FGFR2在肝细胞中功能的研究

发布时间：2018-03-09 18:19

本文选题：胆汁酸　切入点：FXR　出处：《复旦大学》2012年硕士论文　论文类型：学位论文

【摘要】：第一部分FXR在肝细胞对FGFR2调控的研究核受体法尼醇受体(farnesoid X receptor, FXR)在胆汁酸反馈调节机制中起着重要作用。胆汁酸能够激活FXR继而直接促进寡核受体(short heterodimer partner-1, SHP)的转录活性抑制胆汁酸经典合成步骤的第一步限速酶7α-胆固醇羟化酶(cholesterol7a-hydroxylase, CYP7A1)的表达,从而抑制胆汁酸的合成。此外,FXR也可以通过直接调控纤维生长因子FGF15(鼠)/FGF19(人)的表达来调节CYP7A1介导的胆汁酸合成通路。本文首次发现纤维细胞因子受体(Fibroblast growth factor receptor2,FGFR2)能够被FXR调控表达。用FXR的激动剂处理Hep3B细胞以及小鼠原代肝细胞4h和24h后,FGFR2的mRNA都显著上升并且在处理24h后的小鼠原代肝细胞中检测到FGFR2蛋白水平也明显增加。在体内试验表明,灌胃喂食FXR激动剂WAY-362450一天和一周的C57BL/6J小鼠中肝脏中检测到FGFR2的mRNA的表达量增加,而一周后FGFR2的蛋白也有明显的升高。而在FXR-KO小鼠里,FGFR2的表达量并没有太大变化。但是荧光素酶试验在FGFR2基因启动子上并没有找到FXR转录结合位点,可能存在其他位置。综上所述,本研究发现,在肝细胞中胆汁酸激活的核受体FXR能够调控FGFR2的表达,可能影响胆汁酸代谢以及肝细胞增殖。进一步实验目前正在进行中。第二部分FGFR2及其配体FGF7在肝细胞中调控CYP7A1的研究胆汁酸在对营养物质的消化,吸收及转运过程中发挥重要作用。胆汁酸的稳态,对维持正常的生理功能具有重要意义。7α-胆固醇羟化酶(cholesterol7α-hydroxylase, CYP7A1)作为胆汁酸经典调控第一步限速酶,其转录活性能够被许多核受体以及细胞因子调控,进而调节胆汁酸的合成。纤维细胞生长因子7(Fibroblast growth factor7,FGF7)属于细胞生长因子家族,能够特异性的激活受体FGFR2,能够促进表皮细胞的生长,血管新生以及伤口愈合。有文献研究,肝急性损伤以及肝纤维化中激活的肝星状细胞能够分泌FGF7,促进肝细胞增殖与存活。本研究首先发现激活的肝星状细胞(hepatic stellate cell,HSC)分泌的FGF7能够抑制肝细胞中CYP7A1的表达水平。首先,在小鼠CC14诱导的肝纤维化模型中,观察到FGF7显著增加,其受体FGFR2没有变化,而CYP7A1表达降低。体外FGF7处理小鼠肝原代肝细胞以及肝癌细胞Hep3B,都能够显著抑制CYP7A1的表达,并且存在明显的浓度和时间依赖性。敲除Hep3B中的FGFR2,FGF7对CYP7A1的抑制效果被解除。加入JNK1/2抑制剂也能够废除该抑制作用；同时FGF7处理Hep3B也能明显检测到JNK1/2的磷酸化显著增加。进一步研究表明,人源肝星状细胞株LX2检测到能够分泌FGF7,其培液培养的Hep3B能够抑制其中CYP7A1的表达,而加入FGF7中和性抗体该抑制效果消失。以上结果提示,肝星状细胞分泌的FGF7能够通过FGFR2及下游的JNK1/2途径抑制CYP7A1的表达。本文发现了在肝纤维化病理条件下一条胆汁酸合成调控的新途径,这对抑制胆汁酸过高引起的毒性作用以及保护肝再生修复可能起到重要作用。
[Abstract]:Study on the regulation of FGFR2 by hepatocytes in part one of FXR
Nuclear receptor farnesoid X receptor (farnesoid X, receptor, FXR) plays an important role in the feedback regulation mechanism of bile acid. The bile acid can activate FXR and then directly promote the oligo nuclear receptor (short heterodimer partner-1, SHP) inhibited the transcription activity of bile acid classic synthesis the first step enzyme 7 alpha hydroxylase (cholesterol7a-hydroxylase, cholesterol CYP7A1) expression, thereby inhibiting the synthesis of bile acids. In addition, FXR can also be directly regulated by fibroblast growth factor FGF15 (rat) /FGF19 (people) to regulate the expression of bile acid mediated CYP7A1 synthesis pathway.
The paper found that the fiber cell factor receptor (Fibroblast growth factor receptor2, FGFR2) can be regulated by FXR. The expression of primary FXR agonist treatment of Hep3B cells and mouse 4H and 24h liver cells after FGFR2, mRNA and 24h were significantly increased in the treatment of primary liver cells in mice were detected in the protein levels of FGFR2 increased significantly. In vivo tests showed that increased expression of FGFR2 mRNA detected the liver gavage feeding FXR agonist WAY-362450 one day and one week in C57BL/6J mice, and a week after the FGFR2 protein also increased significantly. In FXR-KO mice, the expression of FGFR2 did not change much but in the FGFR2 test. The luciferase gene promoter and found no transcription of FXR binding sites, there may be other positions.
In conclusion, this study found that bile acid activated nuclear receptor FXR can regulate the expression of FGFR2 in hepatocytes, which may affect bile acid metabolism and hepatocyte proliferation. Further experiments are in progress.
The study of second part FGFR2 and its ligand FGF7 in the regulation of CYP7A1 in liver cells
Bile acids on nutrient digestion, absorption and play an important role in the process of transportation. The steady state of bile acids,.7 has an important significance of alpha hydroxylase cholesterol to maintain normal physiological function (cholesterol7 -hydroxylase, CYP7A1) as the first step in regulating bile acid classic rate limiting enzyme, the transcriptional activity of nuclear receptors and can be many cytokine regulation and regulation of bile acid synthesis.
Fibroblast growth factor 7 (Fibroblast growth, factor7, FGF7) belongs to the fibroblast growth factor family, specifically activated receptor FGFR2, can promote skin cell growth, angiogenesis and wound healing. The present study, the activation of hepatic stellate cells in acute liver injury and liver fibrosis can promote liver cell proliferation and secretion of FGF7. And survival.
This paper found that the activation of hepatic stellate cells (hepatic stellate cell, HSC) expression of FGF7 can inhibit the secretion of CYP7A1 in liver cells. First of all, in the model of liver fibrosis induced by CC14 in mice, observed a significant increase of FGF7, its receptor FGFR2 did not change, but the expression of CYP7A1 decreased in FGF7 treated mice liver in vitro. Primary hepatocytes and hepatoma cells Hep3B expression can significantly inhibit CYP7A1, and there was concentration and time dependent manner. Knockdown of Hep3B in FGFR2. The inhibitory effect of FGF7 on CYP7A1 was released. The addition of the JNK1/2 inhibitor can abolish the inhibitory effect of FGF7 treatment at the same time; Hep3B also significantly detected JNK1/2 phosphorylation increased significantly. Further study showed that human hepatic stellate cell line LX2 can detect the secretion of FGF7, the culture liquid of cultured Hep3B can inhibit the expression of CYP7A1, while FGF7 neutralizing anti The effect of this inhibition was disappearing.
These results suggest that the secretion of hepatic stellate cells FGF7 can inhibit expression of CYP7A1 via JNK1/2 pathway and downstream of FGFR2. This paper found a new pathway in liver fibrosis under the condition of a bile acid biosynthesis, the inhibition of bile acid toxicity caused by excessive and protective liver regeneration may play an important role.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R333.4

【共引文献】