MeCP2对类风湿关节炎中PTCH1的调控作用及橙皮素衍生物XIV号抗炎机制的研究
发布时间：2018-09-05 16:03
【摘要】：类风湿性关节炎(rheumatoid arthritis,RA)属于炎症性的自身免疫疾病,其特点是滑膜组织的增厚,软骨与骨组织的破坏。成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)在发病过程中异常增殖,同时产生多种炎症因子。炎症因子IL~(-1),IL-6,IL-8,TNF-α,基质金属蛋白酶和胶原酶形成了一个复杂的网络,造成关节损伤与病变。RA具有较高的发病率和致残率,发病原因与致病机理仍不可知,不仅严重影响了人们的日常生活,引起病人的不适,而且严重者会丧失劳动能力。因此,寻找能有效治疗RA的疗法,成为一个亟待解决的问题。甲基化是表观遗传学修饰的一种,通过抑制转录来调控蛋白的表达。研究发现RA发病的进程中伴随着特定基因甲基化的过程。甲基化Cp G结合蛋白2(Methylated Cp G binding protein 2,MeCP2)是MBD家族的成员,可以特异性的识别和绑定在甲基化的DNA序列上,从而抑制基因的表达。有研究发现MeCP2在RA中高表达,参与调控特定基因的甲基化。文献报道,Hedgehog信号通路在组织修复及持续性的炎症中发挥重要作用,Patch1(PTCH1)在此通路中是一个负调控基因,在多种癌症中,其启动子发生高甲基化,蛋白的表达下降,而PTCH1在RA中如何改变,还不明确。目前甾体抗炎药,非甾体抗炎药,免疫抑制剂以及糖皮质激素等用于治疗RA,但是具有毒性和副作用。橙皮苷(hesperidin,HDN)属于二氢黄酮苷类化合物,本实验室前期的研究结果表明,HDN具有较强的抗炎活性,抗氧化活性,对佐剂性关节炎大鼠(Adjuvant Arthritis,AA)具有治疗作用。但是由于其半衰期较短,水溶性较差,口服给药后失效过快,限制了其在临床上的应用。目前国内外大多侧重于对HDN的结构改造,通过对其结构的改造和修饰,明显改善了其半衰期,水溶性及稳定性。本实验室针对HDN的结构改造和修饰,得到了一系列的橙皮素衍生物(Hesperidin derivatives,HDND),并且进行了抗炎活性的筛选。发现橙皮素衍生物XIV号,即5-(4-氯苯乙基)亚胺基-7-氧-乙酰-4-氯苯乙胺基橙皮素(HDND-XIV)具有较好的抗炎活性。本课题选用与RA具有相似病理特征和免疫学变化的AA大鼠为动物模型,以FLS为细胞研究对象。实验中采用甲基化抑制剂5-杂氮-2-脱氧胞苷(5-hybrid-4-deoxycytidine,5-Azad C)和基因沉默技术,研究MeCP2对PTCH1蛋白表达的调控和其对炎症因子的影响,同时离体观察HDND-XIV对FLS炎症的影响,及可能存在的机制。主要研究内容包括两部分:1.MeCP2对类风湿关节炎中PTCH1表达的调控及机制研究研究MeCP2对PTCH1的调控作用及机制。使用弗氏完全佐剂(Freund's complete adjuvant,FCA)诱导AA大鼠模型,正常组大鼠给予等量的PBS。造模24天后提取原代FLS。体外使用5-azadc作用于FLS,体外采用基因沉默技术沉默MeCP2,观察对PTCH1的调控以及以上对炎症指标的影响。采用HE染色,观察大鼠关节的病理变化,同时检测大鼠继发侧足肿胀度。免疫荧光,免疫组化检测MeCP2,PTCH1蛋白的表达,Western blot法检测MeCP2,PTCH1,Gli1和Shh蛋白的表达,酶联免疫吸附法(Enzyme linked immunosorbent assay,ELISA)检测相应炎症指标,甲基化特异性PCR(Methylation-Specific PCR,MSP)和焦磷酸测序检测甲基化程度。病理结果表明AA大鼠模型成立,膝关节明显病变与肿胀。免疫组化,免疫荧光,Western blot结果发现无论组织上还是细胞上,AA大鼠的MeCP2蛋白表达均升高,PTCH1蛋白表达均降低,同时AA FLS中Gli1,Shh表达升高,提示AA组Hedgehog信号通路激活。体外给予AA FLS 5-azadc后,相比较于AA组,细胞炎症指标均下降,细胞水平MeCP2,Gli1,Shh蛋白表达下降,PTCH1蛋白表达上升。MSP和焦磷酸测序显示AA FLS经5-azadc刺激后,PTCH1基因甲基化水平下降。细胞水平上成功沉默MeCP2后,PTCH1表达升高,并伴随着炎症指标的降低。提示,在类风湿关节炎中,MeCP2通过DNA甲基化来调控PTCH1的表达,为RA的防治与研究提供了新的靶点。2.橙皮素衍生物XIV号对成纤维滑膜细胞炎症的抑制作用及机制研究研究HDND-XIV对FLS炎症的影响及作用机制。使用FCA诱导AA大鼠模型,MTT法检测HDND-XIV的半抑制浓度(Half maximal inhibitory concentration,IC50),ELISA检测每组相应的炎症指标,Western blot法检测FLS中MeCP2,PTCH1,Gli1和Shh蛋白的表达,焦磷酸测序法定量检测甲基化程度。结果发现HDND-XIV的IC50值为161μmol·L~(-1),鉴于药物的毒性,在小于0.5倍IC50的范围里选择五个药物浓度(80μmol·L~(-1),40μmol·L~(-1),20μmol·L~(-1),10μmol·L~(-1),5μmol·L~(-1))加药刺激,结果发现药物浓度为10μmol·L~(-1)时抑制炎症的作用最强,然后以10μmol·L~(-1)为加药组浓度。AA FLS经HDND-XIV作用后,MeCP2,Gli1和Shh蛋白表达下降,PTCH1蛋白表达上升,焦磷酸测序结果显示其PTCH1基因的甲基化程度较AA FLS明显降低。在AA FLS上过表达MeCP2,之后加HDND-XIV刺激,结果显示成功过表达后,PTCH1蛋白表达下降,炎症指标上升。提示HDND-XIV抑制FLS的炎症,其作用机制可能是通过抑制MeCP2的表达,降低PTCH1基因的甲基化程度,最终下调炎性细胞因子的分泌。
[Abstract]:Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by thickening of synovial tissue, destruction of cartilage and bone tissue. Fibroblast-like synoviocytes (FLS) proliferate abnormally in the course of the disease and produce a variety of inflammatory factors. Inflammatory factors IL-1, IL-6, IL-8, TNF-like synoviocytes (FLS) produce a variety of inflammatory factors. A. Matrix metalloproteinases and collagenases form a complex network, resulting in joint injury and disease. RA has a high incidence and disability rate. The pathogenesis and pathogenesis of RA are still unknown. It not only seriously affects people's daily life, causes patients'discomfort, but also seriously loses their ability to work. Methylation is an epigenetic modification that regulates protein expression by suppressing transcription. Studies have found that the pathogenesis of RA is accompanied by the methylation of specific genes. Methylated Cp G binding protein 2 (MeCP2) is a member of the MBD family. MeCP2 is found to be highly expressed in RA and involved in the regulation of specific gene methylation. The Hedgehog signaling pathway has been reported to play an important role in tissue repair and persistent inflammation. Patch 1 (PTCH1) is a negative regulator in this pathway. Control genes, in a variety of cancers, its promoter hypermethylation, protein expression decreased, and PTCH1 in RA how to change, is not clear. Currently steroidal anti-inflammatory drugs, non-steroidal anti-inflammatory drugs, immunosuppressive agents and glucocorticoids used to treat RA, but has toxicity and side effects. Hesperidin (HDN) belongs to the dihydroflavonoid glycosides. Previous studies in our laboratory have shown that HDN has strong anti-inflammatory and antioxidant activities and has therapeutic effects on adjuvant arthritis rats (AA). The structure modification and modification of HDN have improved the half-life, water-solubility and stability of HDN. A series of hesperidin derivatives (HDND) have been obtained and their anti-inflammatory activities have been screened. 5-(4-chlorophenylethyl) imino-7-oxo-acetyl-4-chlorophenylethylamine hesperidin (HDND-XIV) has good anti-inflammatory activity. In this study, AA rats with similar pathological characteristics and immunological changes as RA were selected as animal models, and FLS was used as the research object. 5-hybrid-4-deoxyc-deoxycytidine (5-hybrid-4-deoxyc) was used as methylation inhibitor in the experiment. Ytidine, 5-Azad C) and gene silencing techniques were used to study the regulation of MeCP2 on PTCH1 protein expression and its effect on inflammatory factors. The effects of HDND-XIV on FLS inflammation and possible mechanisms were observed in vitro. The main contents of this study include two parts: 1. MeCP2 on the regulation and mechanism of PTCH1 expression in rheumatoid arthritis; 2. Regulation and mechanism of CH1. Freund's complete adjuvant (FCA) was used to induce AA rat model. Normal group rats were given the same amount of PBS. After 24 days of modeling, primary FLS was extracted. 5-azadc was used to treat FLS in vitro. MeCP2 was silenced by gene silencing technique in vitro. The regulation of PTCH1 and the above effects on inflammatory indexes were observed. The expression of MeCP2, PTCH1, Gli1 and Shh proteins was detected by Western blot, and the corresponding inflammatory markers were detected by enzyme linked immunosorbent assay (ELISA). Methylation-specific PCR (MSP) and pyrophosphatic acid sequencing were used to detect the degree of methylation. The expression of Gli1 and Shh in AA FLS decreased, suggesting that Hedgehog signaling pathway was activated in AA group. Compared with AA group, the expression of MeCP2, Gli1, Shh protein and PTCH1 protein were decreased, and the expression of PTCH1 protein was increased in AA FLS stimulated by 5-azadc. MSP and pyrophosphatic acid sequencing showed that PTCH1 gene methyl was activated by 5-azadc. After successful silencing of MeCP2 at the cellular level, the expression of PTCH1 increased with the decrease of inflammatory markers. It is suggested that in rheumatoid arthritis, MeCP2 regulates the expression of PTCH1 through DNA methylation, which provides a new target for the prevention and treatment of RA. 2. Inhibitory effect of hesperidin derivative XIV on inflammation of fibrosynoviocytes. To study the effect and mechanism of HDND-XIV on inflammation of FLS. FCA-induced AA rat model was used. Half maximum inhibitory concentration (IC50) of HDND-XIV was detected by MTT method, corresponding inflammation indexes were detected by ELISA, the expressions of MeCP2, PTCH1, Gli1 and Shh proteins in FLS were detected by Western blot, and pyrophosphatic acid sequencing was performed. The results showed that the IC50 value of HDND-XIV was 161 micromol (-1). In view of the toxicity of HDND-XIV, five drug concentrations (80 micromol (-1), 40 micromol (-1), 20 micromol (-1), 10 micromol (-1), 5 micromol (-1)) were selected to stimulate HDND-XIV. The results showed that the concentration of HDND-XIV was 10 micromol (-1)) and 5 micromol 65507 After treatment with HDND-XIV, the expression of MeCP2, Gli 1 and Shh protein decreased, and the expression of PTCH1 protein increased. Pyrophosphate sequencing showed that the methylation of PTCH1 gene was significantly lower than that of AA FLS. The results suggest that HDND-XIV can inhibit FLS inflammation by inhibiting the expression of MeCP2 and decreasing the methylation of PTCH1 gene, and ultimately down-regulating the secretion of inflammatory cytokines.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965
本文编号：2224766
[Abstract]:Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by thickening of synovial tissue, destruction of cartilage and bone tissue. Fibroblast-like synoviocytes (FLS) proliferate abnormally in the course of the disease and produce a variety of inflammatory factors. Inflammatory factors IL-1, IL-6, IL-8, TNF-like synoviocytes (FLS) produce a variety of inflammatory factors. A. Matrix metalloproteinases and collagenases form a complex network, resulting in joint injury and disease. RA has a high incidence and disability rate. The pathogenesis and pathogenesis of RA are still unknown. It not only seriously affects people's daily life, causes patients'discomfort, but also seriously loses their ability to work. Methylation is an epigenetic modification that regulates protein expression by suppressing transcription. Studies have found that the pathogenesis of RA is accompanied by the methylation of specific genes. Methylated Cp G binding protein 2 (MeCP2) is a member of the MBD family. MeCP2 is found to be highly expressed in RA and involved in the regulation of specific gene methylation. The Hedgehog signaling pathway has been reported to play an important role in tissue repair and persistent inflammation. Patch 1 (PTCH1) is a negative regulator in this pathway. Control genes, in a variety of cancers, its promoter hypermethylation, protein expression decreased, and PTCH1 in RA how to change, is not clear. Currently steroidal anti-inflammatory drugs, non-steroidal anti-inflammatory drugs, immunosuppressive agents and glucocorticoids used to treat RA, but has toxicity and side effects. Hesperidin (HDN) belongs to the dihydroflavonoid glycosides. Previous studies in our laboratory have shown that HDN has strong anti-inflammatory and antioxidant activities and has therapeutic effects on adjuvant arthritis rats (AA). The structure modification and modification of HDN have improved the half-life, water-solubility and stability of HDN. A series of hesperidin derivatives (HDND) have been obtained and their anti-inflammatory activities have been screened. 5-(4-chlorophenylethyl) imino-7-oxo-acetyl-4-chlorophenylethylamine hesperidin (HDND-XIV) has good anti-inflammatory activity. In this study, AA rats with similar pathological characteristics and immunological changes as RA were selected as animal models, and FLS was used as the research object. 5-hybrid-4-deoxyc-deoxycytidine (5-hybrid-4-deoxyc) was used as methylation inhibitor in the experiment. Ytidine, 5-Azad C) and gene silencing techniques were used to study the regulation of MeCP2 on PTCH1 protein expression and its effect on inflammatory factors. The effects of HDND-XIV on FLS inflammation and possible mechanisms were observed in vitro. The main contents of this study include two parts: 1. MeCP2 on the regulation and mechanism of PTCH1 expression in rheumatoid arthritis; 2. Regulation and mechanism of CH1. Freund's complete adjuvant (FCA) was used to induce AA rat model. Normal group rats were given the same amount of PBS. After 24 days of modeling, primary FLS was extracted. 5-azadc was used to treat FLS in vitro. MeCP2 was silenced by gene silencing technique in vitro. The regulation of PTCH1 and the above effects on inflammatory indexes were observed. The expression of MeCP2, PTCH1, Gli1 and Shh proteins was detected by Western blot, and the corresponding inflammatory markers were detected by enzyme linked immunosorbent assay (ELISA). Methylation-specific PCR (MSP) and pyrophosphatic acid sequencing were used to detect the degree of methylation. The expression of Gli1 and Shh in AA FLS decreased, suggesting that Hedgehog signaling pathway was activated in AA group. Compared with AA group, the expression of MeCP2, Gli1, Shh protein and PTCH1 protein were decreased, and the expression of PTCH1 protein was increased in AA FLS stimulated by 5-azadc. MSP and pyrophosphatic acid sequencing showed that PTCH1 gene methyl was activated by 5-azadc. After successful silencing of MeCP2 at the cellular level, the expression of PTCH1 increased with the decrease of inflammatory markers. It is suggested that in rheumatoid arthritis, MeCP2 regulates the expression of PTCH1 through DNA methylation, which provides a new target for the prevention and treatment of RA. 2. Inhibitory effect of hesperidin derivative XIV on inflammation of fibrosynoviocytes. To study the effect and mechanism of HDND-XIV on inflammation of FLS. FCA-induced AA rat model was used. Half maximum inhibitory concentration (IC50) of HDND-XIV was detected by MTT method, corresponding inflammation indexes were detected by ELISA, the expressions of MeCP2, PTCH1, Gli1 and Shh proteins in FLS were detected by Western blot, and pyrophosphatic acid sequencing was performed. The results showed that the IC50 value of HDND-XIV was 161 micromol (-1). In view of the toxicity of HDND-XIV, five drug concentrations (80 micromol (-1), 40 micromol (-1), 20 micromol (-1), 10 micromol (-1), 5 micromol (-1)) were selected to stimulate HDND-XIV. The results showed that the concentration of HDND-XIV was 10 micromol (-1)) and 5 micromol 65507 After treatment with HDND-XIV, the expression of MeCP2, Gli 1 and Shh protein decreased, and the expression of PTCH1 protein increased. Pyrophosphate sequencing showed that the methylation of PTCH1 gene was significantly lower than that of AA FLS. The results suggest that HDND-XIV can inhibit FLS inflammation by inhibiting the expression of MeCP2 and decreasing the methylation of PTCH1 gene, and ultimately down-regulating the secretion of inflammatory cytokines.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965
【参考文献】
相关期刊论文 前1条
1 何俊锋;刘剑平;;表观遗传学在类风湿关节炎发病机制中的研究进展[J];中华临床医师杂志(电子版);2013年13期
，本文编号：2224766
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2224766.html
最近更新
教材专著
