泛素水解酶22在同型半胱氨酸诱导胶原蛋白I表达中的作用研究

发布时间：2018-12-11 19:05

【摘要】：目的:高同型半胱氨酸血症(Hyperhomocystinemia,HHCY)和肝脏纤维化、脂肪肝等肝脏疾病相关联;然而其内在的机制还不为人们所了解。在本文中,我们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症诱导的肝脏纤维化中的表达及沉默USP22基因对人肝星状细胞(Human hepatic stellate cell-LX2,HSC-LX2)周期蛋白的影响。方法:1.18只C57BL/6小鼠随机分成3组:正常对照组(Con,喂养国家标准鼠饲料)、2%高蛋氨酸饲料喂养1周组、2%高蛋氨酸饲料喂养2周组;2.分别检测每组中小鼠血浆中同型半胱氨酸(Homocysteine,HCY)的含量,选取2%高蛋氨酸饲料喂养2周组作为模型组(Model),Con组和Model组的肝脏组织采用HE、Massion染色,Western blot检测肝脏组织中胶原蛋白I、USP22的蛋白表达,免疫组化检测胶原蛋白I、USP22的表达情况;3.在体外实验中,培养人肝星状细胞LX2,用不同剂量的同型半胱氨酸(0,6μmol/L,12μmol/L,24μmol/L,48μmol/L)处理,且分别作用不同时间(0h,12h,24h,48h,72h);4.Western blot检测不同剂量,不同浓度Hcy刺激下的人肝星状细胞LX2(human hepatic stellate cell-LX2,HSC-LX2)胶原蛋白I、USP22的表达情况,选取Hcy刺激下的最佳作用时间和最佳作用浓度;5.转染分组:正常对照组(未予处理的人肝星状细胞)、Hcy刺激组(最佳浓度,最佳时间刺激下的人肝星状细胞)、siRNA组(siRNA USP22沉默Hcy刺激组),siNC组(si RNA NC沉默Hcy刺激组);6.Western blot检测各组中胶原蛋白I(Collagen I,COL I)、USP22、P53、P27、P21、CyclinB1蛋白的表达情况。结果:发现高蛋氨酸饲料喂养的小鼠和正常饲料喂养的小鼠相比,血浆中同型半胱氨酸的水平升高了,小鼠肝脏组织中胶原蛋白I、泛素水解酶22基因呈高表达状态。在体外培养的人肝星状细胞中,同型半胱氨酸刺激后,胶原蛋白I、USP22的表达呈剂量和时间依赖性,且敲除USP22基因后,可诱导其凋亡,并导致细胞周期被阻滞在G2期以内。结论:1.2%高蛋氨酸饲料喂养的小鼠可以形成高同型半胱氨酸血症;2.同型半胱氨酸可以诱导胶原蛋白I的表达,导致肝脏纤维化的形成,其机制与USP22的高表达有关。3.USP22可能通过P53-P27-P21-CyclinB1通路促进肝星状细胞增殖,诱导肝脏纤维化。
[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.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2;R54

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