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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IC50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],G2/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pSilenc-er3.1-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 silence MDR1 gene expression in drug-resistant HT9 cells by short hairpin RNA (shRNA) expression vector and reverse the drug resistance of human promyelocytic leukemia cell line HT9 to allicin. Methods: shRNA fragments were designed according to the sequence of MDR1 gene, pSilencer3.1-shMDR1 expression vector targeting MDR1 gene was constructed and stably transfected into HT9 cells. Real-time PCR was used to detect the expression of MDR1 mRNA. Western blotting was used to detect P- Glycoprotein (MDR1 gene encoding) expression, MTT assay cell viability, agarose gel electrophoresis after allicin HT9 cell apoptosis, electron microscopy ultrastructure changes, flow cytometry cell cycle. Results: The MDR1 expression vector pSilencer3.1-shMDR1 was successfully constructed and the HT9-shMDR1 cell line was stably transfected into HT9-shMDR1 cells. The MDR1 mRNA expression was significantly decreased in HT9-shMDR1 cells [(0.027 ± 0.002) vs (0.110 ± 0.005) , P <0.01], P-glycoprotein expression was significantly lower (0.856 ± 0.014 vs 1.454 ± 0.027, P <0.05). The IC50 of allicin on HT9-shMDR1 cells was significantly lower than that of untransfected HT9 cells [(26.66 ± 0.59) vs (52.75 ± 0.64) μg / ml, P <0.01]. Allicin-resistant HT9-shMDR1 cells The relative reversal rate was (49.45 ± 1.86)%. Compared with untransfected HT9 cells, the apoptotic DNA fragments of HT9-shMDR1 cells treated with allicin were more obvious. The formation of hemomedia was observed under electron microscope. Allicin treatment did not affect the cell cycle of HT9 cells (HT9-neo cells) transfected with HT9 and untransfected HT9 cells, but allicin reduced the proportion of S phase cells in HT9-shMDR1 cells [(31.40 ± 2.13) % vs (53.80 ± 1.87)%, P <0.01]. The proportion of cells in G2 / M phase increased (35.62 ± 2.06)% vs (9.37 ± 2.09)%, P <0.01. CONCLUSION: pSilenc-er3.1-shMDR1 targeting MDR1 can inhibit MDR1 gene expression and reverse HT9 cell resistance to allicin.