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目的探讨 mdr1短发夹 RNA(mdr1 shRNA)对人红白血病耐阿霉素细胞系 K562/ADM的耐药逆转作用。方法编码设计合成靶位 mdr1基因 mRNA 具有19个碱基发夹结构互补的寡核苷酸模板,构建2个 shRNA 表达载体 pSilencer~(TM)3.1-H1 neo mdr1-A和 mdr1-B,将其稳定转染 K562/ADM细胞。用 RT-PCR 法检测转染后 K562/ADM 细胞 mdr1 mRNA 表达,Western blot 检测 P-糖蛋白(P-gp)表达,流式细胞术和 MTT 法分别检测 K562/ADM 细胞凋亡和对阿霉素的敏感性,用激光共聚焦荧光显微镜观察并测定细胞内柔红霉素的积累。结果在 pSilencer~(TM)3.1-H11 neo mdr1-A 和 mdr1-B shRNA表达载体稳定转染的 K562/ADM 细胞,mdr1 mRNA 表达分别减少到转染前的35.9%(P<0.05)和27.5%(P<0.01);同时 Western blot 结果显示 P-gp 表达被明显而特异地抑制,对阿霉素的耐药性由79倍分别减低到38倍和30倍;并且,细胞内荧光强度与对照组相比显著增加(P<0.05),与阿霉素联合应用凋亡细胞百分率分别增加至18.1%(P<0.05)和54.4%(P<0.01)。结论靶向 mdr1基因shRNA 表达载体可有效逆转耐药,使耐药的肿瘤细胞恢复对化疗药物的敏感性。
Objective To investigate the reversal effect of mdr1 short hairpin RNA (shRNA) against human erythroleukemia-resistant adriamycin-resistant cell line K562 / ADM. Methods The mRNA of mdr1 gene was designed and synthesized. The oligonucleotide template with 19 base hairpin structure was designed. Two shRNA expression vectors, pSilencer TM 3.1-H1 neo mdr1-A and mdr1-B, were constructed, Stably transfected K562 / ADM cells. The expression of mdr1 mRNA in K562 / ADM cells was detected by RT-PCR and the expression of P-glycoprotein (P-gp) was detected by Western blot. The apoptosis of K562 / ADM cells was detected by flow cytometry and MTT assay respectively. The sensitivities were observed by laser scanning confocal microscopy and the intracellular daunorubicin accumulation was measured. Results In K562 / ADM cells stably transfected with pSilencer TM 3.1-H11 neo mdr1-A and mdr1-B shRNA expression vectors, mdr1 mRNA expression was reduced to 35.9% (P <0.05) and 27.5% (P <0.01). At the same time, Western blot results showed that the expression of P-gp was obviously and specifically inhibited, the resistance to doxorubicin decreased from 79 fold to 38 fold and 30 fold, respectively; (P <0.05). The percentage of apoptotic cells in combination with doxorubicin increased to 18.1% (P <0.05) and 54.4% (P <0.01), respectively. Conclusion The mdr1 gene targeting shRNA expression vector can effectively reverse the drug resistance and make the resistant tumor cells recover the sensitivity to chemotherapeutic drugs.