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目的:研究谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPx1)在调控肺腺癌A549微球体细胞增殖和凋亡中的作用,并探讨其可能的机制。方法:采用无血清悬浮法培养A549微球体细胞,应用FCM法检测A549微球体细胞中CD133~+CD44~+细胞所占的比例,蛋白质印迹法检测A549微球体细胞中干细胞标志物Sox2和Nanog蛋白的表达情况,以及GPx1蛋白的表达水平。采用质量浓度为5 mg/m L的顺铂(cisplatin,DDP)分别处理A549微球体细胞和亲本A549细胞,CCK-8法检测48 h内细胞的存活率;利用比色法检测A549微球体细胞和亲本A549细胞中谷胱甘肽(glutathione,GSH)的含量和GPx1的活性,蛋白质印迹法检测GPx1蛋白的表达情况;FCM法检测A549微球体细胞和亲本A549细胞中活性氧(reactive oxygen species,ROS)的水平。将特异性针对GPx1基因的GPx1-si RNA转入A549微球体细胞中,分别采用实时荧光定量PCR和蛋白质印迹法检测GPx1 m RNA及蛋白的表达水平;FCM法、蛋白质印迹法和细胞成球实验分别检测沉默GPx1基因表达后对A549微球体细胞中ROS水平、Sox2和Nanog蛋白表达水平以及A549微球体细胞成球能力的影响。应用FCM法、CCK-8法以及蛋白质印迹法检测转入GPx1-si RNA并联合DDP(5mg/m L)干预后,对细胞的生存率和凋亡水平以及磷酸化p38(phospho-p38,p-p38)、磷酸化激活转录因子2(phospho-activating transcription factor 2,p-ATF2)、p53和Bax蛋白表达水平的影响。结果:培养获得A549微球体细胞,A549微球体细胞中CD133+CD44+细胞所占比例为(11.7±0.6)%,明显高于亲本A549细胞的(2.2±0.3)%。Sox2和Nanog蛋白在A549微球体细胞中的表达水平明显高于在亲本A549细胞中的表达水平(P值均<0.05);DDP(5 mg/mL)处理48 h后,对A549微球体细胞的生存率无明显影响(P>0.05)。A549微球体细胞中GSH含量、GPx1的活性和蛋白的表达水平均明显高于亲本A549细胞(P值均<0.05),而ROS含量在A549微球体细胞中明显低于亲本A549细胞(P<0.05)。转入GPx1-siRNA可明显下调A549微球体细胞中GPx1 mRNA和蛋白的表达水平、并抑制Sox2蛋白的表达水平和微球体的形成(P值均<0.05)。下调GPx1表达并联合DDP处理后,A549微球体细胞的存活率明显下调(P<0.05),而细胞凋亡率明显升高(P<0.05)。此外,下调GPx1表达并联合DDP处理后,A549微球体细胞中p-p38、p-ATF2、p53和Bax蛋白的表达水平均明显上调(P值均<0.05)。结论:下调GPx1可能通过抑制Sox2、上调ROS和激活p38-p53通路发挥抑制细胞增殖、促进凋亡等作用,其可成为肺癌治疗的潜在靶点。
Objective: To investigate the role of glutathione peroxidase 1 (GPx1) in the regulation of proliferation and apoptosis of lung adenocarcinoma A549 microspheres and to explore its possible mechanism. Methods: A549 microspheres were cultured in serum-free suspension. The proportion of CD133 ~ + CD44 ~ + cells in A549 cells was detected by FCM. Sox2 and Nanog proteins were detected by Western blotting in A549 microspheres The expression of GPx1 protein and the level of expression. The A549 microspheres and their parental A549 cells were treated with 5 mg / mL cisplatin (DDP) respectively. The cell viability was measured by CCK-8 assay within 48 h. The colorimetric assay was used to detect the expression of A549 microspheres And glutathione (GSH) content and GPx1 activity of A549 cells were measured by Western blotting. The expression of GPx1 protein was detected by Western blotting. The expression of GPx1 protein in A549 cells and parental A549 cells was detected by FCM. )s level. GPx1-si RNA specific to GPx1 gene was transfected into A549 microspheres cells, and GPx1 m RNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting respectively. FCM, Western blotting and cell spheres The effects of silencing GPx1 gene expression on the level of ROS, the expression of Sox2 and Nanog in A549 microspheres and the ability of A549 microspheres to form cells were detected. The survival rate and apoptosis of cells transfected with GPx1-si RNA and DDP (5 mg / mL) were measured by FCM, CCK-8 and Western blotting, and the phosphorylation of p38 (phospho-p38, p -p38), phospho-activating transcription factor 2 (p-ATF2), p53 and Bax protein expression. Results: The percentage of CD133 + CD44 + cells in A549 microspheres was (11.7 ± 0.6)%, which was significantly higher than that of the parental A549 cells (2.2 ± 0.3)%. The expression of Sox2 and Nanog proteins in A549 microspheres was significantly higher than that in A549 cells (P <0.05). After treated with DDP (5 mg / mL) for 48 h, Survival rate had no significant effect (P> 0.05). The content of GSH, the activity of GPx1 and the expression of protein in A549 microspheres were significantly higher than those in parental A549 cells (P <0.05), while the content of ROS in A549 microspheres was significantly lower than that of the parental A549 cells (P <0.05 ). GPx1-siRNA downregulated the expression of GPx1 mRNA and protein in A549 microspheres and inhibited the expression of Sox2 protein and the formation of microspheres (all P <0.05). After down-regulating the expression of GPx1 and treating with DDP, the survival rate of A549 microspheres was significantly decreased (P <0.05), while the apoptosis rate was significantly increased (P <0.05). In addition, the expression of p-p38, p-ATF2, p53 and Bax in A549 microspheres cells were significantly up-regulated after GPx1 down-regulation and DDP treatment (all P <0.05). Conclusion: Down-regulation of GPx1 may play a potential therapeutic target for lung cancer through inhibition of Sox2, upregulation of ROS and activation of p38-p53 pathway in inhibiting cell proliferation and promoting apoptosis.