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用核酶技术阻断或降低抗凋亡蛋白 Bcl- 2的表达以促进化疗药物紫杉醇诱导的食管癌细胞凋亡 ,探索克服耐药、提高紫杉醇疗效的新途径 .将特异性切割 Bcl- 2 m RNA的核酶克隆至含MTII启动子并可为 Zn SO4 诱导表达的真核表达载体中 ,通过脂质体转入食管癌鳞状上皮细胞系Eca 1 0 9中 ,经 G41 8筛选得到稳定抗性细胞株 X1 0 9R,挑取单细胞株扩大培养 ,1 40μmol/L Zn SO4诱导 3d,用 Northern- blot、免疫荧光、流式细胞仪鉴定核酶及 Bcl- 2蛋白表达情况 ,用 TUNEL标记及流式细胞术检测凋亡细胞的比例 .bcl- 2核酶在不同单细胞株中有不同程度的表达 ,其中一株X1 0 9R1 4表达最高 .测定其中 Bcl- 2蛋白含量 ,发现 Bcl- 2蛋白表达大为降低 .加入紫杉醇后 ,TUNEL标记及凋亡峰测定结果都表明同一条件下凋亡率升高 .结果提示 ,转入特异性切割 bcl- 2m RNA的核酶可有效地阻断 Bcl- 2蛋白合成 .Bcl- 2蛋白表达降低可明显促进紫杉醇诱导的细胞凋亡 .说明 Bcl- 2蛋白在细胞产生耐药过程中起着重要作用
Using ribozyme technology to block or reduce the expression of anti-apoptotic protein Bcl-2 to promote paclitaxel-induced apoptosis of esophageal cancer cells, to explore new ways to overcome drug resistance and improve the efficacy of paclitaxel. To specifically cut Bcl-2 m The RNA ribozyme was cloned into eukaryotic expression vector containing the MTII promoter and induced by ZnSO4 and transfected into esophageal squamous epithelial cell line Eca109 via liposome and screened with G41 8 for stable resistance. Strain X1 0 9R, a single cell strain, was picked up and cultured. After induction with 1 40 μmol/L ZnSO4 for 3 days, the expression of ribozyme and Bcl-2 protein was detected by Northern-blot, immunofluorescence and flow cytometry, and labeled with TUNEL. The percentage of apoptotic cells was detected by flow cytometry. bcl-2 ribozymes were expressed in different degrees in different cell lines. Among them, one strain had the highest expression of X1 0 9R1 4 and the Bcl-2 protein content was detected. Bcl- 2 The protein expression was greatly reduced. After adding paclitaxel, the TUNEL labeling and apoptotic peak assay results showed that the apoptotic rate increased under the same conditions. The results suggest that the transfer of ribozymes that specifically cleave bcl-2m RNA can effectively block Bcl-2 protein synthesis. Bcl-2 eggs The decrease of white expression can significantly promote paclitaxel-induced apoptosis, indicating that Bcl-2 plays an important role in cell resistance.