中文摘要：

目的构建针对mdr1和mcl1基因的短发夹RNA（shRNA）干扰表达质粒，并探讨联合转染对K562／A02细胞耐药的逆转作用。方法根据mdr1和mcl1基因表达序列设计有效的RNA干扰片段，分别将其构建入质粒表达空载体中，以获得两种基因特异性shRNA干扰表达质粒；然后在脂质体介导下分别和联合转染K562／A02细胞，用G418和（或）HygroB筛选出稳定表达的细胞克隆。用RT—PCR分析mdr1和mcl1mRNA的表达；MTT法检测阿霉素对K562／A02细胞的半数抑制浓度（IC50）；流式细胞术测定细胞P-糖蛋白表达水平以及细胞凋亡率。结果成功构建两个基因的shRNA干扰表达质粒。mdr1、mcl1shRNA干扰表达质粒单独和联合转染K562／A02细胞可有效封闭相应基因表达，联合转染组mdr1基因和mcl1基因的mRNA相对表达水平分别是未转染细胞的52％，44％。mdr1、mcl1shRNA干扰表达质粒单独和联合转染后K562／A02细胞耐药逆转率分别为63，8％，71.1％，83.1％，转染两种质粒组对K562／A02细胞耐药逆转率最高，P糖蛋白相对表达量由未转染组的19.70±1．15降至6．40±0，92（P〈0．01），阿霉素诱导的细胞凋亡率由（1．53±0．42）％提高至（7，77±0．42）％（P〈0．01）。联合转染两种质粒组和单独转染组比较，细胞对阿霉素敏感性和阿霉素诱导的细胞凋亡率差异亦有统计学意义（P〈0.05）。结论转染mdr1或mcl1基因的shRNA干扰表达质粒可有效抑制相应基因表达，皆不同程度逆转K562／A02细胞对阿霉素的耐药性；联合转染两种质粒可显著增加逆转耐药的效果。mcl1基因可能与K562／A02耐药相关。

英文摘要：

Objective To construct two recombinant plasmids of mdr1 and mcl1 shRNA, and to investigate their reversal effect on drug resistance in K562 adriamycin resistant cell lines （K562/A02）. Methods Two oligonucleotides of mdr1 and mcl1 gene were designed referring to that of GenBank, double-stranded DNA was derived through annealing, and cloned into pRNAT vector digested by two restricted endoenzymes. K562/A02 cells were transfected with the recombinant plasmids. The mdr1 mRNA expression and its protein product P-glycoprotein （P-gp） were detected by RT-PCR and flow cytometry. The expression of mcl1 gene was detected by RT-PCR. 50% inhibition concentration （ IC50 ） of adriamycin （ ADM ） on K562/A02 cells was determined by MTT method. Cells apoptosis was analyzed by flow cytometry. Results Comparing with K562/A02 cells, the shRNA of mdr1 or mcl1 gene in vitro can remarkably increase the sensitivity of K562/A02 to adriamycin, down-regulate mdr1 or mcl1 gene expression, increase the K562/A02 cells apoptosis rates induced by adriamycin. Cotransfection of mdr1 and mcl1 genes shRNA can also down-regulate the expression of their gene, more remarkably increase the sensitivity and apoptosis of K562/A02 to adriamycin. Conclusion Transfection of mdr1 or mcl1 gene shRNA can promote the sensitivity of K562/A02 to adriamycin and cotransfection of the two shRNA can more remarkably do so. The mcl1 gene might be involved in adriamycin resistant in K562/A02 cells.