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目的通过构建人视网膜母细胞瘤Y79细胞的依托泊苷耐药株,比较耐药株的药物敏感性和细胞生长情况,观察耐药相关基因及细胞信号通路的变化,从而阐释人视网膜母细胞瘤对依托泊苷的耐药机制。方法以不同浓度梯度的依托泊苷处理Y79细胞,采用浓度递增不间断刺激法构建依托泊苷耐药株,观察耐药株细胞的形态学变化和细胞生长增殖情况以及对依托泊苷细胞毒作用的敏感性;以caspase 3/7酶活性来评估耐药株的细胞凋亡程度,并通过Western blot方法研究与细胞增殖和耐药相关蛋白的表达变化。结果成功构建耐受依托泊苷的人视网膜母细胞瘤耐药株Y79/EDR,最大耐药浓度为500 nmol/L。和亲本细胞相比,耐药细胞具有较强的生长增殖能力,倍增时间缩短14.5 h(P<0.001);且对依托泊苷的敏感性显著下降,耐药指数达29.47。Caspase 3/7活性检测结果显示,给予亲本及耐药细胞不同浓度的依托泊苷后,耐药细胞中caspase 3/7活性明显低于亲本细胞,凋亡减少,且呈药物浓度依赖性。Western blot结果表明,耐药细胞中磷酸化AKT(p-AKT)表达明显高于亲本细胞,提示其p-AKT的活性明显增强,p-AKT促进其下游蛋白MDM2的磷酸化从而抑制p53的磷酸化,而凋亡相关蛋白Bax/Bcl-2的比值明显降低。多药耐药性蛋白P-糖蛋白(P-gp)的表达下调,Rb1蛋白表达水平无明显变化,提示耐药株Y79/EDR的耐药并不依赖于P-gp而产生,且与Rb1蛋白无关,而可能通过增加肿瘤驱动蛋白AKT的磷酸化,激活其所介导的下游信号通路,增加靶蛋白MDM2的表达,从而抑制抑癌蛋白p53的表达;同时增加抗凋亡蛋白Bcl-2和降低抑凋亡蛋白Bax的表达水平;最终导致细胞通过增殖显著增强和凋亡明显减少而产生耐药。结论成功构建依托泊苷耐药株Y79/EDR,初步结果表明,其耐药细胞株可能通过促进细胞生长与增殖,同时抑制细胞凋亡而导致耐药,这将为深入研究人视网膜母细胞瘤的病变机制,阐释其对依托泊苷的耐药机制,寻找有效的耐药逆转方案提供一定的前期实验基础。
OBJECTIVE: To construct a chemotherapeutic drug of etoposide-resistant strain of human retinoblastoma Y79 cells to compare the drug sensitivity and cell growth of drug-resistant strains and to observe the changes of drug-resistance-related genes and cell signaling pathways so as to elucidate human retinoblastoma Resistance to etoposide mechanism. Methods Y79 cells were treated with different concentrations of etoposide. The esophageal cancer cells were treated with increasing concentrations of uninterrupted stimulation to observe the morphological changes of cells and cell growth and proliferation as well as the effects of etoposide on cytotoxicity . The caspase 3/7 enzyme activity was used to evaluate the degree of apoptosis of the drug-resistant strains. Western blot was used to investigate the expression of protein related to cell proliferation and drug resistance. Results The resistant cell line Y79 / EDR of etoposide-resistant human retinoblastoma was successfully constructed and the maximum drug concentration was 500 nmol / L. Compared with the parental cells, the resistant cells had stronger ability of growth and proliferation, and the doubling time was shortened by 14.5 h (P <0.001). The sensitivity to etoposide was significantly decreased, and the drug resistance index was 29.47. Caspase 3/7 activity test results showed that after given different concentrations of etoposide, the activity of caspase 3/7 in drug-resistant cells was significantly lower than that of the parental cells, and apoptosis was reduced in a dose-dependent manner. The results of Western blot showed that the expression of phosphorylated AKT (p-AKT) in drug-resistant cells was significantly higher than that of parental cells, suggesting that the activity of p-AKT was significantly increased. P-AKT promoted the phosphorylation of its downstream protein MDM2 and inhibited the phosphorylation of p53 While the ratio of Bax / Bcl-2 in apoptosis-related protein was significantly decreased. The expression of P-glycoprotein (P-gp) was down-regulated and the level of Rb1 protein was not changed, which indicated that the resistance of Y79 / EDR was not dependent on P-gp, Protein may play an important role in inhibiting the expression of the tumor suppressor protein p53 by increasing the phosphorylation of AKT and activating its downstream signaling pathway to increase the expression of MDM2. At the same time, the anti-apoptotic protein Bcl-2 And decrease the expression of the anti-apoptotic protein Bax. Eventually lead to cell proliferation through a significant increase and a significant reduction in apoptosis caused by drug resistance. Conclusion The successful construction of the drug resistant Y79 / EDR of etoposide indicated that the drug-resistant cell lines could induce drug resistance through promoting the cell growth and proliferation and inhibiting the apoptosis of cells. This study will be helpful for the further study of human retinoblastoma Of the pathological mechanism to explain its mechanism of resistance to etoposide, looking for an effective drug reversal program to provide a certain pre-experimental basis.