IL-17RC低甲基化影響葡萄膜炎的機(jī)制研究及中藥治療葡萄膜炎的臨床體會(huì)
發(fā)布時(shí)間：2018-08-23 11:40
【摘要】：背景： 結(jié)節(jié)病,是一種累及多系統(tǒng)并以非干酪樣肉芽腫為病理特點(diǎn)的慢性疾病,是引發(fā)眼局部炎癥的常見(jiàn)原因之一,高達(dá)24%的眼結(jié)節(jié)病患者會(huì)出現(xiàn)嚴(yán)重視力損害,其中10%可視力致殘甚至失明,但早期干預(yù)可明顯改善預(yù)后。眼結(jié)節(jié)病最主要的表現(xiàn)為葡萄膜炎。目前眼結(jié)節(jié)病的診斷仍存在諸多難點(diǎn),皮膚、外周淋巴結(jié)以及肺部病理組織活檢是診斷金標(biāo)準(zhǔn),但侵入性方式無(wú)疑會(huì)給病人帶來(lái)一定的傷害。因此,發(fā)現(xiàn)新的診斷標(biāo)志物,用于提高診斷安全性和準(zhǔn)確性,并應(yīng)用于治療,具有重要的臨床意義。 表觀遺傳學(xué)方法的出現(xiàn),為研究疾病的發(fā)病機(jī)制開(kāi)啟了新的篇章,DNA甲基化是高等動(dòng)物中普遍存在的基因表觀遺傳修飾方式之一。IL-17RC以跨膜蛋白形式存在于細(xì)胞膜上,作為共受體與IL-17亞型特異性結(jié)合,可以激活多條信號(hào)轉(zhuǎn)導(dǎo)通路,促進(jìn)炎癥反應(yīng)的發(fā)生。最新文獻(xiàn)表明IL-17RC啟動(dòng)子區(qū)低甲基化在眼局部炎癥、滲出、新生血管形成的過(guò)程中發(fā)揮了一定作用。我們推測(cè),IL-17RC啟動(dòng)子區(qū)的DNA甲基化異常可能參與眼結(jié)節(jié)病的發(fā)生發(fā)展。 葡萄膜炎是眼結(jié)節(jié)病最主要的表現(xiàn)形式,目前治療以局部或者全身應(yīng)用糖皮質(zhì)激素為主,可聯(lián)合免疫抑制劑以及生物制劑,但副作用均較明顯,且易產(chǎn)生激素依賴性以及停藥后反跳。中醫(yī)藥在治療葡萄膜炎方面具有明顯優(yōu)勢(shì),值得中醫(yī)工作者繼承和發(fā)揚(yáng)。 目的： 第一部分：分析IL-17RC啟動(dòng)子區(qū)DNA甲基化狀態(tài)與眼結(jié)節(jié)病相關(guān)性,檢測(cè)IL-17誘導(dǎo)IL-17RC表達(dá)的能力,初步探討IL-17RC啟動(dòng)子區(qū)甲基化在眼結(jié)節(jié)病分子水平機(jī)制中的作用。 第二部分：葡萄膜炎是眼結(jié)節(jié)病的最主要臨床表現(xiàn),目前西藥治療手段有限,故第二部分內(nèi)容總結(jié)中醫(yī)藥治療葡萄膜炎的臨床經(jīng)驗(yàn),為治療提供幫助。 方法： 第一部分：以眼結(jié)節(jié)病組和健康對(duì)照組為觀察對(duì)象,采用甲基化敏感性限制性內(nèi)切酶(Methylation sensitive restriction endonuclease, MSRE)PCR法觀察兩組IL-17RC啟動(dòng)子區(qū)DNA甲基化的水平；采用流式細(xì)胞術(shù)(Flow cytomerty)比較眼結(jié)節(jié)病患者和正常人外周血主要免疫細(xì)胞表面IL-17RC蛋白的表達(dá)；采用酶聯(lián)免疫吸附試驗(yàn)(Enzyme linked immunosorbent assay, ELISA)比較眼結(jié)節(jié)病患者和正常人血清中IL-17的濃度；采用蘇木精-伊紅染色法(Hematoxylin-eosin staining, HE)觀察靜止期和炎癥活躍期眼結(jié)節(jié)病患者眼組織的形態(tài)結(jié)構(gòu),采用免疫組化染色(Immunohistochemistry, IHC)和逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(Reverse transcription polymerase chain reaction, RT-PCR)檢測(cè)視網(wǎng)膜中IL-17RC蛋白和mRNA的表達(dá)；以抗CD3/抗CD28抗體活化增殖眼結(jié)節(jié)病患者和正常人CD8+T細(xì)胞并體外培養(yǎng)3天,其中刺激組和空白組分別加入IL-17、培養(yǎng)基,以流式細(xì)胞術(shù)比較其表面IL-17RC表達(dá)量的差異。 第二部分：1.檢索并閱讀葡萄膜炎的中西醫(yī)文獻(xiàn),研讀金師發(fā)表的論文和著作。2.跟隨金師門診抄方,學(xué)習(xí)并記錄老師臨證時(shí)診療思路、用藥特點(diǎn),將其治療葡萄膜炎的經(jīng)驗(yàn)進(jìn)行總結(jié)。 結(jié)果： 第一部分：1.相較于健康對(duì)照組,眼結(jié)節(jié)病患者IL-17RC啟動(dòng)子區(qū)DNA存在低甲基化修飾,差異有明顯統(tǒng)計(jì)學(xué)意義(P0.01)；2.外周血主要免疫細(xì)胞中,眼結(jié)節(jié)病患者CD8+T細(xì)胞表面表達(dá)的IL-17RC較健康對(duì)照組的表達(dá)量升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；3.與健康對(duì)照組相比,眼結(jié)節(jié)病患者中血清中IL-17濃度升高,結(jié)果具有顯著統(tǒng)計(jì)學(xué)差異(P0.01)；4.HE染色提示眼結(jié)節(jié)病患者眼組織中存在典型肉芽腫病變,且活躍的炎癥組織有脈絡(luò)膜視網(wǎng)膜瘢痕(Chorioretinal scar, C-R scar), IHC染色提示炎癥活躍期視網(wǎng)膜組織可見(jiàn)IL-17RC的沉積,RT-PCR檢測(cè)證實(shí)炎癥活躍期患者的視網(wǎng)膜組織IL-17RC mRNA轉(zhuǎn)錄水平明顯增高,差異具有顯著統(tǒng)計(jì)學(xué)意義(P0.01)；5.相較于未用IL-17刺激的空白組,經(jīng)IL-17刺激后的眼結(jié)節(jié)病患者以及正常人的CD8+T細(xì)胞表面表達(dá)的IL-17RC均有所增多,但只有正常人刺激組的結(jié)果具有統(tǒng)計(jì)學(xué)差異(P0.01)。 第二部分：金明教授臨證時(shí)主張以“西醫(yī)辨病、中醫(yī)辨證”為中心,用西醫(yī)系統(tǒng)檢查、眼科專科儀器與中醫(yī)“望、聞、問(wèn)、切”四診并行,獲取臨床第一手病史及資料,且提倡切勿局限于眼部,應(yīng)將眼病與整體相聯(lián),綜合審視。葡萄膜炎是一類非常復(fù)雜之疾病,病因涉及外邪侵襲、濕熱毒瘀內(nèi)蘊(yùn)、臟腑虛弱等,病位主要為肝膽腎,病機(jī)繁復(fù)、病情纏綿。在診斷方面,金明教授提出不僅要依據(jù)中醫(yī)理論辨證分型,還應(yīng)將病程細(xì)分為各期,并結(jié)合專科儀器,宏觀辨證與微觀檢查互參,多層面把握病情。在辨證分型方面,金師不拘泥于目前常用分型,而是根據(jù)具體證候和病程,隨機(jī)應(yīng)變。在治療方面,金師提出早期干預(yù)改善預(yù)后,在五輪之中尤重肝腎,并靈活運(yùn)用龍膽瀉肝湯(丸)、六味地黃類方,收效甚好。此外,金師還根據(jù)葡萄膜炎的激素應(yīng)用特點(diǎn),總結(jié)中藥協(xié)同使用的規(guī)律：早期聯(lián)合中藥發(fā)揮類激素作用,可減輕激素用量,阻止疾病進(jìn)展,此期中藥多治以疏散風(fēng)熱、祛風(fēng)勝濕,以散邪為主。發(fā)作期的特點(diǎn)是以大劑量口服激素控制炎癥,其重型需加用免疫抑制劑,中藥可減少由激素和免疫抑制劑帶來(lái)的毒副作用,縮短病程,治則多為清瀉肝膽實(shí)火和濕熱、活血化瘀、滲濕解毒,攻補(bǔ)兼施。在后期的激素減量過(guò)程中,針對(duì)此時(shí)較易產(chǎn)生的激素依賴性以及突然停藥帶來(lái)的反跳,可突出中醫(yī)藥的扶正特性,調(diào)節(jié)機(jī)體免疫功能,協(xié)助激素平穩(wěn)減量,鞏固療效,降低復(fù)發(fā)率及并發(fā)癥的發(fā)生,此期因患者久服激素,正氣虧虛,故中藥多治以培補(bǔ)元?dú)�、滋補(bǔ)肝腎。 結(jié)論： 第一部分：1.眼結(jié)節(jié)病患者IL-17RC啟動(dòng)子區(qū)DNA存在低甲基化修飾,提示其與眼結(jié)節(jié)病的具有相關(guān)性,IL-17RC的低甲基化可作為眼結(jié)節(jié)病的表觀遺傳學(xué)標(biāo)記物；2.外周血增多的IL-17RC+CD8+T細(xì)胞參與了眼結(jié)節(jié)病的發(fā)生發(fā)展過(guò)程；3. IL-17RC含量與炎癥的活躍程度或疾病的嚴(yán)重程度相關(guān)聯(lián)；4.眼結(jié)節(jié)病患者IL-17RC的表達(dá)增高可能是由于外周血高濃度的IL-17刺激所導(dǎo)致。 第二部分：針對(duì)葡萄膜炎的治療,應(yīng)中西合璧,揚(yáng)長(zhǎng)避短,多管齊下,從而達(dá)到最優(yōu)效果。
[Abstract]:Background:
Sarcoidosis, a chronic disease involving multiple systems and characterized by non-caseous granulomatosis, is one of the common causes of ocular inflammation. Up to 24% of patients with ocular sarcoidosis have severe visual impairment, of which 10% are visually disabled or even blind, but early intervention can significantly improve the prognosis. At present, there are still many difficulties in the diagnosis of ocular sarcoidosis. Skin, peripheral lymph nodes and lung biopsy are the gold standard of diagnosis, but invasive methods will undoubtedly bring some harm to patients. Therefore, the discovery of new diagnostic markers to improve the safety and accuracy of diagnosis, and applied to treatment, with a view to Important clinical significance.
The emergence of epigenetic methods has opened a new chapter in the study of the pathogenesis of diseases. DNA methylation is one of the most prevalent epigenetic modifications in higher animals. IL-17RC exists on the cell membrane in the form of transmembrane proteins. As a co-receptor, IL-17 subtype binds specifically to IL-17 and activates multiple signal transduction pathways. Recent literature suggests that hypomethylation of IL-17RC promoter plays a role in ocular local inflammation, exudation and angiogenesis. We speculate that abnormal DNA methylation of IL-17RC promoter may be involved in the development of ocular sarcoidosis.
Uveitis is the most important manifestation of ocular sarcoidosis. At present, local or systemic application of glucocorticoids is the main treatment, and can be combined with immunosuppressive agents and biological agents, but the side effects are obvious, and prone to hormone dependence and rebound after withdrawal. Workers inherit and carry forward.
Objective:
Part I: To analyze the relationship between DNA methylation in the promoter region of IL-17RC and ocular sarcoidosis, to detect the ability of IL-17 to induce IL-17RC expression, and to explore the role of IL-17RC promoter methylation in the molecular mechanism of ocular sarcoidosis.
The second part: uveitis is the most important clinical manifestation of ocular sarcoidosis. At present, western medicine treatment is limited, so the second part summarizes the clinical experience of Chinese medicine in the treatment of uveitis, to provide help for treatment.
Method:
Part I: Methylation sensitive restriction endonuclease (MSRE) PCR was used to detect the DNA methylation in the promoter region of IL-17RC in the ocular sarcoidosis group and the healthy control group. Flow cytomerty was used to compare the DNA methylation in the promoter region of IL-17RC between the two groups. The expression of IL-17RC protein on the surface of the main immune cells in peripheral blood was detected by enzyme linked immunosorbent assay (ELISA) and hematoxylin-eosin staining (HE). Immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of IL-17RC protein and mRNA in the retina of patients with ocular sarcoidosis, and anti-CD3/anti-CD28 antibodies were used to activate proliferating CD8+T cells in patients with ocular sarcoidosis and normal subjects. The expression of IL-17RC on the surface of the cells was compared by flow cytometry.
The second part: 1. Retrieve and read the literature of Chinese and Western medicine on uveitis, study the papers and works published by Jin Shi. 2. Follow the prescriptions of Jin Shi clinic, learn and record the teacher's thoughts of diagnosis and treatment, medication characteristics, and summarize the experience of treating uveitis.
Result:
Compared with the healthy control group, the DNA of IL-17RC promoter region in patients with ocular sarcoidosis was hypomethylated, and the difference was statistically significant (P 0.01); 2. In the peripheral blood of the main immune cells, the expression of IL-17RC on the surface of CD8 + T cells in patients with ocular sarcoidosis was higher than that in the healthy control group, the difference was statistically significant (P 0.05). 3. Compared with the healthy control group, the serum level of IL-17 in patients with ocular sarcoidosis increased significantly (P 0.01). 4. HE staining indicated that there were typical granulomatous lesions in the ocular tissues of patients with ocular sarcoidosis, and the active inflammatory tissues were chorioretinal scar (C-R scar), IHC staining suggested inflammation. IL-17RC deposition was observed in active retinal tissues. RT-PCR assay showed that the level of IL-17RC mRNA transcription in retinal tissues of patients with active inflammation was significantly increased (P 0.01); 5. Compared with the blank group without IL-17 stimulation, the surface of CD8+T cells in patients with ocular sarcoidosis and normal people after IL-17 stimulation was significantly higher (P 0.01). The expression of IL-17RC increased, but there was a significant difference between the normal group and the normal group (P0.01).
The second part: Professor Jin Ming advocates taking "western medicine differentiates disease, traditional Chinese medicine differentiates syndrome" as the center, using the western medicine system examination, ophthalmology specialized instrument and Chinese medicine "look, smell, ask, cut" four diagnoses concurrently, obtains the clinical first-hand medical history and the material, and advocates not to limit to the eye, should link the ophthalmopathy with the whole, comprehensive examination. Professor Jin Ming pointed out that in the diagnosis, not only the TCM syndrome differentiation should be based on the theory of TCM, but also the course should be divided into different stages, combined with specialized instruments, macroscopic syndrome differentiation and microscopic examination. In the aspect of syndrome differentiation and typing, Jin Shi does not stick to the current common typing, but according to the specific syndrome and course of disease, randomly respond. In the treatment, Jin Shi proposed early intervention to improve the prognosis, especially in the five rounds of the liver and kidney, and flexible use of Longdan Xiegan Tang (pills), Liuwei Dihuang recipe, the effect is very good. In addition, Jin Shi also according to Portugal. The characteristics of hormone application of uveitis were summarized, and the law of synergistic use of traditional Chinese medicine was summarized: early combination of traditional Chinese medicine to play the role of hormones can reduce hormone dosage, prevent disease progression, this period of traditional Chinese medicine to evacuate wind-heat, dispel wind over dampness, mainly to disperse evil. Chinese medicine can reduce the toxic and side effects caused by hormones and immunosuppressive agents and shorten the course of disease. The treatment is mostly purging liver and gallbladder fire and damp-heat, activating blood circulation and removing blood stasis, permeating dampness and detoxifying, and taking both supplements. Characteristic, regulate the immune function of the body, help hormone steady reduction, consolidate curative effect, reduce recurrence rate and complications, this period because patients take hormones for a long time, deficiency of positive qi, so Chinese medicine to nourish the liver and kidney.
Conclusion:
Part I: 1. Hypomethylation of IL-17RC promoter DNA in patients with ocular sarcoidosis suggests its association with ocular sarcoidosis. Hypomethylation of IL-17RC may be an epigenetic marker of ocular sarcoidosis; 2. Increased IL-17RC + CD8 + T cells in peripheral blood participate in the occurrence and development of ocular sarcoidosis; 3. IL-17RC content. The increased expression of IL-17RC in patients with ocular sarcoidosis may be due to high concentration of IL-17 stimulation in peripheral blood.
The second part: for the treatment of uveitis, should be a combination of Chinese and Western, make full use of advantages and avoid disadvantages, multi-pronged, so as to achieve the best results.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R773.9
本文編號(hào)：2198976
[Abstract]:Background:
Sarcoidosis, a chronic disease involving multiple systems and characterized by non-caseous granulomatosis, is one of the common causes of ocular inflammation. Up to 24% of patients with ocular sarcoidosis have severe visual impairment, of which 10% are visually disabled or even blind, but early intervention can significantly improve the prognosis. At present, there are still many difficulties in the diagnosis of ocular sarcoidosis. Skin, peripheral lymph nodes and lung biopsy are the gold standard of diagnosis, but invasive methods will undoubtedly bring some harm to patients. Therefore, the discovery of new diagnostic markers to improve the safety and accuracy of diagnosis, and applied to treatment, with a view to Important clinical significance.
The emergence of epigenetic methods has opened a new chapter in the study of the pathogenesis of diseases. DNA methylation is one of the most prevalent epigenetic modifications in higher animals. IL-17RC exists on the cell membrane in the form of transmembrane proteins. As a co-receptor, IL-17 subtype binds specifically to IL-17 and activates multiple signal transduction pathways. Recent literature suggests that hypomethylation of IL-17RC promoter plays a role in ocular local inflammation, exudation and angiogenesis. We speculate that abnormal DNA methylation of IL-17RC promoter may be involved in the development of ocular sarcoidosis.
Uveitis is the most important manifestation of ocular sarcoidosis. At present, local or systemic application of glucocorticoids is the main treatment, and can be combined with immunosuppressive agents and biological agents, but the side effects are obvious, and prone to hormone dependence and rebound after withdrawal. Workers inherit and carry forward.
Objective:
Part I: To analyze the relationship between DNA methylation in the promoter region of IL-17RC and ocular sarcoidosis, to detect the ability of IL-17 to induce IL-17RC expression, and to explore the role of IL-17RC promoter methylation in the molecular mechanism of ocular sarcoidosis.
The second part: uveitis is the most important clinical manifestation of ocular sarcoidosis. At present, western medicine treatment is limited, so the second part summarizes the clinical experience of Chinese medicine in the treatment of uveitis, to provide help for treatment.
Method:
Part I: Methylation sensitive restriction endonuclease (MSRE) PCR was used to detect the DNA methylation in the promoter region of IL-17RC in the ocular sarcoidosis group and the healthy control group. Flow cytomerty was used to compare the DNA methylation in the promoter region of IL-17RC between the two groups. The expression of IL-17RC protein on the surface of the main immune cells in peripheral blood was detected by enzyme linked immunosorbent assay (ELISA) and hematoxylin-eosin staining (HE). Immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of IL-17RC protein and mRNA in the retina of patients with ocular sarcoidosis, and anti-CD3/anti-CD28 antibodies were used to activate proliferating CD8+T cells in patients with ocular sarcoidosis and normal subjects. The expression of IL-17RC on the surface of the cells was compared by flow cytometry.
The second part: 1. Retrieve and read the literature of Chinese and Western medicine on uveitis, study the papers and works published by Jin Shi. 2. Follow the prescriptions of Jin Shi clinic, learn and record the teacher's thoughts of diagnosis and treatment, medication characteristics, and summarize the experience of treating uveitis.
Result:
Compared with the healthy control group, the DNA of IL-17RC promoter region in patients with ocular sarcoidosis was hypomethylated, and the difference was statistically significant (P 0.01); 2. In the peripheral blood of the main immune cells, the expression of IL-17RC on the surface of CD8 + T cells in patients with ocular sarcoidosis was higher than that in the healthy control group, the difference was statistically significant (P 0.05). 3. Compared with the healthy control group, the serum level of IL-17 in patients with ocular sarcoidosis increased significantly (P 0.01). 4. HE staining indicated that there were typical granulomatous lesions in the ocular tissues of patients with ocular sarcoidosis, and the active inflammatory tissues were chorioretinal scar (C-R scar), IHC staining suggested inflammation. IL-17RC deposition was observed in active retinal tissues. RT-PCR assay showed that the level of IL-17RC mRNA transcription in retinal tissues of patients with active inflammation was significantly increased (P 0.01); 5. Compared with the blank group without IL-17 stimulation, the surface of CD8+T cells in patients with ocular sarcoidosis and normal people after IL-17 stimulation was significantly higher (P 0.01). The expression of IL-17RC increased, but there was a significant difference between the normal group and the normal group (P0.01).
The second part: Professor Jin Ming advocates taking "western medicine differentiates disease, traditional Chinese medicine differentiates syndrome" as the center, using the western medicine system examination, ophthalmology specialized instrument and Chinese medicine "look, smell, ask, cut" four diagnoses concurrently, obtains the clinical first-hand medical history and the material, and advocates not to limit to the eye, should link the ophthalmopathy with the whole, comprehensive examination. Professor Jin Ming pointed out that in the diagnosis, not only the TCM syndrome differentiation should be based on the theory of TCM, but also the course should be divided into different stages, combined with specialized instruments, macroscopic syndrome differentiation and microscopic examination. In the aspect of syndrome differentiation and typing, Jin Shi does not stick to the current common typing, but according to the specific syndrome and course of disease, randomly respond. In the treatment, Jin Shi proposed early intervention to improve the prognosis, especially in the five rounds of the liver and kidney, and flexible use of Longdan Xiegan Tang (pills), Liuwei Dihuang recipe, the effect is very good. In addition, Jin Shi also according to Portugal. The characteristics of hormone application of uveitis were summarized, and the law of synergistic use of traditional Chinese medicine was summarized: early combination of traditional Chinese medicine to play the role of hormones can reduce hormone dosage, prevent disease progression, this period of traditional Chinese medicine to evacuate wind-heat, dispel wind over dampness, mainly to disperse evil. Chinese medicine can reduce the toxic and side effects caused by hormones and immunosuppressive agents and shorten the course of disease. The treatment is mostly purging liver and gallbladder fire and damp-heat, activating blood circulation and removing blood stasis, permeating dampness and detoxifying, and taking both supplements. Characteristic, regulate the immune function of the body, help hormone steady reduction, consolidate curative effect, reduce recurrence rate and complications, this period because patients take hormones for a long time, deficiency of positive qi, so Chinese medicine to nourish the liver and kidney.
Conclusion:
Part I: 1. Hypomethylation of IL-17RC promoter DNA in patients with ocular sarcoidosis suggests its association with ocular sarcoidosis. Hypomethylation of IL-17RC may be an epigenetic marker of ocular sarcoidosis; 2. Increased IL-17RC + CD8 + T cells in peripheral blood participate in the occurrence and development of ocular sarcoidosis; 3. IL-17RC content. The increased expression of IL-17RC in patients with ocular sarcoidosis may be due to high concentration of IL-17 stimulation in peripheral blood.
The second part: for the treatment of uveitis, should be a combination of Chinese and Western, make full use of advantages and avoid disadvantages, multi-pronged, so as to achieve the best results.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R773.9
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