背景与目的:通过siRNA干扰沉默ERCC2基因,观察食管癌细胞株对紫杉醇敏感性的改变,初步探讨新的紫杉醇耐药可能机制.方法:体外合成靶向ERCC2的SiRNA(si-ERCC2);应用脂质体法瞬时转染ERCC2高表达细胞株KYSE150;应用RT-PCR和流式细胞术(FCM)分别检测转染组细胞点ERCC2 ndlNA和蛋白的表达水平:应用MTT法检测转染前后细胞对紫杉醇敏感性的变化.结果:si-ERCC2组在转染后24、48、72 h均未测出ERCC2特异性条带且ERCC2蛋白表达量分别下调了31.2%,51.6%和60.0%.si-ERCC2组紫杉醇IC_(50)值为6.3±0.9 μg/mL,较对照组降低49%.结论:siRNA成功封闭了目的基因ERCC2在转录和翻译水平的表达,封闭ERCC2表达能部分逆转紫杉醇耐药现象.%Background and purpose: ERCC2 gene silence by siRNA interference was observed in esophageal cancer cell line and it could change cell sensitivity to taxol. This study was to investigate the biological mechanism of paclitaxel-resistance in esophageal cancer cells. Methods: ERCC2 targeting siRNA (si-ERCC2) has been synthesized. The constructor were transfected into ERCC2 cell lines high-KYSE150(bigh expression of ERCC2) through lipofectamine. RT-PCR and flow cytometry (FCM) were used to detect the ERCC2 mRNA and protein expression levels. MTr assay was used to estimate the paclitaxel sensitivity of the cells. Results: In si-ERCC2 group, ERCC2 could not be detected and ERCC2 protein expression were reduced by 31.2%, 51.6% and 60.0%, respectively, 24,48,72 b after transfection. Paclitaxel IC_(50) value for si-ERCC2 group was 6.32±0.87 μg/mL, lower than the control group(49%). Conclusion: siRNA could successfully silence the target gene ERCC2 at the level of transcription and translation of the gene, the reduction of ERCC2 expression may reverse taxol resistance of the cells.
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