泛素水解酶22在同型半胱氨酸誘導(dǎo)膠原蛋白I表達(dá)中的作用研究
發(fā)布時(shí)間：2018-12-11 19:05
【摘要】：目的:高同型半胱氨酸血癥(Hyperhomocystinemia,HHCY)和肝臟纖維化、脂肪肝等肝臟疾病相關(guān)聯(lián);然而其內(nèi)在的機(jī)制還不為人們所了解。在本文中,我們主要研究泛素水解酶22(ubiquitin carboxyl—terminal hydrolase 22,USP22)基因在高同型半胱氨酸血癥誘導(dǎo)的肝臟纖維化中的表達(dá)及沉默USP22基因?qū)θ烁涡菭罴?xì)胞(Human hepatic stellate cell-LX2,HSC-LX2)周期蛋白的影響。方法:1.18只C57BL/6小鼠隨機(jī)分成3組:正常對(duì)照組(Con,喂養(yǎng)國(guó)家標(biāo)準(zhǔn)鼠飼料)、2%高蛋氨酸飼料喂養(yǎng)1周組、2%高蛋氨酸飼料喂養(yǎng)2周組;2.分別檢測(cè)每組中小鼠血漿中同型半胱氨酸(Homocysteine,HCY)的含量,選取2%高蛋氨酸飼料喂養(yǎng)2周組作為模型組(Model),Con組和Model組的肝臟組織采用HE、Massion染色,Western blot檢測(cè)肝臟組織中膠原蛋白I、USP22的蛋白表達(dá),免疫組化檢測(cè)膠原蛋白I、USP22的表達(dá)情況;3.在體外實(shí)驗(yàn)中,培養(yǎng)人肝星狀細(xì)胞LX2,用不同劑量的同型半胱氨酸(0,6μmol/L,12μmol/L,24μmol/L,48μmol/L)處理,且分別作用不同時(shí)間(0h,12h,24h,48h,72h);4.Western blot檢測(cè)不同劑量,不同濃度Hcy刺激下的人肝星狀細(xì)胞LX2(human hepatic stellate cell-LX2,HSC-LX2)膠原蛋白I、USP22的表達(dá)情況,選取Hcy刺激下的最佳作用時(shí)間和最佳作用濃度;5.轉(zhuǎn)染分組:正常對(duì)照組(未予處理的人肝星狀細(xì)胞)、Hcy刺激組(最佳濃度,最佳時(shí)間刺激下的人肝星狀細(xì)胞)、siRNA組(siRNA USP22沉默Hcy刺激組),siNC組(si RNA NC沉默Hcy刺激組);6.Western blot檢測(cè)各組中膠原蛋白I(Collagen I,COL I)、USP22、P53、P27、P21、CyclinB1蛋白的表達(dá)情況。結(jié)果:發(fā)現(xiàn)高蛋氨酸飼料喂養(yǎng)的小鼠和正常飼料喂養(yǎng)的小鼠相比,血漿中同型半胱氨酸的水平升高了,小鼠肝臟組織中膠原蛋白I、泛素水解酶22基因呈高表達(dá)狀態(tài)。在體外培養(yǎng)的人肝星狀細(xì)胞中,同型半胱氨酸刺激后,膠原蛋白I、USP22的表達(dá)呈劑量和時(shí)間依賴性,且敲除USP22基因后,可誘導(dǎo)其凋亡,并導(dǎo)致細(xì)胞周期被阻滯在G2期以內(nèi)。結(jié)論:1.2%高蛋氨酸飼料喂養(yǎng)的小鼠可以形成高同型半胱氨酸血癥;2.同型半胱氨酸可以誘導(dǎo)膠原蛋白I的表達(dá),導(dǎo)致肝臟纖維化的形成,其機(jī)制與USP22的高表達(dá)有關(guān)。3.USP22可能通過(guò)P53-P27-P21-CyclinB1通路促進(jìn)肝星狀細(xì)胞增殖,誘導(dǎo)肝臟纖維化。
[Abstract]:Aim: hyperhomocysteinemia (Hyperhomocystinemia,HHCY) is associated with liver diseases such as liver fibrosis, fatty liver, etc. In this paper, we studied the expression of ubiquitin hydrolase 22 (ubiquitin carboxyl-terminal hydrolase 22) gene in hyperhomocysteinemia induced hepatic fibrosis and the effect of silencing USP22 gene on (Human hepatic stellate cell-LX2, of human hepatic stellate cells. HSC-LX2) the effect of cyclin. Methods: 1.Eighteen C57BL/6 mice were randomly divided into three groups: normal control group (Con, feed), 2% high methionine diet for 1 week, 2% high methionine diet for 2 weeks, and 2% high methionine diet for 2 weeks. The content of homocysteine (Homocysteine,HCY) in plasma of each group was determined, and the liver tissues of (Model), Con group and Model group were fed with 2% high methionine diet for 2 weeks. The liver tissues were stained with HE,Massion. Western blot was used to detect the expression of collagen Igna USP22 in liver tissue, and immunohistochemistry was used to detect the expression of collagen Igna USP22. 3. In vitro, the cultured human hepatic stellate cells (LX2,) were treated with homocysteine (0 ~ 6 渭 mol/L,12 渭 mol/L,24 渭 mol/L,48 渭 mol/L) at different time (0 h, 12 h, 24 h, 48 h, 72 h). 4.Western blot was used to detect the expression of collagens Ignus P22 in human hepatic stellate cells (LX2 (human hepatic stellate cell-LX2,HSC-LX2) stimulated with different doses and different concentrations of Hcy. The optimal time and concentration of Hcy stimulation were selected. Transfection group: normal control group (untreated human hepatic stellate cell), Hcy stimulation group (optimal concentration, best time stimulation of human hepatic stellate cell), siRNA group (siRNA USP22 silencing Hcy stimulation group), siNC group (si RNA NC silencing Hcy stimulation group); The expression of collagen I (Collagen Icon I), USP22,P53,P27,P21,CyclinB1 protein was detected by 6.Western blot. Results: the levels of homocysteine in plasma of mice fed with high methionine diet were higher than that of mice fed with normal diet. The expression of collagen I and ubiquitin hydrolase 22 gene in liver tissue of mice showed high expression. In cultured human hepatic stellate cells, homocysteine stimulated the expression of collagen Ion-USP22 in a dose-and time-dependent manner, and after knockout of USP22 gene, it could induce apoptosis, resulting in cell cycle arrest within G2 phase. Conclusion: 1.2% high methionine diet could induce hyperhomocysteinemia in mice. Homocysteine can induce the expression of collagen I and the formation of hepatic fibrosis, which is related to the high expression of USP22. 3.USP22 may promote the proliferation of hepatic stellate cells and induce hepatic fibrosis through P53-P27-P21-CyclinB1 pathway.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2;R54
本文編號(hào)：2373069
[Abstract]:Aim: hyperhomocysteinemia (Hyperhomocystinemia,HHCY) is associated with liver diseases such as liver fibrosis, fatty liver, etc. In this paper, we studied the expression of ubiquitin hydrolase 22 (ubiquitin carboxyl-terminal hydrolase 22) gene in hyperhomocysteinemia induced hepatic fibrosis and the effect of silencing USP22 gene on (Human hepatic stellate cell-LX2, of human hepatic stellate cells. HSC-LX2) the effect of cyclin. Methods: 1.Eighteen C57BL/6 mice were randomly divided into three groups: normal control group (Con, feed), 2% high methionine diet for 1 week, 2% high methionine diet for 2 weeks, and 2% high methionine diet for 2 weeks. The content of homocysteine (Homocysteine,HCY) in plasma of each group was determined, and the liver tissues of (Model), Con group and Model group were fed with 2% high methionine diet for 2 weeks. The liver tissues were stained with HE,Massion. Western blot was used to detect the expression of collagen Igna USP22 in liver tissue, and immunohistochemistry was used to detect the expression of collagen Igna USP22. 3. In vitro, the cultured human hepatic stellate cells (LX2,) were treated with homocysteine (0 ~ 6 渭 mol/L,12 渭 mol/L,24 渭 mol/L,48 渭 mol/L) at different time (0 h, 12 h, 24 h, 48 h, 72 h). 4.Western blot was used to detect the expression of collagens Ignus P22 in human hepatic stellate cells (LX2 (human hepatic stellate cell-LX2,HSC-LX2) stimulated with different doses and different concentrations of Hcy. The optimal time and concentration of Hcy stimulation were selected. Transfection group: normal control group (untreated human hepatic stellate cell), Hcy stimulation group (optimal concentration, best time stimulation of human hepatic stellate cell), siRNA group (siRNA USP22 silencing Hcy stimulation group), siNC group (si RNA NC silencing Hcy stimulation group); The expression of collagen I (Collagen Icon I), USP22,P53,P27,P21,CyclinB1 protein was detected by 6.Western blot. Results: the levels of homocysteine in plasma of mice fed with high methionine diet were higher than that of mice fed with normal diet. The expression of collagen I and ubiquitin hydrolase 22 gene in liver tissue of mice showed high expression. In cultured human hepatic stellate cells, homocysteine stimulated the expression of collagen Ion-USP22 in a dose-and time-dependent manner, and after knockout of USP22 gene, it could induce apoptosis, resulting in cell cycle arrest within G2 phase. Conclusion: 1.2% high methionine diet could induce hyperhomocysteinemia in mice. Homocysteine can induce the expression of collagen I and the formation of hepatic fibrosis, which is related to the high expression of USP22. 3.USP22 may promote the proliferation of hepatic stellate cells and induce hepatic fibrosis through P53-P27-P21-CyclinB1 pathway.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2;R54
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 Maria Rosa Biagini;Alessandro Tozzi;Rossella Marcucci;Rita Paniccia;Sandra Fedi;Stefano Milani;Andrea Galli;Elisabetta Ceni;Marco Capanni;Raffaele Manta;Rosanna Abbate;Calogero Surrenti;;Hyperhomocysteinemia and hypercoagulability in primary biliary cirrhosis[J];World Journal of Gastroenterology;2006年10期
2 周秀敏;林菊生;孫雪梅;唐望先;張文英;袁順玉;艾莉;;肝硬化高同型半胱氨酸血癥與 MTHFR 基因 C667T 多態(tài)性的關(guān)系[J];中華肝臟病雜志;2005年12期
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