MeCP2對(duì)類風(fēng)濕關(guān)節(jié)炎中PTCH1的調(diào)控作用及橙皮素衍生物XIV號(hào)抗炎機(jī)制的研究
發(fā)布時(shí)間：2018-09-05 16:03
【摘要】：類風(fēng)濕性關(guān)節(jié)炎(rheumatoid arthritis,RA)屬于炎癥性的自身免疫疾病,其特點(diǎn)是滑膜組織的增厚,軟骨與骨組織的破壞。成纖維樣滑膜細(xì)胞(fibroblast-like synoviocytes,FLS)在發(fā)病過程中異常增殖,同時(shí)產(chǎn)生多種炎癥因子。炎癥因子IL~(-1),IL-6,IL-8,TNF-α,基質(zhì)金屬蛋白酶和膠原酶形成了一個(gè)復(fù)雜的網(wǎng)絡(luò),造成關(guān)節(jié)損傷與病變。RA具有較高的發(fā)病率和致殘率,發(fā)病原因與致病機(jī)理仍不可知,不僅嚴(yán)重影響了人們的日常生活,引起病人的不適,而且嚴(yán)重者會(huì)喪失勞動(dòng)能力。因此,尋找能有效治療RA的療法,成為一個(gè)亟待解決的問題。甲基化是表觀遺傳學(xué)修飾的一種,通過抑制轉(zhuǎn)錄來調(diào)控蛋白的表達(dá)。研究發(fā)現(xiàn)RA發(fā)病的進(jìn)程中伴隨著特定基因甲基化的過程。甲基化Cp G結(jié)合蛋白2(Methylated Cp G binding protein 2,MeCP2)是MBD家族的成員,可以特異性的識(shí)別和綁定在甲基化的DNA序列上,從而抑制基因的表達(dá)。有研究發(fā)現(xiàn)MeCP2在RA中高表達(dá),參與調(diào)控特定基因的甲基化。文獻(xiàn)報(bào)道,Hedgehog信號(hào)通路在組織修復(fù)及持續(xù)性的炎癥中發(fā)揮重要作用,Patch1(PTCH1)在此通路中是一個(gè)負(fù)調(diào)控基因,在多種癌癥中,其啟動(dòng)子發(fā)生高甲基化,蛋白的表達(dá)下降,而PTCH1在RA中如何改變,還不明確。目前甾體抗炎藥,非甾體抗炎藥,免疫抑制劑以及糖皮質(zhì)激素等用于治療RA,但是具有毒性和副作用。橙皮苷(hesperidin,HDN)屬于二氫黃酮苷類化合物,本實(shí)驗(yàn)室前期的研究結(jié)果表明,HDN具有較強(qiáng)的抗炎活性,抗氧化活性,對(duì)佐劑性關(guān)節(jié)炎大鼠(Adjuvant Arthritis,AA)具有治療作用。但是由于其半衰期較短,水溶性較差,口服給藥后失效過快,限制了其在臨床上的應(yīng)用。目前國(guó)內(nèi)外大多側(cè)重于對(duì)HDN的結(jié)構(gòu)改造,通過對(duì)其結(jié)構(gòu)的改造和修飾,明顯改善了其半衰期,水溶性及穩(wěn)定性。本實(shí)驗(yàn)室針對(duì)HDN的結(jié)構(gòu)改造和修飾,得到了一系列的橙皮素衍生物(Hesperidin derivatives,HDND),并且進(jìn)行了抗炎活性的篩選。發(fā)現(xiàn)橙皮素衍生物XIV號(hào),即5-(4-氯苯乙基)亞胺基-7-氧-乙酰-4-氯苯乙胺基橙皮素(HDND-XIV)具有較好的抗炎活性。本課題選用與RA具有相似病理特征和免疫學(xué)變化的AA大鼠為動(dòng)物模型,以FLS為細(xì)胞研究對(duì)象。實(shí)驗(yàn)中采用甲基化抑制劑5-雜氮-2-脫氧胞苷(5-hybrid-4-deoxycytidine,5-Azad C)和基因沉默技術(shù),研究MeCP2對(duì)PTCH1蛋白表達(dá)的調(diào)控和其對(duì)炎癥因子的影響,同時(shí)離體觀察HDND-XIV對(duì)FLS炎癥的影響,及可能存在的機(jī)制。主要研究?jī)?nèi)容包括兩部分:1.MeCP2對(duì)類風(fēng)濕關(guān)節(jié)炎中PTCH1表達(dá)的調(diào)控及機(jī)制研究研究MeCP2對(duì)PTCH1的調(diào)控作用及機(jī)制。使用弗氏完全佐劑(Freund's complete adjuvant,FCA)誘導(dǎo)AA大鼠模型,正常組大鼠給予等量的PBS。造模24天后提取原代FLS。體外使用5-azadc作用于FLS,體外采用基因沉默技術(shù)沉默MeCP2,觀察對(duì)PTCH1的調(diào)控以及以上對(duì)炎癥指標(biāo)的影響。采用HE染色,觀察大鼠關(guān)節(jié)的病理變化,同時(shí)檢測(cè)大鼠繼發(fā)側(cè)足腫脹度。免疫熒光,免疫組化檢測(cè)MeCP2,PTCH1蛋白的表達(dá),Western blot法檢測(cè)MeCP2,PTCH1,Gli1和Shh蛋白的表達(dá),酶聯(lián)免疫吸附法(Enzyme linked immunosorbent assay,ELISA)檢測(cè)相應(yīng)炎癥指標(biāo),甲基化特異性PCR(Methylation-Specific PCR,MSP)和焦磷酸測(cè)序檢測(cè)甲基化程度。病理結(jié)果表明AA大鼠模型成立,膝關(guān)節(jié)明顯病變與腫脹。免疫組化,免疫熒光,Western blot結(jié)果發(fā)現(xiàn)無論組織上還是細(xì)胞上,AA大鼠的MeCP2蛋白表達(dá)均升高,PTCH1蛋白表達(dá)均降低,同時(shí)AA FLS中Gli1,Shh表達(dá)升高,提示AA組Hedgehog信號(hào)通路激活。體外給予AA FLS 5-azadc后,相比較于AA組,細(xì)胞炎癥指標(biāo)均下降,細(xì)胞水平MeCP2,Gli1,Shh蛋白表達(dá)下降,PTCH1蛋白表達(dá)上升。MSP和焦磷酸測(cè)序顯示AA FLS經(jīng)5-azadc刺激后,PTCH1基因甲基化水平下降。細(xì)胞水平上成功沉默MeCP2后,PTCH1表達(dá)升高,并伴隨著炎癥指標(biāo)的降低。提示,在類風(fēng)濕關(guān)節(jié)炎中,MeCP2通過DNA甲基化來調(diào)控PTCH1的表達(dá),為RA的防治與研究提供了新的靶點(diǎn)。2.橙皮素衍生物XIV號(hào)對(duì)成纖維滑膜細(xì)胞炎癥的抑制作用及機(jī)制研究研究HDND-XIV對(duì)FLS炎癥的影響及作用機(jī)制。使用FCA誘導(dǎo)AA大鼠模型,MTT法檢測(cè)HDND-XIV的半抑制濃度(Half maximal inhibitory concentration,IC50),ELISA檢測(cè)每組相應(yīng)的炎癥指標(biāo),Western blot法檢測(cè)FLS中MeCP2,PTCH1,Gli1和Shh蛋白的表達(dá),焦磷酸測(cè)序法定量檢測(cè)甲基化程度。結(jié)果發(fā)現(xiàn)HDND-XIV的IC50值為161μmol·L~(-1),鑒于藥物的毒性,在小于0.5倍IC50的范圍里選擇五個(gè)藥物濃度(80μmol·L~(-1),40μmol·L~(-1),20μmol·L~(-1),10μmol·L~(-1),5μmol·L~(-1))加藥刺激,結(jié)果發(fā)現(xiàn)藥物濃度為10μmol·L~(-1)時(shí)抑制炎癥的作用最強(qiáng),然后以10μmol·L~(-1)為加藥組濃度。AA FLS經(jīng)HDND-XIV作用后,MeCP2,Gli1和Shh蛋白表達(dá)下降,PTCH1蛋白表達(dá)上升,焦磷酸測(cè)序結(jié)果顯示其PTCH1基因的甲基化程度較AA FLS明顯降低。在AA FLS上過表達(dá)MeCP2,之后加HDND-XIV刺激,結(jié)果顯示成功過表達(dá)后,PTCH1蛋白表達(dá)下降,炎癥指標(biāo)上升。提示HDND-XIV抑制FLS的炎癥,其作用機(jī)制可能是通過抑制MeCP2的表達(dá),降低PTCH1基因的甲基化程度,最終下調(diào)炎性細(xì)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R965
本文編號(hào)：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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R965
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 何俊鋒;劉劍平;;表觀遺傳學(xué)在類風(fēng)濕關(guān)節(jié)炎發(fā)病機(jī)制中的研究進(jìn)展[J];中華臨床醫(yī)師雜志(電子版);2013年13期
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