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槲皮素逆转人肝癌MDR相关基因的研究

韦艳   

  1. 武警广西总队医院
  • 收稿日期:2011-10-13 修回日期:2012-04-13 出版日期:2012-10-15 发布日期:2012-10-15
  • 通讯作者: 韦艳

  • Received:2011-10-13 Revised:2012-04-13 Published:2012-10-15 Online:2012-10-15

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摘要： 摘 要 目的: 通过定量PCR芯片技术在基因转录水平上探讨槲皮素逆转人肝癌多药耐药( multi drug resistance, MDR)的作用机制。方法:采用体外培养人肝癌细胞Bel-7402及其耐5-氟尿嘧啶细胞Bel-FU, MTT法测定槲皮素的体外细胞毒性及浓度依赖的逆转耐药作用。定量PCR芯片检测BEL-7402细胞及Bel-FU细胞之间的差异基因,以及槲皮素作用于Bel-FU细胞前后的基因表达差异,实时定量PCR和western-blot法检测细胞中mdr1、H-ras基因以及P-gp蛋白的表达变化以验证基因芯片分析的结果。结果: 槲皮素对Bel-7402、Bel-FU细胞的IC10分别为59.2、58.2μM, IC50分别为226.7、193.9μM。16.5、33.0、49.5 μM 的槲皮素对Bel-FU 的耐药逆转倍数分别为1.14、1.68、2.38倍；与Bel-7402比较, Bel-FU细胞中的基因H-ras、Bcl-2、PDGFA、MDR1、BCRP表达上调（P＜0.05）表达上调。经槲皮素作用后，耐药细胞中基因H-ras、EGFR、FGF、VEGFA、PDGFA、MDR1、Erk1表达下调。实时定量PCR和western-blot证实了槲皮素可下调MDR1、H-ras基因以及P-gp蛋白在耐药细胞中高表达，与芯片检测到的基因水平变化一致。结论：槲皮素可逆转耐药细胞中耐药差异基因的高表达量,从而逆转人肝癌细胞的多药耐药性。

关键词: 槲皮素, 定量PCR芯片, 肝细胞癌, 多药耐药

Abstract: Abstract Objective: To investigate the mechanism of reversing effect of Quercetin on MDR of human hepatocellular carcinoma ( HCC) in the course of genetic transcription by q-PCR microarray.Methods: Human hepatocellular carcinoma cell line Bel-7402 and 5-FU resistant cell line Bel-FU were cultured in vitro. The cytotoxicity and dose-dependen reverse effect of Quercetin were detected by MTT assay. The different gene expression profiles between Bel-7402 and Bel-FU cells ,and those between Bel-FU and Bel-FU cells treated with Quercetin were detected by q-PCR microarray . Then the gene expression of H-ras，MDR1 were detected by real-time quantitative PCR and the expression of P-gp were detected by western-blot to confirm the results of q-PCR microarray .Results: The 10% inhibitory concentration(IC10) as to Bel-7402 and Bel-FU cells were 59.2、58.2μM ，and IC50 were226.7、193.9μM respectively。16.5、33.0、49.5 μM Quercetin reversed the MDR by1.14、1.68、2.38 folds respectively . Compared with those in Bel-7402, and the gene expression of H-ras，Bcl-2，PDGFA，MDR1，BCRP were up-regulated （P＜0.05），The gene expression of H-ras、EGFR、FGF、VEGFA、PDGFA、MDR1、Erk1 were down-regulated in Bel-FU treated with Quercetin（P＜0.05），the real-time quantitative PCR and western-blot comfirmed that gene H-ras，mdr1 and P-gp expression were were down-regulated by Quercetin，they were in accordanc with the result of q-PCR microarray . Conclusion: Quercetin can reverse the up-regulated expression of difference gene in resistant cell line so as to reverse the MDR of human HCC cells.

Key words: Quercetin, q-PCR microarray, hepatocellular carcinoma, multidrug resistance