IL-17RC低甲基化影响葡萄膜炎的机制研究及中药治疗葡萄膜炎的临床体会
发布时间：2018-08-23 11:40
【摘要】：背景： 结节病,是一种累及多系统并以非干酪样肉芽肿为病理特点的慢性疾病,是引发眼局部炎症的常见原因之一,高达24%的眼结节病患者会出现严重视力损害,其中10%可视力致残甚至失明,但早期干预可明显改善预后。眼结节病最主要的表现为葡萄膜炎。目前眼结节病的诊断仍存在诸多难点,皮肤、外周淋巴结以及肺部病理组织活检是诊断金标准,但侵入性方式无疑会给病人带来一定的伤害。因此,发现新的诊断标志物,用于提高诊断安全性和准确性,并应用于治疗,具有重要的临床意义。 表观遗传学方法的出现,为研究疾病的发病机制开启了新的篇章,DNA甲基化是高等动物中普遍存在的基因表观遗传修饰方式之一。IL-17RC以跨膜蛋白形式存在于细胞膜上,作为共受体与IL-17亚型特异性结合,可以激活多条信号转导通路,促进炎症反应的发生。最新文献表明IL-17RC启动子区低甲基化在眼局部炎症、渗出、新生血管形成的过程中发挥了一定作用。我们推测,IL-17RC启动子区的DNA甲基化异常可能参与眼结节病的发生发展。 葡萄膜炎是眼结节病最主要的表现形式,目前治疗以局部或者全身应用糖皮质激素为主,可联合免疫抑制剂以及生物制剂,但副作用均较明显,且易产生激素依赖性以及停药后反跳。中医药在治疗葡萄膜炎方面具有明显优势,值得中医工作者继承和发扬。 目的： 第一部分：分析IL-17RC启动子区DNA甲基化状态与眼结节病相关性,检测IL-17诱导IL-17RC表达的能力,初步探讨IL-17RC启动子区甲基化在眼结节病分子水平机制中的作用。 第二部分：葡萄膜炎是眼结节病的最主要临床表现,目前西药治疗手段有限,故第二部分内容总结中医药治疗葡萄膜炎的临床经验,为治疗提供帮助。 方法： 第一部分：以眼结节病组和健康对照组为观察对象,采用甲基化敏感性限制性内切酶(Methylation sensitive restriction endonuclease, MSRE)PCR法观察两组IL-17RC启动子区DNA甲基化的水平；采用流式细胞术(Flow cytomerty)比较眼结节病患者和正常人外周血主要免疫细胞表面IL-17RC蛋白的表达；采用酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)比较眼结节病患者和正常人血清中IL-17的浓度；采用苏木精-伊红染色法(Hematoxylin-eosin staining, HE)观察静止期和炎症活跃期眼结节病患者眼组织的形态结构,采用免疫组化染色(Immunohistochemistry, IHC)和逆转录聚合酶链反应(Reverse transcription polymerase chain reaction, RT-PCR)检测视网膜中IL-17RC蛋白和mRNA的表达；以抗CD3/抗CD28抗体活化增殖眼结节病患者和正常人CD8+T细胞并体外培养3天,其中刺激组和空白组分别加入IL-17、培养基,以流式细胞术比较其表面IL-17RC表达量的差异。 第二部分：1.检索并阅读葡萄膜炎的中西医文献,研读金师发表的论文和著作。2.跟随金师门诊抄方,学习并记录老师临证时诊疗思路、用药特点,将其治疗葡萄膜炎的经验进行总结。 结果： 第一部分：1.相较于健康对照组,眼结节病患者IL-17RC启动子区DNA存在低甲基化修饰,差异有明显统计学意义(P0.01)；2.外周血主要免疫细胞中,眼结节病患者CD8+T细胞表面表达的IL-17RC较健康对照组的表达量升高,差异有统计学意义(P0.05)；3.与健康对照组相比,眼结节病患者中血清中IL-17浓度升高,结果具有显著统计学差异(P0.01)；4.HE染色提示眼结节病患者眼组织中存在典型肉芽肿病变,且活跃的炎症组织有脉络膜视网膜瘢痕(Chorioretinal scar, C-R scar), IHC染色提示炎症活跃期视网膜组织可见IL-17RC的沉积,RT-PCR检测证实炎症活跃期患者的视网膜组织IL-17RC mRNA转录水平明显增高,差异具有显著统计学意义(P0.01)；5.相较于未用IL-17刺激的空白组,经IL-17刺激后的眼结节病患者以及正常人的CD8+T细胞表面表达的IL-17RC均有所增多,但只有正常人刺激组的结果具有统计学差异(P0.01)。 第二部分：金明教授临证时主张以“西医辨病、中医辨证”为中心,用西医系统检查、眼科专科仪器与中医“望、闻、问、切”四诊并行,获取临床第一手病史及资料,且提倡切勿局限于眼部,应将眼病与整体相联,综合审视。葡萄膜炎是一类非常复杂之疾病,病因涉及外邪侵袭、湿热毒瘀内蕴、脏腑虚弱等,病位主要为肝胆肾,病机繁复、病情缠绵。在诊断方面,金明教授提出不仅要依据中医理论辨证分型,还应将病程细分为各期,并结合专科仪器,宏观辨证与微观检查互参,多层面把握病情。在辨证分型方面,金师不拘泥于目前常用分型,而是根据具体证候和病程,随机应变。在治疗方面,金师提出早期干预改善预后,在五轮之中尤重肝肾,并灵活运用龙胆泻肝汤(丸)、六味地黄类方,收效甚好。此外,金师还根据葡萄膜炎的激素应用特点,总结中药协同使用的规律：早期联合中药发挥类激素作用,可减轻激素用量,阻止疾病进展,此期中药多治以疏散风热、祛风胜湿,以散邪为主。发作期的特点是以大剂量口服激素控制炎症,其重型需加用免疫抑制剂,中药可减少由激素和免疫抑制剂带来的毒副作用,缩短病程,治则多为清泻肝胆实火和湿热、活血化瘀、渗湿解毒,攻补兼施。在后期的激素减量过程中,针对此时较易产生的激素依赖性以及突然停药带来的反跳,可突出中医药的扶正特性,调节机体免疫功能,协助激素平稳减量,巩固疗效,降低复发率及并发症的发生,此期因患者久服激素,正气亏虚,故中药多治以培补元气、滋补肝肾。 结论： 第一部分：1.眼结节病患者IL-17RC启动子区DNA存在低甲基化修饰,提示其与眼结节病的具有相关性,IL-17RC的低甲基化可作为眼结节病的表观遗传学标记物；2.外周血增多的IL-17RC+CD8+T细胞参与了眼结节病的发生发展过程；3. IL-17RC含量与炎症的活跃程度或疾病的严重程度相关联；4.眼结节病患者IL-17RC的表达增高可能是由于外周血高浓度的IL-17刺激所导致。 第二部分：针对葡萄膜炎的治疗,应中西合璧,扬长避短,多管齐下,从而达到最优效果。
[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.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R773.9
本文编号：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.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R773.9
【参考文献】
相关期刊论文 前10条
1 曾樨良;;眼病与湿[J];成都中医学院学报;1981年01期
2 罗耀红;朱惠安;;中西医结合治疗前葡萄膜炎39例临床观察[J];中医药导报;2007年08期
3 吴奇志;李梅;;龙胆泻肝配方颗粒治疗急性前葡萄膜炎31例总结[J];湖南中医杂志;2011年01期
4 赵经梅;王志;;试论瞳神紧小症[J];辽宁中医杂志;1989年02期
5 温海玲;;中西医结合治疗急性前葡萄膜炎45例临床观察[J];内蒙古中医药;2012年06期
6 周澜华;从肝论治虹膜睫状体炎[J];辽宁中医杂志;1997年09期
7 应慧星;;十珍汤眼科治验举隅[J];辽宁中医药大学学报;2007年03期
8 陆萍;邹菊生运用和营清热法治疗葡萄膜炎的经验[J];上海中医药杂志;2005年06期
9 陈亚琴;丹栀逍遥散加味治疗慢性顽固性葡萄膜炎[J];陕西中医;2000年04期
10 孙华;李健辉;;五原学说在色素膜炎治疗上的应用[J];实用医技杂志;2007年24期
相关博士学位论文 前1条
1 郭维;消痰散结方对胃癌P16基因甲基化的影响[D];第二军医大学;2010年
，本文编号：2198976
本文链接：https://www.wllwen.com/yixuelunwen/yank/2198976.html
最近更新
教材专著
