【摘要】：目的:探討紫外線(ultraviolet,UV)、雌激素及人類內(nèi)源性逆轉(zhuǎn)錄病毒(human endogenous retroviruses,HERVs)加重系統(tǒng)性紅斑狼瘡(systemic lupus erythematosus,SLE)的作用機(jī)制。方法:(1)收集35名SLE患者及15名正常人外周血CD4+T細(xì)胞,采用不同劑量UVB照射,流式細(xì)胞儀檢測(cè)DNA甲基化水平,實(shí)時(shí)定量PCR及Western Blotting檢測(cè)DNA甲基轉(zhuǎn)移酶1(DNA methyltransferase 1,DNMT1)表達(dá),熒光法檢測(cè)DNMT1酶催化活性。(2)收集30名女性SLE患者及15名正常女性外周血CD4+T細(xì)胞,采用17β-雌二醇干預(yù)培養(yǎng),流式細(xì)胞儀檢測(cè)DNA甲基化水平,實(shí)時(shí)定量PCR及Western Blotting檢測(cè)DNMT1表達(dá),ELISA方法檢測(cè)血漿17β-雌二醇水平。(3)收集15名SLE患者及10名正常人外周血CD4+T細(xì)胞進(jìn)行HERVs表達(dá)研究。實(shí)時(shí)定量PCR檢測(cè)6種HERVs的表達(dá),DNA亞硫酸鹽處理及克隆測(cè)序方法檢測(cè)HERVs長(zhǎng)末端重復(fù)序列(long terminal repeats,LTRs)甲基化水平。5-氮雜胞苷及UVB處理外周血CD4+T細(xì)胞,檢測(cè)LTRs甲基化及HERVs表達(dá)。結(jié)果:(1)SLE患者外周血CD4+T細(xì)胞DNA甲基化及DNMT1表達(dá)顯著低于正常人。UVB照射劑量依賴性地抑制SLE患者DNA甲基化,但對(duì)正常人無(wú)影響。UVB照射對(duì)SLE患者及正常人DNMT1表達(dá)無(wú)影響。UVB照射劑量依賴性地抑制SLE患者DNMT1酶催化活性。(2)17β-雌二醇抑制女性SLE患者外周血CD4+T細(xì)胞中DNMT1表達(dá)及DNA甲基化。SLE患者及正常人血漿17β-雌二醇水平無(wú)明顯差異。SLE患者外周血CD4+T細(xì)胞雌激素受體α(estrogen receptorα,ERα)m RNA表達(dá)顯著高于正常人。在ER拮抗劑存在條件下,17β-雌二醇對(duì)SLE患者DNMT1表達(dá)及DNA甲基化的抑制作用被逆轉(zhuǎn)。(3)SLE患者外周血CD4+T細(xì)胞HERV-E m RNA表達(dá)顯著高于正常人,并與疾病活動(dòng)性呈正相關(guān)。SLE患者外周血CD4+T細(xì)胞HERV-E LTR甲基化水平顯著低于正常人,并與HERV-E m RNA表達(dá)呈負(fù)相關(guān)。5-氮雜胞苷及UVB抑制SLE患者外周血CD4+T細(xì)胞HERV-E LTR甲基化,上調(diào)其m RNA表達(dá)。結(jié)論:(1)UVB劑量依賴性地抑制SLE患者外周血CD4+T細(xì)胞DNMT1酶催化活性,加重DNA低甲基化。(2)17β-雌二醇通過(guò)高表達(dá)的ERα抑制女性SLE患者外周血CD4+T細(xì)胞DNMT1表達(dá),加重DNA低甲基化。(3)HERV-E參與SLE發(fā)病。SLE患者外周血CD4+細(xì)胞HERV-E LTR甲基化水平調(diào)控其轉(zhuǎn)錄表達(dá)。
[Abstract]:Aim: to investigate the mechanism of ultraviolet (ultraviolet,UV), estrogen and human endogenous retrovirus (human endogenous retroviruses,HERVs) in exacerbating systemic lupus erythematosus (systemic lupus erythematosus,SLE). Methods: (1) Peripheral blood CD4 T cells were collected from 35 patients with SLE and 15 normal controls. DNA methylation level was detected by flow cytometry, DNA methyltransferase 1 (DNA methyltransferase 1 was detected by real-time PCR and Western Blotting. (2) 30 female SLE patients and 15 normal female peripheral blood CD4 T cells were collected and cultured with 17 尾 -estradiol, and DNA methylation level was detected by flow cytometry. DNMT1 expression was detected by real-time quantitative PCR and Western Blotting, and plasma 17 尾 -estradiol was detected by ELISA. (3) HERVs expression of CD4 T cells in peripheral blood of 15 SLE patients and 10 normal controls was studied. Real-time quantitative PCR was used to detect the expression of six kinds of HERVs, DNA sulfite treatment and clone sequencing were used to detect the methylation level of HERVs long terminal repeat (long terminal repeats,LTRs. 5-azacytidine and UVB were used to treat CD4 T cells in peripheral blood. LTRs methylation and HERVs expression were detected. Results: (1) DNA methylation and DNMT1 expression of CD4 T cells in peripheral blood of SLE patients were significantly lower than those of normal controls. UVB irradiation inhibited DNA methylation in SLE patients in a dose-dependent manner. UVB irradiation had no effect on the expression of DNMT1 in SLE patients and normal subjects. UVB irradiation inhibited the activity of DNMT1 enzyme catalytic activity in SLE patients in a dose-dependent manner. (2) 17 尾 -estradiol inhibited CD4 T cells in peripheral blood of female SLE patients. There was no significant difference in plasma 17 尾 -estradiol levels between SLE patients and normal subjects. The estrogen receptor 偽 (estrogen receptor 偽 of CD4 T cells in peripheral blood of SLE patients had no significant difference. The expression of ER 偽) m RNA was significantly higher than that of normal subjects. In the presence of ER antagonist, the inhibitory effect of 17 尾 -estradiol on DNMT1 expression and DNA methylation in SLE patients was reversed. (3) the HERV-E m RNA expression of CD4 T cells in peripheral blood of SLE patients was significantly higher than that of normal controls. The HERV-E LTR methylation level of CD4 T cells in peripheral blood of SLE patients was significantly lower than that of normal controls. 5-azacytidine and UVB inhibited HERV-E LTR methylation of CD4 T cells in peripheral blood of SLE patients and upregulated the expression of m RNA. Conclusion: (1) UVB inhibited the DNMT1 catalytic activity of CD4 T cells in SLE patients in a dose-dependent manner and aggravated DNA hypomethylation. (2) 17 尾 -estradiol inhibited the DNMT1 expression of CD4 T cells in female SLE patients by high expression of ER 偽. (3) HERV-E was involved in the pathogenesis of SLE. HERV-E LTR methylation level of CD4 cells in peripheral blood of SLE patients regulated its transcription and expression.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.241

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1 張嵐;郭豫杰;魯維飛;李清風(fēng);;雌激素受體研究進(jìn)展[J];上海畜牧獸醫(yī)通訊;2006年05期

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