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目的观察多药耐药基因1(MDR1)在耐砷的人肝细胞(L-02)中的表达情况,探讨其与L-02耐砷性的关系。方法培养L-02和耐砷的L-02,用real-time PCR和免疫组织化学法检测细胞MDR1 mRNA和P糖蛋白的表达情况;2种细胞单用亚砷酸钠(NaAsO2)2.5,5.0,10 mmol/L及NaAsO2与MDR1抑制剂环胞菌素D衍生物(PSC833)联合作用24 h后收集细胞,四甲基偶氮噻唑蓝(MTT)法和石墨炉原子吸收光谱法检测细胞生存率和细胞内总砷浓度。结果MDR1 mRNA及P糖蛋白在耐砷的L-02中的表达强度分别为(8.890±0.968),(68.76±3.81)%,高于L-02的(1.014±0.189),(24.44±4.03)%,差异有统计学意义(P<0.001);NaAsO2单用时,低、中、高剂量组耐砷的L-02生存率分别为(109.630±0.511)%,(95.469±0.054)%,(78.890±0.024)%,明显高于L-02的(104.841±0.015)%,(91.534±0.026)%,(64.811±0.079)%;而耐砷的L-02内总砷浓度分别为(0.682±0.004),(1.355±0.005),(3.274±0.010)μg/L,明显低于L-02的(1.165±0.010),(3.661±0.002),(7.529±0.006)μg/L,差异有统计学意义(P<0.001);NaAsO2与PSC833联合作用则可增加细胞内总砷浓度、降低细胞存活率(P<0.001)。结论L-02细胞的耐砷性与MDR1高表达有关,在mRNA和蛋白水平均有体现。
Objective To investigate the expression of multidrug resistance-1 (MDR1) gene in arsenic-tolerant human hepatocytes (L-02) and its relationship with arsenic tolerance of L-02. Methods The expression of MDR1 mRNA and P-glycoprotein in L-02 and A-resistant L-02 cells was detected by real-time PCR and immunohistochemistry. The two cells were treated with NaAsO2 2.5, 5.0 , 10 mmol / L NaAsO2 and MDR1 inhibitor cyclosporin D derivatives (PSC833) were collected 24 h after the cells were collected, MTT assay and graphite furnace atomic absorption spectrometry were used to detect cell viability Rate and total intracellular arsenic concentration. Results The expression intensity of MDR1 mRNA and P-glycoprotein in arsenic resistant L-02 cells were (8.890 ± 0.968), (68.76 ± 3.81)%, (1.014 ± 0.189) and (24.44 ± 4.03) higher than L-02 %, The difference was statistically significant (P <0.001). When NaAsO2 was used alone, the survival rates of L-02 in low, medium and high dose groups were 109.630 ± 0.511%, 95.469 ± 0.054%, 78.890% ± 0.024)%, which was significantly higher than that of L-02 (104.841 ± 0.015)%, (91.534 ± 0.026)% and (64.811 ± 0.079)%, respectively. (1.355 ± 0.005) and (3.274 ± 0.010) μg / L, respectively, which were significantly lower than those of L-02 (1.165 ± 0.010), (3.661 ± 0.002) and (7.529 ± 0.006) μg / L, (P <0.001). The combination of NaAsO2 and PSC833 could increase the intracellular total arsenic concentration and decrease the cell survival rate (P <0.001). Conclusion The arsenic resistance of L-02 cells is related to the high expression of MDR1, which is reflected in the mRNA and protein levels.