本文選題：膽汁酸　切入點(diǎn)：FXR　出處：《復(fù)旦大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分FXR在肝細(xì)胞對(duì)FGFR2調(diào)控的研究 核受體法尼醇受體(farnesoid X receptor, FXR)在膽汁酸反饋調(diào)節(jié)機(jī)制中起著重要作用。膽汁酸能夠激活FXR繼而直接促進(jìn)寡核受體(short heterodimer partner-1, SHP)的轉(zhuǎn)錄活性抑制膽汁酸經(jīng)典合成步驟的第一步限速酶7α-膽固醇羥化酶(cholesterol7a-hydroxylase, CYP7A1)的表達(dá),從而抑制膽汁酸的合成。此外,FXR也可以通過(guò)直接調(diào)控纖維生長(zhǎng)因子FGF15(鼠)/FGF19(人)的表達(dá)來(lái)調(diào)節(jié)CYP7A1介導(dǎo)的膽汁酸合成通路。 本文首次發(fā)現(xiàn)纖維細(xì)胞因子受體(Fibroblast growth factor receptor2,FGFR2)能夠被FXR調(diào)控表達(dá)。用FXR的激動(dòng)劑處理Hep3B細(xì)胞以及小鼠原代肝細(xì)胞4h和24h后,FGFR2的mRNA都顯著上升并且在處理24h后的小鼠原代肝細(xì)胞中檢測(cè)到FGFR2蛋白水平也明顯增加。在體內(nèi)試驗(yàn)表明,灌胃喂食FXR激動(dòng)劑WAY-362450一天和一周的C57BL/6J小鼠中肝臟中檢測(cè)到FGFR2的mRNA的表達(dá)量增加,而一周后FGFR2的蛋白也有明顯的升高。而在FXR-KO小鼠里,FGFR2的表達(dá)量并沒(méi)有太大變化。但是熒光素酶試驗(yàn)在FGFR2基因啟動(dòng)子上并沒(méi)有找到FXR轉(zhuǎn)錄結(jié)合位點(diǎn),可能存在其他位置。 綜上所述,本研究發(fā)現(xiàn),在肝細(xì)胞中膽汁酸激活的核受體FXR能夠調(diào)控FGFR2的表達(dá),可能影響膽汁酸代謝以及肝細(xì)胞增殖。進(jìn)一步實(shí)驗(yàn)?zāi)壳罢谶M(jìn)行中。 第二部分FGFR2及其配體FGF7在肝細(xì)胞中調(diào)控CYP7A1的研究 膽汁酸在對(duì)營(yíng)養(yǎng)物質(zhì)的消化,吸收及轉(zhuǎn)運(yùn)過(guò)程中發(fā)揮重要作用。膽汁酸的穩(wěn)態(tài),對(duì)維持正常的生理功能具有重要意義。7α-膽固醇羥化酶(cholesterol7α-hydroxylase, CYP7A1)作為膽汁酸經(jīng)典調(diào)控第一步限速酶,其轉(zhuǎn)錄活性能夠被許多核受體以及細(xì)胞因子調(diào)控,進(jìn)而調(diào)節(jié)膽汁酸的合成。 纖維細(xì)胞生長(zhǎng)因子7(Fibroblast growth factor7,FGF7)屬于細(xì)胞生長(zhǎng)因子家族,能夠特異性的激活受體FGFR2,能夠促進(jìn)表皮細(xì)胞的生長(zhǎng),血管新生以及傷口愈合。有文獻(xiàn)研究,肝急性損傷以及肝纖維化中激活的肝星狀細(xì)胞能夠分泌FGF7,促進(jìn)肝細(xì)胞增殖與存活。 本研究首先發(fā)現(xiàn)激活的肝星狀細(xì)胞(hepatic stellate cell,HSC)分泌的FGF7能夠抑制肝細(xì)胞中CYP7A1的表達(dá)水平。首先,在小鼠CC14誘導(dǎo)的肝纖維化模型中,觀察到FGF7顯著增加,其受體FGFR2沒(méi)有變化,而CYP7A1表達(dá)降低。體外FGF7處理小鼠肝原代肝細(xì)胞以及肝癌細(xì)胞Hep3B,都能夠顯著抑制CYP7A1的表達(dá),并且存在明顯的濃度和時(shí)間依賴性。敲除Hep3B中的FGFR2,FGF7對(duì)CYP7A1的抑制效果被解除。加入JNK1/2抑制劑也能夠廢除該抑制作用；同時(shí)FGF7處理Hep3B也能明顯檢測(cè)到JNK1/2的磷酸化顯著增加。進(jìn)一步研究表明,人源肝星狀細(xì)胞株LX2檢測(cè)到能夠分泌FGF7,其培液培養(yǎng)的Hep3B能夠抑制其中CYP7A1的表達(dá),而加入FGF7中和性抗體該抑制效果消失。 以上結(jié)果提示,肝星狀細(xì)胞分泌的FGF7能夠通過(guò)FGFR2及下游的JNK1/2途徑抑制CYP7A1的表達(dá)。本文發(fā)現(xiàn)了在肝纖維化病理?xiàng)l件下一條膽汁酸合成調(diào)控的新途徑,這對(duì)抑制膽汁酸過(guò)高引起的毒性作用以及保護(hù)肝再生修復(fù)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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R333.4

【共引文獻(xiàn)】

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