论文部分内容阅读
目的探讨表没食子儿茶素没食子酸酯(EGCG)逆转人肝癌细胞耐多药的机制,为进一步在基因转录水平上高通量筛选逆转肝癌耐多药天然药物提供一定的参考依据。方法采用人肝癌细胞Bel-7402及其耐5-氟尿嘧啶Bel-FU细胞,甲基四唑蓝法检测耐药Bel-FU细胞的耐多药性及对EGCG进行体外细胞毒性试验;选择与肝癌发生发展相关途径的42个功能基因作为检测基因,采用实时定量PCR微阵列技术检测Bel-7402组、Bel-FU组、Bel-FU+EGCG作用组基因表达;实时定量PCR检测相同条件下处理的基因Hras、MDR1表达变化以验证微阵列技术结果。结果耐药细胞Bel-FU对5-氟尿嘧啶、阿糖胞苷、长春新碱、阿霉素、柔红霉素的耐药指数分别为:89.6、3.9、37.2、16.5、3.7;EGCG对细胞Bel-FU的IC50、IC10值分别为587.4、73.1μg/mL;实时定量PCR微阵列检测结果显示:与Be-7402组比较,Bel-FU组中有5个基因上调;与Bel-FU组比较,Bel-FU+EGCG作用组有5个基因下调,其中有3个基因为Bel-7402组与Bel-FU组之间的差异基因;实时定量PCR验证结果:Bel-FU组中基因Hras、MDR1表达为Bel-7402组中的2.84、2.74倍,与Bel-FU组比较,Bel-FU+EGCG组作用中基因Hras、MDR1表达分别下调3.21、3.74倍。结论 EGCG对人肝癌耐多药细胞Bel-FU具有一定逆转作用。
Objective To investigate the mechanism of epigallocatechin-3-gallate (EGCG) reversing multidrug resistance in human hepatocellular carcinoma cells, and to provide a reference for further screening of multidrug-resistant natural drugs in liver cancer by high-throughput screening at gene transcription level. Methods Bel-7402 human hepatocellular carcinoma cells and Bel-FU cells resistant to 5-fluorouracil were used to detect the multidrug resistance of Bel-FU cells and the in vitro cytotoxicity of EGCG by methyltetrazolium method. Forty-two functional genes were detected by real-time quantitative PCR. The gene expression of Bel-7402, Bel-FU and Bel-FU + EGCG groups were detected by real-time quantitative PCR. , Changes in MDR1 expression to verify microarray technology results. Results The resistant index of Bel-FU to 5-fluorouracil, cytarabine, vincristine, doxorubicin and daunorubicin were 89.6, 3.9, 37.2, 16.5 and 3.7 respectively. -FU with IC50 values of 587.4 and 73.1 μg / mL, respectively. Real-time quantitative PCR showed that five genes were up-regulated in Bel-FU group compared with Be-7402 group. Compared with Bel-FU group, Five genes were down-regulated in Bel-FU + EGCG group, of which 3 were Bel-7402 group and Bel-FU group. Real-time PCR results showed that the expression of Hras and MDR1 gene in Bel- Compared with Bel-FU group, the expression of Hras and MDR1 genes in Bel-FU + EGCG group were down-regulated by 3.21 and 3.74 times, respectively. Conclusion EGCG has a certain reversal effect on Bel-FU human multidrug-resistant human hepatocellular carcinoma cells.