目的： 利用短发夹RNA（short hairpin RNA，shRNA）表达载体沉默耐药细胞HT9中 MDR1 基因表达，从而逆转人早幼粒白血病细胞株HT9对大蒜素的耐药性。 方法： 根据 MDR1 基因序列设计shRNA片段，构建靶向 MDR1 基因的pSilencer3.1-shMDR1表达载体，稳定转染HT9细胞，real-time PCR检测细胞中 MDR1 mRNA的表达，Western blotting检测HT9细胞中P-糖蛋白（ MDR1 基因编码）的表达，MTT法检测细胞存活率，琼脂糖凝胶电泳检测经大蒜素处理后HT9细胞的凋亡，电镜观察细胞超微结构变化，流式细胞术检测细胞周期。 结果： 成功构建靶向 MDR1 的表达载体pSilencer3.1-shMDR1，稳定转染HT9细胞形成HT9-shMDR1细胞系，HT9-shMDR1细胞中 MDR1 mRNA表达显著降低\[（0.027±0.002） vs （0.110±0005），P<0.01\]，P-糖蛋白表达也明显降低\[（0.856±0014） vs （1.454±0.027），P<0.05\]。大蒜素对HT9-shMDR1细胞的IC50较对未转染组HT9细胞明显降低\[（26.66±0.59） vs （52.75±0.64）μg/ml，P<0.01\]，HT9-shMDR1细胞对大蒜素耐药的相对逆转率为（49.45±1.86）%。与未转染组HT9细胞相比，经大蒜素处理后HT9-shMDR1细胞凋亡的DNA片段更为明显，电镜下可见细胞凋亡特有的半月体形成。大蒜素处理不影响未转染组HT9细胞和对照质粒转染后HT9细胞（HT9-neo细胞）的细胞周期，但大蒜素处理使HT9-shMDR1细胞的S期细胞比例减少\[(31.40±2.13)% vs (53.80±1.87)%, P<001\]，G2/M期细胞比例增多\[(35.62±2.06)% vs (9.37±209)%, P<0.01\]。 结论： 靶向 MDR1 的干扰表达载体pSilencer3.1-shMDR1能够抑制 MDR1 基因的表达，从而逆转HT9细胞对大蒜素的耐药性。

Objective:To investigate the effects of short hairpin RNA (shRNA) expression plasmid on silencing of MDR1 gene in drug resistant H79 cells and thus reverse the drug resistance of human promyelocytic leukemia HT9 cells to allicin. Methods: shRNA fragment targeting MDR1 gene was designed and constructed to obtain pSilencer3.1-shMDR1 expression plasmid, which was then stably transfected into HT9 cells. The expression of MDR1 mRNA in HT9 cells was assayed by real-time PCR. The P-gp protein (encoded by the MDR1 gene) expression was assayed by Western blotting. The cell mortality rate of HT9 cells was determined by MTT method. After treated with allicin, the apoptosis of HT9 cells was observed by gel electrophorosis, cell ultrastructure changes were observed under a transmission electron microscope, and the cell cycle was detected by flow cytometry. Results: pSilencer3.1-shMDR1 expression plasmid targeting MDR1 was constructed successfully and then stably transfected into HT9 cells to form the HT9-shMDR1 cell line. The MDR1 mRNA (\[0.027±0.002\] vs \[0.110±0.005\]，P<0.01) and P-gp protein expressions (\[0.856±0.014\] vs \[1.454±0027\], P<005) were significantly decreased in HT9-shMDR1 cells. The IC50 of allicin to HT9-shMDR1 cells significantly decreased compared with that in untransfected HT9 cells (\[26.66±0.59\] vs \[52.75±0.64\] μg/ml，P<0.01). The relative drug resistance reversal rate of HT9-shMDR1 to allicin was (49.45±1.86)%. Compared with untransfected HT9 cells, DNA ladder was more obvious in HT9-shMDR1 cells treated with allicin, and the typical apoptotic body was found by electron microscopy. Allicin treatment did not affect the cell cycle distribution of untransfected HT9 cells and control plasmid transfected HT9 cells (HT9-neo cells). However, the ratios of allicin-treated HT9-shMDR1 cells were significantly decreased in the S phase (\[3140±2.13\]% vs \[53.80±1.87\]%, P<0.01\] and were significantly increased in the G2/M phase (\[35.62±206\]% vs \[9.37±2.09\]%, P<0.01\]. Conclusion: pSilencer3.1-MDR1 expression plasmid targeting MDR1 gene can inhibit the expression of MDR1 gene, therefore reversing drug resistance of HT9 cells to allicin.

黑龙江省自然科学基金资助项目（No. C200624）；黑龙江省教育厅科学技术项目资助（No. 11511447，No.12511611）；齐齐哈尔大学青年教师科研启动基金资助项目（No. 2011K-M38）