【摘要】 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更多还原

【Abstract】 Expression of Bcl 2 was decreased by bcl 2 mRNA targeting ribozyme to investigate whether the decrease could accelerate apoptosis induced by paclitaxel in human esophageal carcinoma cells and overcome the resistance to paclitaxel.The DNA of hammerhead ribozyme specially targeted to bcl 2 mRNA was cloned into expression vector downstream MTII promoter which could be induced by ZnSO 4.The human esophageal carcinoma cell line Eca 109 was transfected with the recombinant vector by LipofectAMINE and screened by G418.Separate clonal transfectants were induced by 140 μmol/L ZnSO 4 for three days.The expression of the ribozyme and Bcl 2 protein were identified by Northern blot,indirect immunofluorescent and flow cytometry.Apoptotic rate was determined by TUNEL assay and cell cycle analysis.Results showed that in X109R14 cells,which had a highest level expression of ribozyme,the amount of Bcl 2 protein was obviously decreased.Under the same condition of treatment with paclitaxel,the apoptotic rate was higher in X109R14 cells than that in cells only transfected with no gene.These data demonstrate that ribozyme transfected into cells can prevent the synthesis of Bcl 2 protein.The decrease of Bcl 2 protein can significantly enhance apoptosis induced by paclitaxel.It is suggested that Bcl 2 is one of important factors resulting in resistance to paclitaxel.更多还原