肝癌组织OATP1B1的表达与调控对其介导药物转运功能及药物疗效影响的研究

OATP1B1, expressed specificly in liver, plays an important role in transport of drugs and clinical treatment, but its expression, function and the effect on drug treatment in hepatocellular carcinoma tissue have not been knowned. Our previous studies have shown that OATP1B1 gene mutation could significantly affect the healthy population or normal cell pharmacokinetic characteristics. Simultaneously, we preliminarily found that the pharmacokinetics of sorafenib (a substrate of OATP1B1) in rats were significantly altered in the pathological conditions of hepatocellular carcinoma. Therefore, we speculate that the gene mutation may significantly decrease the expression of OATP1B1 in hepatocarcinoma tissues, transport function and therapeutic effect in hepatocellular carcinoma tissue. This project firstly intends to explore the expression of OATP1B1*1a/*1b/*15 in hepatocarcinoma patients, and effect of gene mutation on OATP1B1-mediated drug transport characteristics and tumor suppressor effect in hepatocarcinoma cells. Then regulating the expression of OATP1B1 gene in hepatocellular carcinoma cells by LncRNA, so as to investigate the changes of its uptake and transport function, and its-mediated drug tumor suppressor effect. Finally, up-regulation the expression of Oatp2 in hepatocellular carcinoma rat model to explore the drug pharmacokinetic changes and its therapeutic effects on hepatocellular carcinoma. This project will provides new ideas for the effective treatment of hepatocellular carcinoma with targeted drugs , which has important clinical value.

OATP1B1特异表达于肝细胞，对药物的肝脏转运及临床治疗具有重要作用，但其在肝癌组织中的表达、功能及对药物疗效的影响尚未探知。前期研究表明OATP1B1基因突变可显著影响健康人群或细胞的药动学特性；同时，我们预实验发现肝癌病理状况下，索拉菲尼（OATP1B1底物）在大鼠体内的药动学呈显著改变。据此，我们推测基因突变可能进一步加剧降低肝癌组织中OATP1B1的表达、转运功能及介导药物的疗效。本项目拟首先探究肝癌患者OATP1B1*1a、*1b及*15的表达情况、以及基因突变对肝癌细胞药物转运与抑癌效果的影响；然后通过LncRNA调控肝癌细胞中OATP1B1的基因表达，以考察其转运摄取功能的变化及其介导药物抑癌效果的影响；最后通过上调肝癌大鼠模型体内Oatp2的表达，以考察药物在大鼠肝癌模型中的药动学变化及其对肝癌治疗效果的变化。本课题将为肝癌靶向药物的有效治疗提供新思路，具有重要临床价值。

目的：本研究旨在探索OATP1B1在肝癌细胞中介导索拉非尼转运、摄取作用与特性，以及OATP1B1对索拉非尼的体内外药效学的影响。.方法：在HepG2、HepG2- oatp1b1 *1a、HepG2- oatp1b1 *1b、HepG2- oatp1b1 *15、LO2、LO2- oatp1b1 *1a、LO2- oatp1b1 *1b、LO2- oatp1b1 *15细胞模型中以及转染miR-148a模拟物的HepG2细胞模型中探索索拉非尼的的摄取与转运特性，同时观察索拉非尼对细胞活力和凋亡率的影响；最后建立肝癌大鼠模型，探讨调控Oatp2表达后索拉非尼的药代动力学和药效学特性。.结果：OATP1B1的表达变化和基因突变显著影响HepG2和LO2转基因细胞对索拉非尼的摄取，且HepG2细胞对索拉非尼的摄取高于LO2细胞。与HepG2对照组细胞相比，HepG2转基因细胞中的OATP1B1过表达显著增加其对索拉非尼的摄取；基因突变显著影响OATP1B1介导索拉非尼的摄取，与OATP1B1*1a-HepG2细胞相比，索拉非尼浓度分别为10和15μM时，基因突变显著降低了OATP1B1*1b-HepG2细胞和OATP1B1*15-HepG2细胞中索拉非尼的摄入量；与LO2对照组细胞相比，LO2转基因细胞中的OATP1B1过表达显著增加对索拉非尼的摄取；与非突变野生型相比较，基因突变也会减少索拉非尼在OATP1B1*1b-GFP-LO2细胞和OATP1B1*15-GFP-LO2细胞中的摄入量；同时，OATP1B1基因突变显著影响索拉非尼治疗后HepG2细胞的存活率和凋亡率。与对照组相比，miR-148a模拟物转染HepG2细胞后对索拉非尼的摄取减少而细胞活力增加，结果显示加入5、10和15μmol/L索拉非尼后，转染miR-148a后细胞内索拉非尼的积累分别减少46.1%、28.5%和52.5%，相应地，HepG2细胞的存活率分别提高了8.2%、12.1%和19.9%。PCN显著增加大鼠体内Oatp2的表达，并影响索拉非尼的药代动力学，结果显示实验组的消除半衰期和浓度-时间曲线下的面积显著高于对照组，分别增加了约76.28%和73.04%；同时实验组肿瘤邻近组织中的血管内皮生长因子水平和微血管密度显著降低，表明Oatp2表达增加可改善索拉非尼在大鼠肝癌模型中的治疗效果。

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