到抑制,透射电镜及TUNEL法可见细胞出现凋亡。结论成功构建了survivin基因的RNAi真核表达载体,pGenesil-shRNA-survivin重组质粒能有效抑制腺样囊性癌细胞生长。
Objective To investigate the biological behavior of adenoid cystic carcinoma cells after RNAi downregulation of survivin gene expression and its relationship with caspase-3 gene expression. Methods Small interfering RNA (siRNA) was designed and synthesized. The expression vector pGenesil-shRNA-survivin was constructed. The recombinant plasmids were transfected into human adenoid cystic carcinoma cell line ACC-2. The positive clones were screened by G418. The expression of survivin and caspase-3 mRNA and protein in ACC-2 cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. The proliferation of ACC-2 cells was detected by MTT assay , Apoptosis in situ (TUNEL) and transmission electron microscopy. Results The expression vector of pGenesil-shRNA-survivin was successfully constructed. The expression of survivin mRNA and protein in pGenesil-shRNA-survivin cells was significantly decreased (P <0.05) and the expression of caspase-3 mRNA and protein was significantly increased (P < There was a significant negative correlation between survivin and caspase-3 mRNA and protein (r = -0.333, P <0.05). ACC-2 cell proliferation was inhibited, transmission electron microscopy and TUNEL method cells showed apoptosis. Conclusion The RNAi eukaryotic expression vector of survivin gene was successfully constructed. The pGenesil-shRNA-survivin recombinant plasmid can effectively inhibit the growth of adenoid cystic carcinoma cells.