【摘要】：背景:強直性脊柱炎(Ankylosing Spondylitis,AS)屬慢性炎癥性的自身免疫性的疾病,可造成髖關(guān)節(jié)破壞和脊柱強直,嚴(yán)重影響患者的工作能力和生活自理能力,降低患者的生活質(zhì)量。炎癥是AS首要病理改變。輔助性T細胞(Thelper cell 17,Th17)是觸發(fā)AS炎癥的重要效應(yīng)細胞,由初始CD4+T細胞(Naive CD4+T cell)分化而來。研究表明,表觀遺傳調(diào)控機制在CD4+T細胞向Th17分化中扮演著重要角色,組蛋白去甲基化酶JMJD3催化H3K27位點去甲基化,可能是Th17分化的關(guān)鍵調(diào)控因子。在前期臨床研究中,我們發(fā)現(xiàn)清熱利濕活血法緩解AS炎癥的臨床療效滿意。且經(jīng)清熱利濕活血方治療后,AS患者IL-17表達水平顯著下降,Th17數(shù)量及特征性轉(zhuǎn)錄因子RORc表達明顯減少,JAK/STAT通路活化水平顯著下降,表明清熱利濕活血法可以通過干預(yù)Th17分化,發(fā)揮緩解AS炎癥的作用。本研究基于此,進一步探究JMJD3調(diào)控AS Th17分化的表觀遺傳機制及清熱利濕活血法的干預(yù)作用。目的:初步探究AS患者JMJD3表達情況及其去甲基化H3K27對Th17分化的干預(yù)作用;中藥清熱強脊湯治療及中藥單體雷公藤甲素體外干預(yù)PBMC對AS患者JMJD3的表達及Th17分化的調(diào)節(jié)作用。方法:研究一、研究二及研究三以活動期AS患者的血清及PBMC為研究對象,運用Western Blotting檢測蛋白表達情況,Quantitative Real-time PCR等技術(shù)檢測mRNA表達情況,ELISA檢測血清炎性細胞因子分泌情況。分別檢測疾病活動期、疾病相對穩(wěn)定期、中藥治療3個月對AS患者JMJD3表達、H3K27me3的甲基化情況及Th17分化狀況的影響作用。研究四以體外分離培養(yǎng)的活動期AS患者外周血PBMC為研究對象,運用Western Blotting檢測蛋白表達情況,Quantitative Real-time PCR等技術(shù)檢測mRNA表達情況,研究中藥雷公藤有效單體雷公藤甲素體外干預(yù)對AS患者JMJD3表達、JMJD3催化的H3K27去甲基化情況及Th17分化狀況的影響作用。結(jié)果:1.活動期AS患者JMJD3表達情況分析中,發(fā)現(xiàn)活動期AS患者血清中Th17特異性分泌的細胞因子IL-17表達水平較正常對照組顯著增高,且差異具有統(tǒng)計學(xué)意義(p0.001);與患者炎癥指標(biāo)ESR、CRP以及疾病活動指數(shù)BASDAI做了相關(guān)性分析,發(fā)現(xiàn)IL-17、ESR、CRP互相高度相關(guān)(IL-17-ESR:p0.001;IL-17-CRP:p0.01)�；顒悠� AS 患者 PBMC 中,JMJD3 的 mRNA表達水平較正常對照顯著增高,(p0.001);JMJD3與炎癥指標(biāo)ESR、CRP間均顯著相關(guān)(ESR:r= 0.631,p=0.0030.01)(CRP:r= 0.567,p=0.0090.01)。JMJD3與炎性細胞因子IL-17間顯著相關(guān)(IL-17:p0.01),而JMJD3、IL-17與BASDAI均無明顯相關(guān)性關(guān)系。2.活動期AS患者JMJD3去甲基化H3K27及對Th17分化的干預(yù)作用研究中,檢測AS患者(AS-Active)、穩(wěn)定期AS患者(AS-Stable)、正常對照組(N)的JMJD3、H3K27me3、JAK/STAT信號通路及特征性轉(zhuǎn)錄因子RORc蛋白及mRNA表達水平�；顒悠贏S患者的JMJD3蛋白表達水平顯著高于正常對照組與非活動期AS患者(p0.001),而非活動期AS患者的JMJD3蛋白表達水平則與正常對照無明顯差異(p0.05)。在基因水平上,活動期AS患者的JMJD3 mRNA表達水平較正常對照組及與非活動期AS患者均顯著增高,非活動期AS患者較正常對照組則無明顯差異(AS-Active:p0.001;AS-Stable:p0.05),活動期與非活動期AS患者間JMJD3的mRNA表達水平亦存在顯著差異(p0.001)�；顒悠贏S患者的H3K27me3甲基化水平均顯著降低,較正常對照組差異有統(tǒng)計學(xué)意義(p0.01);活動期AS患者與非活動期AS患者的H3K27甲基化水平亦存在明顯差異(p0.001),正常對照組與非活動期AS患者無明顯差異(p0.05)。JAK/STAT信號通路中信號分子STAT3的磷酸化水平(pSTAT3/STAT3)較正常對照顯著升高(AS-Active:p0.001;AS-Stable:p0.01)。與非活動期患者相比較,活動期AS患者JAK2的表達水平雖沒有明顯差異,但磷酸化的JAK2表達水平顯著增高(p=0.0130.05);STAT3的蛋白表達水平及磷酸化水平均較非活動期AS患者明顯增高(STAT3:p0.01;pSTAT3:p0.001)。在基因水平上,活動期AS患者及非活動期AS患者的JAK2及STAT3 mRNA表達水平均明顯高于正常對照組(AS-Active:p0.001;AS-Stable:p0.01);而相較于非活動期AS患者,活動期AS患者上述信號分子的mRNA表達量亦存在明顯差異(p0.001)。活動期AS患者與非活動期AS患者轉(zhuǎn)錄因子RORc表達水平較正常對照組顯著增高(p0.001);活動期AS患者的RORc表達水平較非活動期無明顯差異(p0.05)。在基因表達水平上,相較于正常對照組,活動期AS患者及非活動期AS患者RORc的mRNA均顯著增高(p0.01);活動期AS患者與非活動期AS患者直接亦存在表達差異(p0.01)。3.在清熱利濕活血法對活動期AS患者JMJD3表達及Th17分化的調(diào)節(jié)作用研究中,我們選用本科治療活動期骨痹的臨床有效方劑清熱強脊湯辨證治療濕熱瘀阻證的AS患者,并在服藥3個月后檢測其Th17細胞的活性與功能。經(jīng)清熱強脊湯治療3個月后,患者ESR、CRP、BASDAI均較治療前明顯下降,差異有統(tǒng)計學(xué)意義(p0.001)。治療后患者血清炎性細胞因子IL-17的表達水平較治療前也明顯下降(p0.05);JMJD3的蛋白和mRNA表達情況和相對灰度值變化均較治療前存在顯著差異(p0.05);患者H3K27me3的甲基化水平均較治療前也表現(xiàn)出了顯著下降(p0.001);患者RORc的基因及蛋白表達水平均較治療前顯著下降(p0.001)。4.以雷公藤提取物雷公藤甲素干預(yù)活動期AS患者的PBMC,經(jīng)雷公藤甲素干預(yù)后,AS患者的JMJD3蛋白表達水平較干預(yù)前顯著降低(p0.001);基因水平的檢測得到了相同的結(jié)果(p0.001)。經(jīng)雷公藤甲素干預(yù)后,H3K27甲基化水平較治療前顯著提高,差異具有統(tǒng)計學(xué)意義(p0.001);雷公藤甲素干預(yù)后的H3K27me3水平與正常對照組則無明顯統(tǒng)計學(xué)差異(p=0.080.05)。經(jīng)雷公藤甲素干預(yù)后,患者JAK2/STAT3信號通路的活化被明顯抑制,各信號分子的磷酸化水平顯著下降(p0.001)�；蛩缴�,JAK2、STAT3的mRNA表達量明顯下降(p0.001)。經(jīng)雷公藤甲素干預(yù)后,RORc的蛋白和mRNA表達量顯著下降(p0.001)。結(jié)論:1.JMJD3是Th17分化及功能的重要調(diào)控因子,參與了活動期AS的炎癥發(fā)生。JMJD3的表達水平與Th17的分化及功能高度相關(guān);JMJD3與AS炎癥指標(biāo)也密切相關(guān),可能是控制AS炎癥有意義的治療靶點之一。2.AS患者存在著JMJD3的高度表達和總體H3K27me3甲基化水平的下降,且在疾病不同活動階段的JMJD3表達水平和H3K27甲基化水平存在明顯差異。同時不同疾病活動階段的患者,其RORc表達、JAK/STAT信號通路活化及IL-17分泌均存在差異。JMJD3調(diào)控的H3K27me3去甲基化,可能是干預(yù)Th17的分化、活化及功能,從而影響AS炎癥及疾病活動機制之一。3.清熱利濕活血法對患者的炎癥指標(biāo)及疾病活動都有較好的控制作用,可干預(yù)JMJD3的表達水平,以及JMJD3催化的H3K27me3去甲基化活性,從而影響表觀遺傳調(diào)控的Th17分化,使RORc的表達水平和JAK/STAT信號通路的活化水平降低,從而干預(yù)了Th17的活化及IL-17的表達。我們推測,清熱利濕活血法對AS炎癥的緩解作用,可能是通過干預(yù)JMJD的活化與功能,從而抑制Th17的分化、活化以及功能而實現(xiàn)的。4.中藥單體雷公藤甲素體外干預(yù),可降低AS患者PBMC中JMJD3的表達水平,影響JMJD3調(diào)控的H3K27me3去甲基化過程,從而使Th17轉(zhuǎn)錄因子RORc、信號通路JAK/STAT及IL-17的轉(zhuǎn)錄抑制和蛋白表達被抑制,可能是調(diào)節(jié)Th17的分化偏移及功能,從而緩解AS炎癥的有意義的治療藥物之一。
[Abstract]:BACKGROUND: Ankylosing Spondylitis (AS) is a chronic inflammatory autoimmune disease, which can cause hip joint destruction and spinal rigidity, seriously affect the working ability and self-care ability of patients, and reduce the quality of life of patients. Inflammation is the primary pathological change of AS. The helper T cell (Th17) is the trigger of A. The important effector cells of S inflammation are differentiated from the initial CD4 + T cells. Studies have shown that epigenetic regulation plays an important role in the differentiation of CD4 + T cells into Th17. Histone demethylase JMJD3 catalyzes the demethylation of H3K27 site and may be a key regulator of Th17 differentiation. They found that the method of clearing away heat and dampness and activating blood circulation has a satisfactory clinical effect on alleviating inflammation of AS. After treatment with the prescription of clearing away heat and removing dampness and activating blood circulation, the level of IL-17 expression, the number of Th17 and the expression of RORc, the activation level of JAK/STAT pathway were significantly decreased in AS patients, indicating that the method of clearing away heat and removing dampness and activating blood circulation could be exerted by interfering the differentiation of Th17. Objective: To investigate the expression of JMJD3 and the effect of demethylated H3K27 on the differentiation of Th17 in AS patients. Methods: Study 1, Study 2 and Study 3 were used to detect the expression of JMJD3 and the differentiation of Th17 in the serum and PBMC of AS patients in active phase. The expression of JMJD3 and Th17 mRNA were detected by Western Blotting, Quantitative Real-time PCR and serum inflammatory cytokines were detected by ELISA. Secretion of JMJD3, methylation of H3K27me3 and differentiation of Th17 in AS patients were detected at active stage, relatively stable stage, and three months after treatment with Chinese herbs. Objective:To study the effect of triptolide on the expression of JMJD3, the demethylation of H3K27 catalyzed by JMJD3 and the differentiation of Th17 in AS patients in vitro. Results:1. The expression of Th17 in AS patients in active phase was detected by the analysis of JMJD3 expression. The expression of cytokine IL-17 in heterosexual secretion was significantly higher than that in normal control group (p0.001), and the difference was statistically significant (p0.001); the correlation analysis with inflammatory markers ESR, CRP and disease activity index BASDAI showed that IL-17, ESR and CRP were highly correlated (IL-17-ESR: p0.001; IL-17-CRP: p0.01). The expression of JMJD3 mRNA was significantly higher than that of normal controls (p0.001); JMJD3 was significantly correlated with inflammatory markers ESR and CRP (ESR: r = 0.631, P = 0.0030.01) (CRP: r = 0.567, P = 0.0090.01). JMJD3 was significantly correlated with inflammatory cytokine IL-17 (IL-17: p0.01), but JMJD3, IL-17 were not significantly correlated with BASDAI.2. The expression of JMJD3, H3K27me3, JAK/STAT signaling pathway and characteristic transcription factor RORc protein and mRNA in AS patients (AS-Active), AS-Stable patients (AS-Stable), normal control group (N), and active AS patients were detected. The expression level of JMJD3 mRNA in active AS patients was significantly higher than that in non-active AS patients and normal AS patients (p 0.001), but not in active AS patients (p 0.05). There were significant differences in the expression of JMJD3 mRNA between active and inactive AS patients (p0.001). The methylation level of H3K27me3 in active AS patients was significantly lower than that in normal control group (p0.01). The methylation level of H3K27 in active AS patients and inactive AS patients was also significantly lower than that in normal control group (p0.01). The phosphorylation level of STAT3 in JAK / STAT signaling pathway (pSTAT3 / STAT3) was significantly higher than that in normal controls (AS-Active: p0.001; AS-Stable: p0.01). Compared with non-active patients, the expression level of JAK2 in active AS patients was not significantly different. However, the expression of phosphorylated JAK2 was significantly increased (p=0.0130.05), and the expression of STAT3 protein and phosphorylation were significantly higher than those of inactive AS patients (STAT3:p0.01; pSTAT3:p0.001). At the gene level, the expression of JAK2 and STAT3 mRNA in active AS patients and inactive AS patients were significantly higher than those of normal control group (AS-Active:p0.01). The expression level of RORc in active AS patients and inactive AS patients was significantly higher than that in normal control group (p0.001), while the expression level of RORc in active AS patients was significantly higher than that in inactive AS patients (p0.001). The expression of RORc mRNA was significantly higher in active AS patients and inactive AS patients than in normal control group (p0.01). The expression of JMJD3 and Th17 was also significantly different in active AS patients and inactive AS patients (p0.01). 3. The regulation of heat-clearing, dampness-activating and Blood-Activating Therapy on on JMJD3 expression and Th17 differentiation in active AS patients. In the study of action, we chose Qingre Qiangji Decoction, a clinical effective prescription for active bone arthralgia, to treat AS patients with dampness-heat-stasis syndrome, and detected the activity and function of Th17 cells 3 months after taking the medicine. After treatment, the levels of serum inflammatory cytokine IL-17 were also significantly lower than before treatment (p0.05); JMJD3 protein and mRNA expression and relative gray value changes were significantly different than before treatment (p0.05); H3K27me3 methylation levels in patients were also significantly lower than before treatment (p0.001); The expression of JMJD3 protein in active AS patients treated with triptolide was significantly lower than that before treatment (p0.001). The same result was obtained by gene level detection (p0.001). Dry triptolide was used to treat active AS patients with PBMC. Prognosis, H3K27 methylation level was significantly higher than before treatment, the difference was statistically significant (p0.001); Tripterygium wilfordii intervention H3K27me3 level and normal control group, there was no significant difference (p = 0.080.05). After triptolide intervention, JAK2 / STAT3 signal pathway activation was significantly inhibited, each signal molecule phosphorylated water. At the gene level, JAK2 and STAT3 mRNA expression decreased significantly (p0.001). After triptolide intervention, RORc protein and mRNA expression decreased significantly (p0.001). Conclusion: 1. JMJD3 is an important regulator of Th17 differentiation and function, and participates in the inflammation of active AS. JMJD3 is also closely related to the inflammation index of AS, and may be one of the therapeutic targets for controlling inflammation of AS. 2. There is a high expression of JMJD3 and a decrease of H3K27me3 methylation in AS patients. There are differences in RORc expression, JAK/STAT signaling pathway activation and IL-17 secretion in patients with active disease. H3K27me3 demethylation regulated by JMJD3 may interfere with the differentiation, activation and function of Th17, thus affecting the inflammation and disease activity of AS. 3. Clearing away heat, eliminating dampness and activating blood circulation method has a better effect on inflammatory indexes and disease activity of patients. Controlling effect can interfere with the expression of JMJD3 and the demethylation activity of H3K27me3 catalyzed by JMJD3, thus affecting the Th17 differentiation of epigenetic regulation, reducing the expression of RORc and the activation of JAK/STAT signaling pathway, thus interfering with the activation of Th17 and the expression of IL-17. Mitigation may be achieved by interfering with the activation and function of JMJD, thereby inhibiting the differentiation, activation and function of Th17. 4. Tripterygium Wilfordii in vitro can reduce the expression of JMJD3 in PBMC of AS patients, and affect the H3K27me3 demethylation process regulated by JMJD3, thus making Th17 transcription factor RORc, JAK/STAT signaling pathway. Inhibition of transcriptional and protein expression of IL-17 and IL-17 may be one of the significant therapeutic drugs to alleviate AS inflammation by regulating the differentiation and function of Th17.
【學(xué)位授予單位】：中國中醫(yī)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R259

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