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目的探讨r Ad-p53基因对耐化疗药物MGC-803胃癌细胞的抑制作用。方法体外培养经紫杉醇处理的MGC-803细胞(MGC-803/PTX细胞),将其分为观察组和耐药对照组:观察组分别加入50、100、200、400、800、1600MOI r Ad-p53注射液,耐药对照组加入相应体积的培养液RPMI-1640;应用MTT法检测细胞增殖抑制情况,应用流式细胞仪检测观察期细胞凋亡率和细胞周期。采用“爬片法”收集细胞,将其分为r Ad-p53处理组,敏感对照组及耐药对照组,r Ad-p53处理组加入400 MOI r Ad-p53注射液,敏感对照组及耐药对照组均加入对应量的RPMI-1640培养基。3组分别采用免疫组化法在光学显微镜下观察细胞内多耐药基因表达蛋白(MDR1-Pgp蛋白)染色颗粒,及免疫印迹法检测MDR1-Pgp蛋白表达量(灰度值)。结果 MTT法检测结果显示,r Ad-p53对MGC-803/PTX细胞增殖的抑制作用显著,其抑制率随着时间的增加而逐渐上升;流式细胞术检测结果显示,100、200、400 MOI r Adp53处理组MGC-803/PTX细胞周期被阻滞在G2/M期,且其效果具有剂量依赖性;不同浓度的r Ad-p53处理后,MGC-803/PTX细胞凋亡率显著高于耐药对照组(P均<0.05)且具有剂量依赖性。免疫印迹法表明,r Ad-p53处理组MDR1-Pgp蛋白质灰度值显著高于敏感对照组,显著低于耐药对照组(P均<0.05);免疫组化法显示r Ad-p53处理组细胞核蓝染,可见少量阳性颗粒,而耐药对照组细胞内见大量深染颗粒,敏感对照组无阳性颗粒。结论 r Adp53对胃癌MGC-803/PTX细胞的抑制效果显著,因此可降低化疗治疗中的多药耐药性,为胃癌治疗提供了新的治疗途径。
Objective To investigate the inhibitory effect of Ad-p53 gene on gastric cancer cell line MGC-803. Methods Paclitaxel-treated MGC-803 cells (MGC-803 / PTX cells) were cultured in vitro and divided into observation group and drug-resistant control group. The observation group was treated with 50,100,200,400,800,1600MOI r Ad- p53 injection, drug-resistant control group were added to the corresponding volume of culture medium RPMI-1640; application of MTT assay inhibition of cell proliferation, the application of flow cytometry observation period apoptosis rate and cell cycle. Ad-p53 treatment group, sensitive control group and drug-resistant control group were treated with Ad-p53 injection and 400 MOI r Ad-p53 injection and control group And resistant control group were added to the corresponding amount of RPMI-1640 medium. Immunohistochemistry was used to observe the expression of multidrug resistance gene (MDR1-Pgp) staining particles in the three groups and the expression of MDR1-Pgp protein (gray value) by immunoblotting. Results MTT assay showed that the inhibitory effect of r-Ad-p53 on the proliferation of MGC-803 / PTX cells was significant, and the inhibition rate of r-Ad-p53 increased gradually with the increase of time. The results of flow cytometry showed that at 100, 200 and 400 MOI The cell cycle of MGC-803 / PTX treated with Adp53 was arrested in G2 / M phase and the effect was dose-dependent. The apoptotic rate of MGC-803 / PTX cells was significantly higher than that of Ad-p53 treated group Resistant control group (all P <0.05) and in a dose-dependent manner. Immunoblotting showed that the gray value of MDR1-Pgp protein in r Ad-p53 treatment group was significantly higher than that in the sensitive control group (P <0.05), and the expression of Ad-p53 in rAd-p53 treatment group The nuclei were stained blue, showing a few positive particles, but a large number of deep-staining particles were seen in the cells of the resistant control group, and no positive particles in the sensitive control group. Conclusions r Adp53 has a significant inhibitory effect on gastric cancer MGC-803 / PTX cells, thus reducing the multidrug resistance in chemotherapy and providing a new therapeutic approach for the treatment of gastric cancer.