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目的:探讨RNA干扰人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)的表达对大肠癌细胞SW480凋亡的影响。方法:构建携带hTERT小发夹干扰RNA(small hairpin RNA,shRNA)的重组表达载体pGPU6/GFP/Neo-hTERT-shRNA(简称hTERT-shRNA质粒),脂质体法转染SW480细胞,RT-PCR法检测不同转染时间点SW480细胞中hTERTmRNA的表达。TRAP-PCR-ELISA法检测转染后48h SW480细胞的端粒酶活性,透射电镜观察转染后48h SW480细胞超微结构。结果:hTERT-shRNA质粒转染48h时,hTERT-shRNA组SW480细胞hTERT mRNA表达的抑制率显著高于空白组、脂质体组、NC-shRNA组(75.0%vs39.2%、33.3%、28.0%,P<0.05)。hTERT-shRNA转染组SW480细胞端粒酶活性显著低于空白组、脂质体组、NC-shRNA组(2.242±0.285vs2.756±0.089、2.693±0.225、2.691±0.120,P<0.05)。hTERT-shRNA质粒转染后的SW480细胞体积明显缩小、细胞核固缩、染色质不均匀地沿核膜下聚集、空泡形成增多,出现典型的凋亡形态。结论:RNAi可有效沉默SW480细胞中hTERT的表达,降低SW480细胞端粒酶活性,诱导SW480细胞凋亡。
Objective: To investigate the effect of hTERT RNA interference on apoptosis of colorectal cancer cell line SW480. Methods: Recombinant vector pGPU6 / GFP / Neo-hTERT-shRNA (hTERT-shRNA plasmid) carrying hTERT small hairpin RNA (shRNA) was constructed and transfected into SW480 cells by lipofectamine. RT- Method was used to detect the expression of hTERT mRNA in SW480 cells at different time points. The telomerase activity of SW480 cells was detected by TRAP-PCR-ELISA 48 h after transfection. The ultrastructure of SW480 cells was observed by transmission electron microscopy 48 h after transfection. Results: The hTERT mRNA expression in hTERT-shRNA group was significantly higher than that in blank group, liposome group and NC-shRNA group (75.0% vs 39.2%, 33.3%, 28.0 %, P <0.05). The telomerase activity of SW480 cells transfected with hTERT-shRNA was significantly lower than that of the blank group, the liposome group and the NC-shRNA group (2.242 ± 0.285 vs. 2.756 ± 0.089, 2.693 ± 0.225, 2.691 ± 0.120, P <0.05). After transfected with hTERT-shRNA plasmid, the volume of SW480 cells was significantly reduced, the nuclei were pyknotic, the chromatin was unevenly distributed along the nucleus, the formation of vacuoles increased, and the typical apoptotic morphology appeared. Conclusion: RNAi can effectively silence the expression of hTERT in SW480 cells, decrease the telomerase activity in SW480 cells and induce the apoptosis of SW480 cells.