MDR1基因沉默增强三尖杉酯碱诱导的HT9细胞凋亡

MDR1gene silencing enhances harringtonine-induced apoptosis of HT9 cells

目的:探讨靶向P-糖蛋白(又称多药耐药蛋白1,MDR1)基因的shRNA对三尖杉酯碱诱导人耐三尖杉酯碱的前髓细胞白血病HT9细胞凋亡的影响。方法:构建MDR1基因特异的shRNA表达载体pSilencer 3.1-H1 neo-MDR1,稳定电转染HT9细胞,实时荧光定量PCR分析MDR1 mRNA的表达,Western blotting检测细胞MDR1蛋白表达,MTT法检测细胞活力,经三尖杉酯碱处理后琼脂糖凝胶电泳检测凋亡细胞DNA片段化,激光共聚焦观察细胞形态变化,流式细胞术检测细胞周期变化。结果:成功构建了shRNA表达载体pSilencer 3.1-H1neo-MDR1,稳定转染后,HT9细胞MDR1 mRNA和蛋白表达均显著降低,HT9细胞三尖杉酯碱的IC50由(1.184±0.130)mg/L降至(0.711±0.010)mg/L。与HT9细胞相比,经三尖杉酯碱处理后稳定转染细胞HT9/sh-3.1-1的DNA ladder更明显,激光共聚焦显微镜下可见形态不规则的碎片状或梅花状等典型的细胞凋亡形态;细胞的S期细胞增多,并出现明显的凋亡峰。结论:shRNA表达载体pSilencer 3.1-H1 neo-MDR1能够稳定、持久地抑制MDR1基因,并有效增强三尖杉酯碱诱导的HT9细胞凋亡。

AIM： To investigate the effects of P - glycoprotein （multidrug resistance protein 1, MDR1 ） shR- NA on harringtonine - induced apoptosis of human harringtonine - resistant actue premyelocytic leukemia HT9 cells. METHODS： MDR1 shRNA expression plasmid pSilencer 3.1 - H1 neo - MDR1 was constructed and introduced into HT9 cells （HT9/sh- 3.1 -1 cells）. The mRNA of MDR1 was detected by real- time fluorescent quantitative PCR. The pro- tein level of MDR1 was assayed by Western blotting. The viability of HT9 cells was determined by MTY method. After the cells were treated with harringtonine, the DNA fragmentation of the cells were observed by gel electrophoresis. The morpho- logical alteration of the cells was observed under confocal laser scanning microscope. The cell cycle was also analyzed by flow cytometry. RESULTS： MDR1 shRNA expression plasmid was successfully constructed. The expression of MDR1 at mRNA and protein levels was significantly decreased after pSilencer 3.1 - H1 neo - MDR1 was transfected into H39 cells, and ICso of harringtonine was decreased from （1. 184 ~ 0. 130） mg/L to （0. 711 ~ 0. 010）mg/L. Compared with HID cells, DNA ladder was observed in HT9/sh - 3.1 - 1 cells treated with harringtonine, and the typical apeptotic morphology was also found under a confocal laser scanning microscope. The cells in S phase were increased, and apeptotie peak was observed. CONCLUSION： shRNA expression plasmid pSilencer 3.1 - H1 neo - MDR1 permanently and stablely inhibits MDR1 gene expression and promotes the sensitivity of HT9 cells to harringtonine - induced apoptosis.