细胞色素酶P4502E1介导的氧化应激对人肝星状细胞的活化作用

摘要

目的 研究细胞色素酶P4502E1(CYP2E1)介导的氧化应激对人肝星状细胞的活化作用. 方法 以CYP2E1为目的 基因,将CYP2E1表达载体PCI-CYP2E1及空载体PCI-neo转染到人肝癌细胞株HepG2细胞,分别为HepG2/CYP2E1,HepG2/PCI.检测HepG2/CYP2E1,HepG2/PCI及正常HepG23组细胞上清液中丙二醛(MDA)含量.将上述3种细胞分别与人肝星状细胞LX2共培养48 h,分别命名为CYP2E1/LX2组,PCI/LX2组,HepG2/LX2组,提取RNA、上清液,用羟脯氨酸(Hyp)试剂盒检测3组LX2上清液中Hyp含量,用逆转录多聚酶链反应检测LX2细胞内Ⅰ型胶原、基质金属蛋白酶2(MMP2)的mRNA水平,用酶联免疫吸附试验检测3组上清液中LX2分泌的Ⅰ型前胶原羧基肽(PICP)蛋白水平,用明胶酶谱法检测3组上清液中LX2分泌的MMP2酶活性.组间比较用单因素方差分析进行统计学分析. 结果 (1)HepG2/CYP2E1细胞、阴性对照组HepG2细胞及HepG2/PCI细胞的MDA值分别为(6.51±0.25)nmol/ml、(3.07±0.29)nmol/ml和(2.57±0.29)nmol/ml,F值为22.66,P值均<0.01.(2)HepG2/CYP2E1、HepG2、HepG2/PCI 3组细胞培养液中Hyp含量分别为(35.24±3.52)nmol/ml、(24.50±1.37)nmol/ml和(17.77±2.58)nmol/ml,F值为58.89,P值均<0.01; 3组LX2细胞Ⅰ型胶原mRNA水平表达,差异无统计学意义.CYP2E1/LX2组、阴性对照组HepG2组及PCI/LX2组LX2分泌的PICP蛋白吸光度值分别为540.01±11.38、262.57±15.61和231.59±12.76,F值为124.97,P值均<0.01; 3组LX2细胞内MMP2 mRNA水平表达、MMP2的酶活性,差异无统计学意义. 结论 CYP2E1可引起氧化应激,CYP2E可增加LX2细胞外Hyp的合成和分泌.CYP2E1活化了肝星状细胞,可促进其细胞外PICP合成与分泌.%Objective To explore the stimulation of human hepatic stellate cells by Cytochrome P4502El-mediated oxidative stress. Methods HepG2-1ine was transfected with hunman CYP2E1 plasmid (HepG2/CYP2E1) and empty plasmid (HepG2/PCI) respectively. The CYP2E1 expression was evaluated with RT-PCR and Western blot. MDA was measured in culture medium of HepG2 cell lines. LX2 was coincubatied with HepG2/CYP2E1, HepG2/PCI and HepG2 respectively. The level of hydroxyproline in culture medium was examined in 48 hours and the cells were lysated and total RNA and protein were extracted.COL-1 and MMP2 mRNA levels were detected by RT-PCR and analyzed semi-quantitatively. PICP proteins were measured by ELISA. Zymography was performed to investigate MMP2 enzymatic activities. Results(1) MDA from the HepG2 which (HepG2/CYP2E1)express human CYP2E1 (6.51±0.25) was significantly higher than that from the HepG2 which do not (HepG2/PCI)express human CYP2E1 (3.07 ± 0.29)and HepG2 alone (2.57 ± 0.29). (F = 22.66, all P < 0.01). (2) After co-incubatied for 48 hours,the level of hydroxyproline in culture medium (35.24 ± 3.52) excreted from CYP2E 1/LX2 could significantly increase (F = 58.89,P < 0.01). PICP protein (540.01 ± 11.38) excreted from CYP2E1/LX2 was significantly increased (F =124.97, P < 0.01). Zymography showed MMP2 gene expression and enzymatic activities of MMP2 had no difference among the groups (F = 0.29, P > 0.05) (F = 0.33, P > 0.05). Conclusions CYP2E1 derived oxidative stress mediated stimulation of collagen I synthesis by hepatic stellate cells. Hydroxyproline excreted by LX2 was increased by CYP2E1. COL- 1 mRNA had no difference among the groups (F = 0.73, P > 0.05).