摘要目的 观察bcl-2反义寡核苷酸对膀胱癌顺铂(DDP)耐药细胞耐药性的逆转并增强其敏感性的作用.方法 以膀胱癌顺铂耐药细胞株BIU87/DDP为模型,用脂质体转染和电转染的方法,将bcl-2反义寡核苷酸(bcl-2-AODN)转染BIU87/DDP细胞,同时以转染bcl-2正义链(bel-2-SODN)及RPMI 1640作对照.采用DNA凝胶电泳检测转染后是否诱导细胞凋亡,应用免疫组织化学技术检测bcl-2蛋白表达变化,逆转录-聚合酶链反应(RT-PCR)检测bcl-2 mRNA表达水平的变化.噻唑蓝(MTY)比色法检测细胞抑制率与药物半数抑制浓度(IC50).结果 转染AODN的细胞IC50值(20.400 ±2.590)mg/L,较未转染耐药细胞(66.000±4.637)mg/L,差异有统计学意义(P＜0.05);脂质体转染bcl-2反义寡核苷酸12 h后,倒置显微镜下即可见BIU87/DDP有30%细胞出现变化(但不能分辨死亡或凋亡),DNA凝胶电泳出现明显的DNA梯带状条带,对照组无变化.采用电穿孔转染48 h后,细胞在倒置显微镜下的变化不明显,以顺铂10mg/L作用于转染细胞24 h,分别收集细胞检测.bcl-2蛋白表达降低;实验组BIU87/DDP+AODN细胞bcl-2 mRNA表达(0.72 ±0.07)与转染前(2.94±0.09)比较,mRNA表达率下降(P＜0.05).结论 bcl-2反义寡核苷酸可通过增加细胞的凋亡而一定程度地逆转膀胱癌细胞的顺铂耐药性,提高膀胱癌细胞对化学治疗药物的敏感性.

abstractsObjective To observe the influence of bcl-2 antisense oligodeoxynucleotides (ASON)on reversal of cisplatin resistance in BIU87 cell line of bladder carcinoma. Methods Cisplatin resistance bladder carcinoma cells served as models. The bcl-2 antisense oligodeoxynucleotides were transfected to the cells by lipofectamine and electroporation. The sense-and random-oligodeoxynucleotides were used as controls. The expression level of bcl-2 mRNA was detected by using semi-quantitative reverse transcription polymerase chain reaction ( RT-PCR) . The apoptosis of bladder cancer cell lines was examined by DNA ladder assay. The protein level and 50% inhibitory concentration (IC50) were measured by immunofluorescence technique and methyl thiazol tetrazolium (MTT) assay, respectively. Results Transfecting BIU87/DDP cells with antisense oligonucleotides statistically reduced IC50, values of BID/DDP cells from (66. 000 ±4. 637) to (20. 400 ±2. 590) mg/L. Twelve h after transfection by lipofectamine, 30% cells had apoptotic or necrotic changes under the inverted phase contrast microscopy, and DNA " Ladder" was observed with agrose gel electrophoresis in the antisense-transfected cells. Forty-eight h after transfection by electroporation followed by treatment of 10 mg/L cisplatin, RT-PCR showed the expression of bcl-2 mRNA in the transfected cells was significantly lower (0. 72 ± 0. 07 ) than before transfection ( 2. 94 ± 0. 09 ) , and that of bcl-2 protein was decreased, as well as the IC50. Conclusion bcl-2 antisense oligodeoxynucleotides can reverse the resistance to cisplatin in BIU87/DDP cells by inhibiting the expression of bcl-2 protein and increasing the apoptosis.