【作者】 李青；

【导师】 吴新华；

【作者基本信息】 中南大学 ， 妇产科， 2010， 硕士

【摘要】 目的：研究不同浓度17β雌二醇(E2)对L-02人正常肝细胞胆盐输出泵(BSEP)及钠离子牛磺酸共转运子(NTCP)表达的影响以及激活蛋白-1(c-jun/AP-1)抑制剂SP600125在雌激素调节肝细胞BSEP及NTCP表达中的作用。方法：体外培养的人正常肝细胞株L-02,用浓度为500nM的17β-雌二醇干预不同时间(6、12、18、24、30、36小时)；用不同浓度的17β-雌二醇(0.1,1.0,10,100,500,1000nmol/L)干预L-02肝细胞(其中0.1、1.0、10nmol/L为生理浓度)24h；以及不同浓度的SP600125 (5、10、20umon/l)与17β-雌二醇(500nmol/l)共同干预于L-02肝细胞,提取细胞总RNA并收集细胞培养上清液,采用RT-PCR法检测各组肝细胞BSEP与NTCP mRNA的表达情况,用生化方法检测各组培养液中胆汁酸浓度。结果：(1)随着E2作用时间延长,肝细胞BSEP mRNA及NTCP mRNA表达水平明显下降,各组间比较有显著差异性(P<0.05),且呈时间依赖性(R=-0.982,R=-0.959,P<0.05)；而培养液中胆汁酸浓度在6h、12h、18h、24h两两比较无差异性(P>0.05),30小时与24小时相比有差异性(P<0.05),36小时与30小时相比亦无明显差别(P>0.05)；(2)生理浓度的17β-雌二醇(0.1、1.0、10nmol/L)不影响肝细胞BSEP mRNA及NTCP mRNA的表达及胆汁酸的分泌,与空白对照组相比无显著差异性(P>0.05)；而高浓度的E2下调BSEP及NTCP表达(P<0.05),并使肝细胞分泌胆汁酸减少(P<0.05),呈剂量依赖关系(R=-0.884,R=-0.868, P<0.05),且在500nmol/l时抑制作用最明显。(3)c-jun/AP-1抑制剂SP600125可抑制AP-1的活性,明显抑制雌激素下调BSEP及NTCP表达,而使BSEP及NTCP mRNA表达升高,培养液中胆汁酸水平升高,与对照组相比有显著性差异(P<0.05, P<0.05, P<0.05),且呈剂量依赖关系(R=0.643, R=0.539, R=0.693, P<0.05)。结论：生理浓度雌激素不影响肝细胞BSEP及NTCP的表达,而高于生理浓度的雌激素可在转录水平下调肝细胞BSEP及NTCP的表达,其抑制效应有量效相关性；AP-1抑制剂SP600125明显抑制雌激素下调BSEP及NTCP表达,使BSEP及NTCP的mRNA表达增多,胆汁酸的分泌亦增加,其抑制效应亦有量效相关性,提示AP-1可能参与了雌激素下调BSEP及NTCP mRNA的表达。更多还原

【Abstract】 Objective:To study the effect of different concentration level of 17(3 estradiol on the expression of Bile salt export pump (BSEP) and Na+-taurocholatecotransporting polypeptide (NTCP) in L-02 normal human liver cells, as well as the effect of c-jun/AP-1 inhibitor SP600125 on the expression of BSEP and NTCP role in estrogen-regulated liver cell.Methods:Culture human liver cell line L-O2, with 17β-estradiol of 500nM respectively for 6hours,12 hours,18 hours,24 hours,30 hours and 36 hours, as well as with different concentration levels of 17βestradiol (0.1,1.0,10,100,500,1000 nmol/L) for 24h, among which 0.1,1.0,10 nmol/L are the physiological concentration; using RT-PCR to analyze BSEP and NTCP mRNA expression respectively in hepatic cells,then adopt biochemical methods to detect bile acid secretion of liver cells.Results:As the action of 17β-estradiol prolonged,the expression of BSEP and NTCP mRNA was decreased, in each group had significant differences (P<0.05), while bile acid concentration in culture medium at 6 hours,12 hours,18 hours 24h, were no difference (P> 0.05),30 hours and 24 hours difference compared (P<0.05),36 hours and 30 hours showed no significant difference (P> 0.05); Physiological concentration levels of 17(3-estradiol (0.1,1.0,10 nmol/L) does not affect BSEP and NTCP expression and bile acid secretion in liver cells, with no significant difference compared to the control group (P> 0.05); high concentration levels of 17β-estradiol could down regulate the expression of BSEP and NTCP, and up regulate bile acid concentration (P<0.05, P <0.05, P<0.05), which proves to be dose-dependent; JNK inhibitor SP600125 could inhibit AP-1 activity, significantly affecting the down-regulation of estrogen on the expression of BSEP and NTCP. As a result, the level of BSEP and NTCP mRNA expression and bile acid are up-regulated, which are dose-dependent and significantly different compared with the control group.(P<0.05, P<0.05, P<0.05).Conclusions:Physiological concentration levels of 17β-estradiol does not affect BSEP and NTCP expression in liver cells, while concentration above the physiological level can down regulate the expression, which is dose-dependent;AP-1 inhibitor SP600125 can significantly down regulate BSEP and NTCP expression,which is also dose-dependent.更多还原