EGCG、Taurine和Genistein联合抗大鼠肝纤维化作用的关键蛋白验证研究

发布时间：2018-08-19 14:08

【摘要】：目的:以表没食子儿茶素没食子酸酯(EGCG)、牛磺酸(Taurine)和三羟基异黄酮(Genistein)三种具有不同抗纤维化作用的药物组成联合药物干预CC14诱导的肝纤维化大鼠及肝星状细胞HSC-T6后,对前期用i TRAQ标记联合质谱的技术筛选和鉴定出肝星状细胞、大鼠肝组织及血清中差异表达的9个蛋白质Mst4、CDK4、Fgb、Tpi1、FVII、Hdgf、Glud 1、Lamp1、Cathepsin D进行深入的体内外验证;并从中选出感兴趣的蛋白FVII,构建稳定慢病毒载体对HSC-T6中FVII进行基因干扰,筛选到稳转株后,采用联合药物干预,观察细胞行为学变化,以对FVII蛋白在肝纤维化发生发展中的功能进行更深一步的研究。研究结果以期验证联合用药抗肝纤维化作用的关键蛋白,探讨联合用药抗肝纤维化作用的机理,为肝纤维化的药物筛选及早期诊断提供帮助。方法:1.关键蛋白的体内外验证研究:(1)以CC14诱导大鼠肝纤维化模型,在造模成功后给予EGCG、Taurine和Genistein组成的联合药物治疗6周,采集大鼠血清和肝组织,HE染色观察肝组织病理变化。(2)以HSC-T6细胞株为对象,应用联合药物对其进行干预后收集细胞。(3)提取肝组织及细胞的RNA和蛋白质,采用Western blot和RT-qPCR技术分别在mRNA和蛋白水平对9个可能的关键蛋白进行验证,并用ELISA法测定血清中FVII的含量。2.关键蛋白FVII的功能分析:(1)运用RNAi技术构建并筛选出稳定过表达FVII的慢病毒载体,构建FVII基因过表达的稳转细胞株。(2)CCK-8法检测过表达FVII的HSC-T6增殖情况,分析FVII对HSC-T6增殖的影响。(3)联合药物干预过表达FVII的HSC-T6,CCK-8法检测各组细胞的药物抑制率,分析fvii过表达后对联合药物干预hsc-t6的增殖影响。(4)流式细胞术检测联合药物干预的hsc-t6亲本株、fvii过表达hsc-t6细胞株的细胞周期变化,分析fvii过表达后对联合药物干预hsc-t6的细胞周期的影响。结果:1.关键蛋白的体内外验证结果:(1)给药六周后,he染色结果显示,联合用药组能减轻大鼠纤维化程度。(2)westernblot检测发现,联合用药干预后,与模型组相比,大鼠肝组织fⅦ、cdk4、hdgf蛋白表达量显著降低;与对照组相比,药物组肝星状细胞中lamp1、mst4、fⅦ蛋白表达量降低,tpi1蛋白表达量升高,差异有统计学意义。(3)real-timeqpcr检测发现,与模型组相比,联合用药组大鼠肝组织中lamp1、fvii、hdgf、glud1的表达量降低,差异有统计学意义。与对照组相比,联合用药组细胞中fvii的mrna表达水平在用药后降低,fgb、tpi1的mrna表达量升高,差异有统计学意义。(4)elisa结果显示,与模型组相比,药物干预6周后联合用药组大鼠血清中fvii含量降低,差异有统计学意义。2.关键蛋白fvii的功能分析结果:(1)cck-8法绘制hsc-t6亲本株、nc株及fⅦ过表达株细胞的生长曲线,发现fⅦ过表达后,肝星状细胞增殖加快。(2)在fⅦ过表达hsc-t6细胞株中,高剂量药物对细胞增殖抑制显著,低、中剂量组药物对细胞增殖抑制不显著;在hsc-t6亲本株与nc株中,中、高剂量药物对细胞抑制显著;低剂量药物对细胞抑制不显著,说明fⅦ的过表达,阻碍了联合用药抑制hsc-t6的增殖。进一步分析发现,相同浓度的联合药物对hsc-t6株及nc株的抑制率高于lv-fⅦ株,且中剂量药物组差异有统计学意义(p0.01),从而表明在联合用药抗肝纤维化作用中,fⅦ可能降低了联合用药抗肝纤维化的效果。(3)流式细胞仪检测发现,不同浓度药物处理hsc-t6亲本株24h后,细胞的周期发生了变化,与对照组相比,随着药物浓度增加,s期百分比升高,g1期百分比下降,而g2期百分比没有显著改变;中浓度药物组与高浓度药物组细胞与对照组相比,s期及G1期百分比变化有统计学差异,P0.05,低浓度药物组与对照组相比细胞周期各期百分比无明显改变。应用不同浓度药物处理过表达株LV-FⅦ24h后,细胞周期发生了变化,与对照组相比,随着药物浓度增加,S期百分比升高,G2期百分比均下降,G1期百分比变化没有显著差异;中浓度药物组与高浓度药物组细胞与对照组相比,S期及G2期百分比变化有统计学差异,P0.05,低浓度药物组与对照组相比细胞周期各期百分比无明显改变,表明联合药物处理后肝星状细胞增殖被抑制,细胞周期被阻滞于S期。结论:1.应用Western blot和RT-qPCR技术分别在细胞和动物水平对联合用药抗肝纤维化作用的9个候选关键蛋白进行了mRNA和蛋白验证,其中Fgb、Tpi1、CDK4、Mst4、FⅦ的基因和蛋白差异表达水平与前期的研究结果一致,可能为联合用药抗肝纤维化作用的关键蛋白。我们推测联合用药可能是通过调控Fgb、Tpi1、CDK4、Mst4、FⅦ等关键蛋白的表达,从而影响溶酶体、糖酵解、抑制DNA复制和有丝分裂、抗氧化防御系统以及凝血级联反应等多重细胞信号途径起到抗纤维化作用。2.依据验证结果选择FⅦ进一步对其在联合用药抗肝纤维化作用的功能进行分析后,发现FVII过表达后,肝星状细胞增殖被促进。过表达FⅦ会降低肝星状细胞对联合用药的敏感性,能减轻药物对肝星状细胞的抑制,推测可能FⅦ起到药效的负调控的作用。联合药物干预24h后,亲本株及FⅦ过表达株的细胞周期都受到了抑制,推断联合用药可能是通过将细胞周期阻滞于S期,抑制HSC的增殖。相同浓度的药物对FⅦ过表达前后的HSC-T6细胞周期的干预没有明显差异,说明FⅦ可能不是通过调控细胞周期来影响细胞对药物的反应,具体机制还需深入探索。
[Abstract]:OBJECTIVE: To screen and identify hepatic stellate cells (HSC-T6) and CC14-induced hepatic fibrosis in rats with hepatic stellate cells (HSC-T6) treated by epigallocatechin gallate (EGCG), taurine (Taurine) and trihydroxy isoflavone (Genistein). Differentially expressed 9 proteins Mst4, CDK4, Fgb, Tpi1, FVII, Hdgf, Glud 1, Lamp1, Cathepsin D in stellate cells, rat liver tissues and serum were further validated in vitro and in vivo, and interesting proteins FVII were selected from them to construct stable lentiviral vector to interfere with FVII gene in HSC-T6. To observe the changes of cellular behavior and further study the function of FVII protein in the occurrence and development of hepatic fibrosis. The results were expected to verify the key proteins of anti-hepatic fibrosis effect of combination therapy, and to explore the mechanism of anti-hepatic fibrosis effect of combination therapy, so as to provide help for drug screening and early diagnosis of hepatic fibrosis. In vitro and in vivo validation of the key proteins: (1) CC14-induced rat liver fibrosis model, after successful modeling, the rats were treated with EGCG, Taurine and Genistein for 6 weeks. Serum and liver tissues were collected, and the pathological changes of liver tissues were observed by HE staining. (2) HSC-T6 cell line was used as the object, and the combined drugs were used to interfere with it. Collector cells. (3) RNA and protein were extracted from liver tissues and cells. Nine possible key proteins were identified at mRNA and protein levels by Western blot and RT-q PCR. Serum FVII levels were determined by ELISA. Functional analysis of key protein FVII was carried out. (1) Slow over-expression of FVII was constructed and screened by RNAi technique. (2) The proliferation of HSC-T6 overexpressing FVII was detected by CCK-8 method, and the effect of FVII on the proliferation of HSC-T6 was analyzed. (3) The drug inhibition rate of HSC-T6 overexpressing FVII was detected by CCK-8 method, and the effect of FVII overexpressing on the proliferation of HSC-T6 was analyzed. 4) Flow cytometry was used to detect t he cell cycle changes of HSC-T6 parental strains which were interfered with drugs. The effect of FVII overexpression on t he cell cycle of HSC-T6 was analyzed. Results: 1. The results of in vitro and in vivo validation of t he key proteins: (1) After six weeks of administration, HE staining showed that t he combination group could significantly reduce t he cell cycle. (2) Western blot showed that the expression of f_, cdk4, HDGF protein in rat liver tissue was significantly lower than that in model group after treatment, while the expression of lamp1, mst4, f_protein in hepatic stellate cells was significantly lower and the expression of tpi1 protein was significantly higher in drug group than that in control group. (3) Real-time qPCR assay Compared with the model group, the expression of lamp-1, fvii, hdgf, glud-1 in the liver tissue of rats in the combined treatment group was significantly lower than that in the control group. compared with the control group, the expression of FVII mRNA in the cells of the combined treatment group was decreased, and the expression of fgb, tpi-1 mRNA was increased, the difference was statistically significant. (4) ELISA results showed that compared with the model, the expression of FVII mRNA in the cells of the combined treatment group was decreased. Compared with the control group, the content of FVII in the serum of rats in the combined treatment group decreased after 6 weeks of drug intervention, and the difference was statistically significant. 2. functional analysis of the key protein fvii: (1) the growth curve of HSC-T6 parental strain, NC strain and f_overexpression strain was drawn by CCK-8 method, and it was found that the proliferation of hepatic stellate cells was accelerated after f_overexpression. (2) the fine expression of HSC-T6 in f_was observed. In cell lines, high-dose drugs inhibited cell proliferation significantly, while low-dose drugs inhibited cell proliferation insignificantly; in HSC-T6 parental and NC strains, high-dose drugs inhibited cell proliferation significantly; low-dose drugs inhibited cell proliferation insignificantly, suggesting that the over-expression of f_inhibited the proliferation of hsc-t6. It was found that the inhibitory rate of the same concentration of combination drugs on HSC-T6 and NC strains was higher than that of lv-f_strains, and the difference was statistically significant in the middle dose group (p0.01). Thus, in the anti-hepatic fibrosis effect of combination drugs, f_may reduce the anti-hepatic fibrosis effect of combination drugs. (3) Flow cytometry detection found that different concentrations of drug treatment. Compared with the control group, with the increase of drug concentration, the percentage of S phase increased, the percentage of G1 phase decreased, but the percentage of G2 phase did not change significantly. Compared with the control group, the percentage of S phase and G1 phase changed significantly in the middle concentration group and the high concentration group, P 0.05, the percentage of low concentration group. Compared with the control group, the percentage of S phase increased, that of G2 phase decreased, and that of G1 phase did not change significantly. Compared with the control group, the percentage changes of S and G2 phases were statistically significant. P 0.05, there was no significant change in the percentage of cell cycle in the low concentration group compared with the control group, indicating that the proliferation of hepatic stellate cells was inhibited and the cell cycle was blocked in S phase after treatment with combined drugs. CR technique was used to verify the mRNA and protein expression of nine candidate key proteins of anti-hepatic fibrosis effect of combination therapy at the cellular and animal levels. The differential expression levels of Fgb, Tpi1, CDK4, Mst4 and F genes and proteins were consistent with previous studies, which may be the key proteins of anti-hepatic fibrosis effect of combination therapy. The combination of Fgb, Tpi1, CDK4, Mst4, F may play an anti-fibrosis role by regulating the expression of key proteins such as Fgb, Tpi1, CDK4, Mst4, F, thus affecting lysosome, glycolysis, inhibiting DNA replication and mitosis, antioxidant defense system and coagulation cascade reaction. After analyzing the function of anti-hepatic fibrosis, it was found that the proliferation of hepatic stellate cells was promoted after overexpression of FVII. Overexpression of F could reduce the sensitivity of hepatic stellate cells to combination therapy, and alleviate the inhibition of drug on hepatic stellate cells. It was speculated that F might play a negative role in regulating the efficacy of the drug. The cell cycle of HSC-T6 overexpressed strains was inhibited, and it was concluded that the combination therapy might inhibit the proliferation of HSC by blocking the cell cycle at S phase. The specific mechanism needs further exploration.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2

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