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目的:探讨液泡ATPase[vacuolar(H+)-ATPase,V-ATPase]非特异性抑制剂奥美拉唑(omeprazole,OME)逆转人肺腺癌耐药细胞株A549/DDP的耐药性及可能的作用机制。方法:以非细胞毒性浓度4μg/mL的OME预处理A549/DDP细胞24 h,再用2μg/mL顺铂(cisplatin,DDP)处理A549/DDP细胞。MTT法检测细胞的增殖抑制率,激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM)法间接检测细胞内pH值的变化,FCM法检测凋亡相关蛋白Bcl-2和PTEN的表达,RT-PCR法检测VATPase、Bcl-2和PTEN mRNA的表达。结果:OME具有逆转A549/DDP细胞耐药性的作用,逆转倍数为1.45(P<0.01);OME预处理后A549/DDP细胞内pH值明显降低,Bcl-2蛋白表达下降,PTEN蛋白表达增加(P<0.01)。V-ATPase在亲本A549及A549/DDP细胞中相对表达量分别为0.88±0.00和0.99±0.00(P<0.01);A549/DDP细胞中V-ATPase在有无OME预处理的情况下的相对表达量分别为1.09±0.00和1.05±0.02,差异无统计学意义。Bcl-2 mRNA相对表达量下降(P<0.01),PTEN mRNA相对表达量增加(P<0.01)。结论:V-ATPase在A549/DDP细胞中高表达,OME能部分逆转A549/DDP耐药性,其作用机制可能与上调PTEN和下调Bcl-2表达有关。
OBJECTIVE: To investigate the drug resistance and its possible role of omeprazole (OME), a non-specific inhibitor of vacuolar ATPase (H +) -ATPase and V-ATPase, in reversing human lung adenocarcinoma cell line A549 / DDP mechanism. Methods: A549 / DDP cells were pretreated with OME at a non-cytotoxic concentration of 4 μg / mL for 24 h and then treated with 2 μg / mL cisplatin (DDP) for 5 days. The cell proliferation inhibition rate was detected by MTT assay, the intracellular pH value was detected indirectly by laser scanning confocal microscope (LSCM), the expression of Bcl-2 and PTEN was detected by FCM, Method to detect the expression of VATPase, Bcl-2 and PTEN mRNA. Results: OME could reverse the drug resistance of A549 / DDP cells with a reversal multiple of 1.45 (P <0.01). The OME pretreatment significantly decreased the intracellular pH and the expression of Bcl-2 protein and PTEN protein (P <0.01). The relative expression of V-ATPase in parental A549 and A549 / DDP cells was 0.88 ± 0.00 and 0.99 ± 0.00 (P <0.01), respectively. The relative expression of V-ATPase in A549 / DDP cells in the presence or absence of OME pretreatment The amount was 1.09 ± 0.00 and 1.05 ± 0.02, the difference was not statistically significant. The relative expression of Bcl-2 mRNA decreased (P <0.01), and the relative expression of PTEN mRNA increased (P <0.01). Conclusion: V-ATPase is highly expressed in A549 / DDP cells. OME can partially reverse the drug resistance of A549 / DDP. The mechanism may be related to the up-regulation of PTEN and the down-regulation of Bcl-2 expression.