常见诱发因素加重系统性红斑狼疮的机制研究
发布时间：2018-12-10 08:17
【摘要】：目的:探讨紫外线(ultraviolet,UV)、雌激素及人类内源性逆转录病毒(human endogenous retroviruses,HERVs)加重系统性红斑狼疮(systemic lupus erythematosus,SLE)的作用机制。方法:(1)收集35名SLE患者及15名正常人外周血CD4+T细胞,采用不同剂量UVB照射,流式细胞仪检测DNA甲基化水平,实时定量PCR及Western Blotting检测DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)表达,荧光法检测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细胞,采用17β-雌二醇干预培养,流式细胞仪检测DNA甲基化水平,实时定量PCR及Western Blotting检测DNMT1表达,ELISA方法检测血浆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细胞进行HERVs表达研究。实时定量PCR检测6种HERVs的表达,DNA亚硫酸盐处理及克隆测序方法检测HERVs长末端重复序列(long terminal repeats,LTRs)甲基化水平。5-氮杂胞苷及UVB处理外周血CD4+T细胞,检测LTRs甲基化及HERVs表达。结果:(1)SLE患者外周血CD4+T细胞DNA甲基化及DNMT1表达显著低于正常人。UVB照射剂量依赖性地抑制SLE患者DNA甲基化,但对正常人无影响。UVB照射对SLE患者及正常人DNMT1表达无影响。UVB照射剂量依赖性地抑制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细胞中DNMT1表达及DNA甲基化。SLE患者及正常人血浆17β-雌二醇水平无明显差异。SLE患者外周血CD4+T细胞雌激素受体α(estrogen receptorα,ERα)m RNA表达显著高于正常人。在ER拮抗剂存在条件下,17β-雌二醇对SLE患者DNMT1表达及DNA甲基化的抑制作用被逆转。(3)SLE患者外周血CD4+T细胞HERV-E m RNA表达显著高于正常人,并与疾病活动性呈正相关。SLE患者外周血CD4+T细胞HERV-E LTR甲基化水平显著低于正常人,并与HERV-E m RNA表达呈负相关。5-氮杂胞苷及UVB抑制SLE患者外周血CD4+T细胞HERV-E LTR甲基化,上调其m RNA表达。结论:(1)UVB剂量依赖性地抑制SLE患者外周血CD4+T细胞DNMT1酶催化活性,加重DNA低甲基化。(2)17β-雌二醇通过高表达的ERα抑制女性SLE患者外周血CD4+T细胞DNMT1表达,加重DNA低甲基化。(3)HERV-E参与SLE发病。SLE患者外周血CD4+细胞HERV-E LTR甲基化水平调控其转录表达。
[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 伪 (estrogen receptor 伪 of CD4 T cells in peripheral blood of SLE patients had no significant difference. The expression of ER 伪) 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 伪. (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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R593.241
本文编号：2370270
[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 伪 (estrogen receptor 伪 of CD4 T cells in peripheral blood of SLE patients had no significant difference. The expression of ER 伪) 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 伪. (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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R593.241
【参考文献】
相关期刊论文 前1条
1 张岚;郭豫杰;鲁维飞;李清风;;雌激素受体研究进展[J];上海畜牧兽医通讯;2006年05期
，本文编号：2370270
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