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目的:探讨小分子干扰RNA(siRNA)对食管癌EC9706细胞组织蛋白酶B(CB)基因表达的影响。方法:设计并用体外转录方法合成针对CB基因的siRNA,转染EC9706细胞(设10μmol/L、15μmol/L、20μmol/L3个转染浓度),分别培养24h、48h、72h(实验组),采用免疫细胞化学及原位杂交方法检测转染细胞中CB蛋白和mRNA表达的变化,并设正常对照、空白对照、无关对照。结果:不同浓度siRNA转染24h、48h、72hEC9706细胞CB蛋白表达量均较正常、空白及无关对照降低(P<0.05),以转染72h的抑制效应最为明显,但3者相比差异无统计学意义(P>0.05)。3个时间段内随浓度增加siRNA抑制效应增强,3者相比差异有统计学意义(P<0.05),正常对照、空白对照、无关对照均未表现出对CB蛋白表达的抑制效应。转染siRNA后部分细胞形态发生改变。结论:特异性siRNA能有效抑制EC9706细胞中CB蛋白和mRNA表达。
Objective: To investigate the effect of small interfering RNA (siRNA) on the expression of cathepsin B (CB) gene in esophageal cancer EC9706 cells. METHODS: siRNA targeting CB gene was synthesized and synthesized by in vitro transcription method and transfected into EC9706 cells (10μmol/L, 15μmol/L, and 20μmol/L transfection concentrations). The cells were cultured for 24h, 48h, and 72h (experimental group). Immunocytochemistry and in situ hybridization were used to detect the changes of CB protein and mRNA expression in transfected cells. Normal control, blank control and unrelated control were used. RESULTS: The expression of CB protein in EC9706 cells at 24 h, 48 h, and 72 h after transfection with different concentrations of siRNA was significantly lower than that of normal, blank, and irrelevant controls (P<0.05). The inhibitory effect was most obvious at 72 h after transfection, but there was no statistical difference between the three groups. Significance (P>0.05). The inhibitory effect of siRNA increased with increasing concentration in three time periods, and there was a statistically significant difference among the three groups (P<0.05). The normal control, blank control, and irrelevant control showed no inhibitory effect on the expression of CB protein. Some of the cell morphology changes after siRNA transfection. Conclusion: Specific siRNA can effectively inhibit the expression of CB protein and mRNA in EC9706 cells.