白藜芦醇对TNF-α诱导人类风湿关节炎成纤维样滑膜细胞增殖的影响和调节Wnt信号通路的研究

发布时间：2018-03-23 23:24

本文选题：类风湿关节炎　切入点：白藜芦醇　出处：《四川医科大学》2015年硕士论文

【摘要】：背景:类风湿关节炎(rheumatoid arthritis,RA)作为弥漫性结缔组织疾病之一,已成为世界范围内的健康问题,严重影响病人及其家人的生活质量及身心健康。RA是一种慢性系统性自身免疫性疾病,主要以关节滑膜为主要靶组织。介导关节破坏的主要细胞为成纤维样滑膜细胞,主要病理表现为滑膜组织异常增殖、滑膜下层炎症侵润,滑膜炎可引起关节中骨与软骨的持续损害,RA发病机制至今尚未完全明确。RA的发病与多种细胞因子相关,肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)是其中重要参与因子之一。目前,对RA的主要研究集中于滑膜细胞中异常的信号途径,Wnt信号通路在多种细胞增殖与分化方面有着关键调控作用,在RA滑膜炎症以及调节骨代谢过程中起重要作用,其中Wnt/β-catenin信号通路目前研究最为详尽,鉴于Wnt信号通路在RA的FLS中的重要调控作用,干预Wnt信号转导通路中的各个环节成为治疗RA的新靶点。白藜芦醇(resveratrol,Res)广泛存在于虎杖、花生及葡萄等植物当中,是多酚类化合物之一,具有抗氧化、抗衰老、抗炎症及保护心血管的作用,祖国医学发现虎杖根(主要有效成分为白藜芦醇)能治疗关节的炎症。近年研究者发现Res能诱导多种肿瘤细胞的凋亡,而RA中成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)的增殖与分化类似于肿瘤细胞的特性。就此,本课题以滑膜细胞为研究对象,研究TNF-α对成纤维样滑膜细胞的影响,从Wnt信号通路方面进一步研究白藜芦醇对成纤维样滑膜细胞作用可能的分子机制。目的:1.观察TNF-α对人类风湿关节炎成纤维样滑膜细胞(Human Fibroblast-like synoviocyte of rheumatoid arthritis,HFLS-RA)增殖的影响,以及观察不同浓度的白藜芦醇对TNF-α诱导的HFLS-RA增殖的影响。2.观察TNF-α对HFLS-RA中Wnt信号通路的调控,以及不同浓度的Res对TNF-α诱导的HFLS-RA的Wnt信号通路的影响。方法:1.复苏购买的HFLS-RA细胞株后,培养传代至第3-5代备用。2.采用TNF-α(10μg/l)处理HFLS-RA24h后,运用CCK8法观察TNF-α对HFLS-RA增殖的影响。3.不同浓度的Res(50umol/l、100umol/l、200umol/l、400umol/l)干预HFLS-RA后再用10μg/l的TNF-α刺激24h,用CCK8法测定Res对TNF-α诱导的HFLS-RA生长的影响。4.将浓度为10μg/l的TNF-α加入HFLS-RA中共同孵育30min,运用Western-blot检测其与空白对照组的s FRP4、β-catenin及C-myc蛋白的表达水平。5.将不同浓度的Res(50umol/l、100umol/l、200umol/l、400umol/l)与HFLS-RA共同孵育24h后,再用10μg/l的TNF-α刺激30min,用Western-blot检测Res对TNF-α诱导的HFLS-RA中s FRP4、β-catenin及C-myc蛋白表达的影响。结果:1.HFLS-RA由TNF-α诱导后,HFLS-RA增殖明显增强,不同浓度的Res(50umol/l、100umol/l、200umol/l、400umol/l)对TNF-α诱导后的HFLS-RA增殖有抑制作用,并呈剂量依赖性。2.经Western-blot检测发现TNF-α诱导后的HFLS-RA与空白对照组相比较,s FRP4的表达水平降低,β-catenin及C-myc的表达水平增高。(P0.05)3.与TNF-α组相比较,经Western-blot方法分析显示:不同浓度Res(50umol/l、100umol/l、200umol/l、400umol/l)组能降低TNF-α诱导后的HFLS-RA的β-catenin及C-myc的蛋白表达水平,s FRP4的表达水平增高。(P0.05)结论:1.TNF-α有促进HFLS-RA增殖的作用。2.Res能抑制TNF-α诱导HFLS-RA增殖的作用,并呈剂量依赖性。3.TNF-α能明显提高HFLS-RA的Wnt信号通路的活化水平。4.Res能够剂量依耐性的抑制TNF-α诱导后HFLS-RA中的Wnt信号通路的活化水平。
[Abstract]:Background: rheumatoid arthritis (rheumatoid arthritis, RA) as one of the diffuse connective tissue disease, has become a worldwide health problem, seriously affecting the quality of life of patients and their families and health.RA is a chronic systemic autoimmune disease, mainly in the synovial membrane as the main target tissue. The main cell mediated joint destruction for fibroblast like synovial cells, is characterized by abnormal proliferation of synovial tissue, suhsynovial inflammatory infiltration, synovitis can cause sustained damage to bone and cartilage joint, RA pathogenesis has not yet entirely clear and the incidence of.RA related cytokines, tumor necrosis factor alpha (tumor necrosis factor alpha, TNF- alpha) is one of the important factors involved in one. At present, the main research on the RA signal pathway in synovial cells, Wnt signaling pathway in a cell proliferation and A key role of differentiation, play an important role in the regulation of bone metabolism and RA synovitis of the Wnt/ beta -catenin signal pathway on the most detailed, in view of the important role of Wnt signaling pathway in RA FLS, each link of intervention in the Wnt signal transduction pathway has become a new target for treatment of RA. Resveratrol (resveratrol, Res) widely exists in Polygonum cuspidatum, peanut and grape plants, is one of the polyphenolic compounds with antioxidant, anti-aging, anti-inflammatory and cardiovascular protection effect, Chinese Medicine found the root of Polygonum cuspidatum (the main active ingredients of resveratrol can treat inflammation) in recent years, joint. Found that Res can induce apoptosis in various tumor cells, and RA in fibroblast like synoviocytes (fibroblast-like synoviocytes, FLS) the characteristics of proliferation and differentiation of tumor cells. It is similar to this, this topic in synovium Cell as the research object, research TNF- alpha on the proliferation of fibroblast like synovial cells, resveratrol on molecular mechanism of fibroblast like synoviocytes from Wnt signaling pathway may further study. Objective: 1. to observe the effect of TNF- on human rheumatoid arthritis synovial fibroblasts (Human Fibroblast-like synoviocyte of rheumatoid arthritis, HFLS-RA) proliferation and effect observation of different concentrations of resveratrol on TNF- induced proliferation of HFLS-RA.2. to observe the regulation of TNF- alpha on Wnt signaling pathway in HFLS-RA, and the effect of Wnt signaling pathway in different concentrations of Res on TNF- induced by HFLS-RA. Methods: 1. HFLS-RA cells were purchased after recovery. Train passage to the 3-5 generation of alternate.2. by TNF- alpha (10 g/l) after HFLS-RA24h treatment, using CCK8 method to observe the effect on proliferation of HFLS-RA TNF- alpha.3. of different concentrations of Res (50umol/l, 100umol/l, 20 0umol/l, 400umol/l and HFLS-RA) after the intervention of 10 g/l TNF- stimulated 24h, CCK8 method was used to determine the effect of.4. Res on TNF- induced growth of HFLS-RA concentration is 10 g/l TNF- for HFLS-RA with alpha join 30min, using Western-blot to detect the s and control group FRP4, -catenin and beta C-myc protein expression level of.5. in different concentration of Res (50umol/l, 100umol/l, 200umol/l, 400umol/l) HFLS-RA were incubated with 24h, and 10 g/l TNF- stimulated 30min on TNF- induced by Western-blot Res HFLS-RA s FRP4 detection, the expressions of -catenin and C-myc protein beta. Results: 1.HFLS-RA induced by TNF- alpha, HFLS-RA proliferation was significantly enhanced, different concentrations of Res (50umol/l, 100umol/l, 200umol/l, 400umol/l) inhibited TNF- alpha induced HFLS-RA proliferation in a dose-dependent manner.2. was detected on Western-blot induced by TNF- after H FLS-RA compared with the control group, the expression level of s decreased FRP4, increased the expression level of -catenin beta and C-myc. (P0.05) 3. and TNF- alpha group compared with the Western-blot method analysis showed that: different concentrations of Res (50umol/l, 100umol/l, 200umol/l, 400umol/l) expression levels of beta -catenin and C-myc group can reduce TNF- after the induction of HFLS-RA alpha protein, increased the expression level of s FRP4 (P0.05). Conclusion: 1.TNF- can promote the proliferation of HFLS-RA alpha.2.Res can inhibit TNF- induced HFLS-RA proliferation in a dose-dependent manner.3.TNF- can significantly improve the level of.4.Res alpha activation of Wnt signaling pathway to HFLS-RA high dose according to activation the level of Wnt signaling pathway inhibited TNF- induced tolerance in HFLS-RA.

【学位授予单位】：四川医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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