甲泼尼龙对多发性硬化TH17细胞相关细胞因子的影响

发布时间：2018-05-21 13:29

  本文选题：复发缓解型多发性硬化 + 甲泼尼龙　； 参考：《郑州大学》2016年博士论文

【摘要】：多发性硬化(Multiple Sclerosis,MS)是中枢神经系统常见的一种自身免疫性疾病,复发缓解型多发性硬化(RRMS)是MS最常见的类型。该病好发于青壮年劳动力,常反复发病,治疗成本高,易致残。近年研究发现,多发性硬化在国内患病率呈上升趋势。目前,该病的病因和发病机制至今尚未完全明确。随着免疫学研究的进展,新的辅助性T细胞亚群的发现,其与MS发病关系的研究也正成为未来研究的重要方向之一,有望在其中发现新的诊断指标或治疗靶点。新近研究证实,IL-23的致病作用是由于IL-23诱导CD4+T细胞产生IL-17,IL-17才是MS最为关键的致炎细胞因子。但是这些研究均是在实验性自身免疫性脑脊髓炎开展,对于MS的研究较少,且对与其密切相关的IL-17A、IL-23、IL-21、IL-22、IL-6、TGF-β等细胞因子的研究更少。目前国内外对IL-17A、IL-23、IL-6等部分细胞因子在MS患者血清中的含量测定,无对比分析脑脊液和血清中细胞因子的变化,也无MS急性复发期与缓解期细胞因子水平变化的比较,研究的细胞因子较少,设计也不完善。我们同时研究急性期RRMS患者大剂量甲泼尼龙冲击治疗1周后和治疗1月后血清和或脑脊液中细胞因子含量的变化、细胞因子之间的相关性、细胞因子与EDSS相关性、恢复期与急性复发期血清中细胞因子水平的对比研究,研究内容全面,设计合理,为探索MS的发病机制具有重要意义。炎性细胞因子不仅能诱导MS脱髓鞘病变,与MS非脱髓鞘病灶也密切相关。当MS患者轴索、神经元发生损害后,细胞骨架和轴膜上的Tau蛋白、S100蛋白、14-3-3蛋白,神经丝蛋白被释放到脑脊液中,引起继发性免疫反应。这在一定程度能反映损害的程度,提示预后及免疫治疗疗效。目前国内外对ms患者脑脊液和血清中tau蛋白、s100蛋白、14-3-3蛋白或神经丝蛋白的部分研究,但研究的结论并不一致,且未对缓解期ms患者血清中tau蛋白、s100蛋白、14-3-3蛋白或神经丝蛋白的动态观察,也未探寻其与炎性细胞因子之间的关系。本研究通过检测急性复发期及缓解期rrms患者血清中tau蛋白、s100蛋白、14-3-3蛋白、神经丝蛋白水平,探讨甲泼尼龙对其影响tau蛋白、s100蛋白、14-3-3蛋白、神经丝蛋白与edss相关性,探寻炎性细胞因子与ms非脱髓鞘损害相关的依据。在治疗方面,甲泼尼龙仍是目前我国治疗ms急性发作期的常用药物。尽管临床证明甲泼尼龙治疗急性复发期ms有效,但并不能阻止疾病的复发,且治疗机理不明确。本课题中检测rrms患者甲泼尼龙治疗1周前后、1月后两个时间段血清和或脑脊液中il-23、il-17a、il-21、il-22、tgf-β、il-6等th17细胞相关细胞因子水平的变化,以及治疗1月后il-23、il-17a、il-21、il-22、tgf-β、il-6等th17细胞相关细胞因子水平与edss的相关性,进一步探讨甲泼尼龙治疗ms的治疗机理,为探寻ms发病机制、明确治疗靶点以及探索新的治疗靶点有着重要的意义。目的:1、探讨rrms患者甲泼尼龙治疗1周前后il-23、il-17a水平的变化及其与edss的相关性;2、探索rrms患者甲泼尼龙治疗1月前后血清和脑脊液中il-23、il-17a、il-21、il-22、tgf-β、il-6等th17细胞相关细胞因子的变化及这些细胞因子之间的相关性;3、探索rrms患者急性复发期与缓解期血清中il-23、il-17a、il-21、il-22、tgf-β、il-6等th17细胞相关细胞因子的变化及其与rrms再复发的关系;4、探讨甲泼尼龙对rrms患者急性期及恢复期血清中tau蛋白、s100蛋白、14-3-3蛋白、神经丝蛋白水平的影响,及其与edss相关性,与rrms再复发的关系;5、探寻rrms患者血清中il-23、il-17a、il-21、il-22、tgf-β、il-6等th17细胞相关细胞因子导致ms非脱髓鞘损害的依据。方法:本研究分三部分:2011年9月到2014年9月郑州大学第一附属医院住院确诊的rrms患者;发病48h入院,符合2010年mcdonald诊断标准及中华神经科杂志《多发性硬化诊断和治疗中国专家共识(2011版)》诊断标准。共纳入rrms患者104例,其中男35例,女69例,平均年龄31.2±11.5岁;病程2个月到9.5年,平均38.2个月;复发次数2-8次,平均3.9次;脑脊液寡克隆区带阳性55例(阳性率52.88%),104例患者均有新发阳性体征/症状,头颅mri均有新发病灶。第一部分1、设计路线:根据扩展残疾状况量表(edss)把ms分成三组:ms(a)组(edss:4分),ms(b)组(edss:≥4-6分),ms(c)组(edss:≥6-9分),20例神经系统非炎性疾病(nind)患者为对照组,elisa方法检测nind组和大剂量甲泼尼龙冲击治疗rrms患者1周前后血清中il-23和il-17a水平变化,治疗1月后血清和脑脊液中il-23和il-17a水平变化、两者相关性以及其与edss的相关性。2、纳入患者情况:确诊的rrms患者66例根据edss分成三组:ms(a)组27例,其中男8例,女19例,平均年龄(35.20±11.71)岁;ms(b)组28例,其中男11例,女17例,平均年龄(33.40±9.18)岁;ms(c)组11例,其中男3例,女8例,平均年龄(36.60±14.50)岁。3、方法及标本采集:所有纳入患者治疗前采肘静脉血5ml,腰穿抽取脑脊液3ml,立即离心后取上清液分装于分离管内,标记、冻存待测。rrms组患者给予甲泼尼龙针(1.0givggtqd-3,后0.5givgttqd-4),治疗1周时再次采肘静脉血5ml,同样方法保存待测。甲泼尼龙冲击治疗1周后口服强的松片60mg/d,渐递减后1月内停服,同时口服保护胃黏膜保护药,以及补钙、补钾药物治疗,口服强的松治疗3周(共使用激素治疗1月),再次采肘静脉血5ml,腰穿抽取脑脊液3ml,同样方法保存待测。神经系统非炎症疾病(nind)患者20例为对照组。第二部分1、设计路线:检测rrms患者甲泼尼龙冲击治疗前后血清和脑脊液中il-23、il-17a、il-21、il-22、tgf-β、il-6等th17细胞相关细胞因子的变化,以及细胞因子之间的相关性。2、纳入患者情况:共纳入rrms组患者38例,其中男性13例,女性25例,年龄(31.4±9.5)岁。3、方法及标本采集:所有纳入患者治疗前采肘静脉血5ml,腰穿抽取脑脊液3ml,立即离心5后取上清液分装于分离管内,标记、冻存待测。rrms组患者给予甲泼尼龙针(1.0givggtqd-3,后0.5givgttqd-4,后口服强的松片60mg/d,渐递减后1月内停服),同时加用保护胃药物,补钙补钾治疗,口服强的松片治疗3周后(共使用激素治疗1月)再次采肘静脉血5ml,腰穿抽取脑脊液3ml,同样方法保存待测。神经系统非炎症疾病(nind)患者20例作为对照组。第三部分:1、设计方案:参与第一或第二部分研究中的处于缓解期门诊随访的治疗后半年的rrms患者。elisa方法检测nind组和rrms患者治疗前后及缓解期血清中th17细胞相关细胞因子和tau蛋白,s100蛋白,14-3-3蛋白,神经丝蛋白的水平及其与edss相关性。2、纳入患者情况:共纳入rrms随访患者41例,其中男性14例,女性27例,平均年龄(33.6±10.1)岁,41例患者均无新发症状及体征,复查头颅mri均有无发病灶。纳入患者根据edss分成三组:ms(a)组(edss:4分),ms(b)组(edss:≥4-7分),ms(c)组(edss:≥6-9分),其中ms(a)组17例,其中男5例,女12例,平均年龄(33.12±10.28)岁;ms(b)组18例,其中男7例,女11例,平均年龄(36.27±11.29)岁;ms(c)组6例,其中男2例,女4例,平均年龄(35.44±15.70)岁。3、方法及标本采集:所有纳入患者治疗前采肘静脉血5ml,立即经离心后取上清液分装于分离管内,标记、冻存待测。结果:1、甲泼尼龙治疗rrms1周及1月后血清中il-23、il-17a的水平进行性下降,脑脊液中il-23、17a水平较治疗前亦下降,但两者仍高于nind组,rrms患者脑脊液中il-17a水平高于血清;2、甲泼尼龙冲击治疗rrms后,患者edss在ms(a)组和ms(b)组均下降,ms(c)组下降不明显;3、ms组脑脊液中il-17a水平与edss正相关;4、甲泼尼龙治疗rrms后血清和脑脊液中il-17a、il-23、il-21、il-22水平较治疗前下降,仍较nind组高,tgf-β水平较治疗前增高;5、rrms患者治疗前血清和脑脊液中tgf-β水平较nind组低,治疗后脑脊液中tgf-β水平显著高于nind组;6、相关性分析:rrms治疗前血清中il-23与il-17a正相关;脑脊液中il-23、il-17a、il-21两两正相关;7、rrms恢复期,血清和脑脊液中il-23、il-17a、il-21、il-22水平仍高于nind组,tgf-β水平低于nind组,提示恢复期的ms仍存在炎症反应;8、rrms患者血清和脑脊液中il-17a与il-23正相关,脑脊液中il-17a、il-23与il-22正相关,提示这3个炎性细胞因子在rrms发病中存在同向协调关系;9、缓解期rrms患者血清中il-23、il-17a、il-21、il-22水平较急性复发期及治疗后下降,但仍高于nind组;10、rrms患者甲泼尼龙冲击治疗后血清中s100蛋白水平下降,但恢复期仍高于nind组;血清中tau蛋白、14-3-3蛋白、神经丝蛋白水平无明显变化;11、rrms患者血清中il-17a、il-23、il-21、il-22、tgf-β与s100蛋白正相关,s100蛋白水平与edss正相关。结论:1、甲泼尼龙治疗rrms1周和1月后il-23、il-17a水平及edss评分下降,提示il-23、17a与rrms发病相关;2、甲泼尼龙可以通过降低rrms患者il-23、il-17a水平,发挥抗炎作用;3、rrms患者il-17a水平与该疾病严重程度相关;4、rrms患者il-23、il-17a、il-21、il-22、tgf-β与rrms发病相关;5、甲泼尼龙可能通过抑制炎性细胞因子il-23、il-17a、il-21、il-22,上调tgf-β发挥抗炎作用;6、恢复期rrms仍存在高水平的炎性细胞因子,这可能与ms的复发相关;7、rrms患者il-17a、il-23与il-22在rrms发病中存在同向协调关系,提示阻断il-17a、il-23或il-22的炎性通路,有可能阻断疾病进展;8、缓解期rrms患者血清中高水平的il-23、il-17a、il-21、il-22,可能与rrms再复发相关;9、rrms患者血清中s100蛋白水平恢复期仍增高,提示rrms患者持续存在神经胶质细胞增殖反应;10、rrms患者血清中s100蛋白水平能预测rrms患者临床严重程度;11、il-23、il-17a、il-21、il-22等细胞因子与rrms患者炎性脱髓鞘和非脱髓鞘损害均相关。
[Abstract]:Multiple sclerosis (Multiple Sclerosis, MS) is a common autoimmune disease in the central nervous system. Relapsed remission multiple sclerosis (RRMS) is the most common type of MS. This disease occurs in young and middle-aged labor, often recurred, with high cost and disability. Recent studies have found that the prevalence of multiple sclerosis is on the rise in the country. At present, the etiology and pathogenesis of the disease have not yet been fully defined. With the progress of immunology research, the discovery of new auxiliary T cell subsets, the study of its relationship with the pathogenesis of MS is also becoming one of the important directions of future research, and a new diagnostic index or therapeutic target is expected to be found in it. Recent studies have confirmed the pathogeny of IL-23. It is because IL-23 induces the production of IL-17 in CD4+T cells. IL-17 is the most critical inflammatory cytokine of MS. However, these studies are carried out in experimental autoimmune encephalomyelitis. There are few studies on MS, and there are fewer studies on the cytokines such as IL-17A, IL-23, IL-21, IL-22, IL-6, TGF- beta, and so on. IL-17A, IL-23, IL-6 and other cytokines were measured in the serum of MS patients. There was no comparison of the changes in cytokines in the cerebrospinal fluid and serum, nor the comparison of the changes in the level of the MS in the acute and remission stage of the cytokine. The cytokine was less in the study and the design was not perfect. At the same time, we studied the large dose of large dose of nail in the acute phase of RRMS patients. The changes in the content of cytokines in serum and cerebrospinal fluid after 1 weeks and after the treatment of prednisolone after January, the correlation between cytokines, the correlation of cytokines and EDSS, the comparison of the level of cytokines in the recovery period and the acute recurrence period of serum, the content of the research is comprehensive and the design is reasonable, which is important to explore the pathogenesis of MS. Inflammatory cytokines can not only induce MS demyelinating lesions, but also be closely related to non demyelinating lesions of MS. When the axons of MS patients are damaged, the Tau protein, S100 protein, 14-3-3 protein, and neurofilament protein on the cytoskeleton and the axial membrane are released into the cerebrospinal fluid and lead to secondary immune responses. This can reflect the damage to a certain extent. Degree, suggesting prognosis and immunotherapy effect. The research on tau protein, S100 protein, 14-3-3 protein or neurofilament protein in cerebrospinal fluid and serum of MS patients at home and abroad is not consistent, and the dynamic observation of tau protein, S100 egg white, 14-3-3 protein or neurofilament protein in serum of MS patients in remission period has not been observed, nor is the dynamic observation of the serum protein, S100 egg white, 14-3-3 protein or neurofilament protein in the patients with MS To explore the relationship between tau protein, S100 protein, 14-3-3 protein and neurofilament protein in the serum of RRMS patients in the acute recurrence and remission period, and to explore the correlation of tau protein, S100 protein, 14-3-3 protein, neurofilament protein and EDSS, and to explore the non - inflammatory cytokines and MS non - In the treatment, methylprednisolone is still a common drug for the treatment of acute attack of MS in our country. Although it is proved that methylprednisolone is effective in the treatment of acute recurrence of MS, but it does not prevent the recurrence of the disease, and the mechanism of treatment is not clear. In this study, the treatment of methylprednisolone for RRMS patients 1 weeks before and after January, after January The changes in the level of IL-23, IL-17A, IL-21, IL-22, tgf- beta, IL-6 and other cytokines in the serum and cerebrospinal fluid of the two time periods, and the correlation of the level of cytokines related to the cytokines of IL-23, IL-17A, IL-21, IL-22, tgf- beta, and IL-6 after January, and further explore the mechanism of the treatment of methylprednisolone. The pathogenesis, the clear target of treatment and the exploration of new therapeutic targets are of great significance. 1, to explore the changes of IL-23, IL-17A level and the correlation with EDSS before and after the treatment of methylprednisolone in RRMS patients. 2, to explore IL-23, IL-17A, IL-21, IL-22, tgf- beta, IL-6, etc. in the serum and cerebrospinal fluid of RRMS patients before and after January Change of cell related cytokines and the correlation between these cytokines. 3, explore the changes in serum IL-23, IL-17A, IL-21, IL-22, tgf- beta, IL-6 and other Th17 cell related cytokines in the serum of RRMS patients at acute recurrence and remission stage, and the relationship with RRMS re recurrence; 4, to explore the tau of methylprednisolone in the acute and convalescent serum of RRMS patients. The effect of protein, S100 protein, 14-3-3 protein, neurofilament level and its correlation with EDSS and the recurrence of RRMS; 5, explore the basis of IL-23, IL-17A, IL-21, IL-22, tgf- beta, IL-6 and other Th17 cell related cytokines in the serum of RRMS patients. Methods: This study was divided into three parts: September 2011 to September 2014 The hospitalized RRMS patients in the First Affiliated Hospital of State University were hospitalized with 48h admission, which met the 2010 McDonald diagnostic criteria and the Chinese Journal of Neurology, the Chinese expert consensus (2011 Edition) for multiple sclerosis diagnosis and treatment. 104 cases of RRMS patients were included, including 35 males and 69 females with an average age of 31.2 + 11.5 years; the course of disease was 2 months to 9.5 years. The average 38.2 months were 2-8 times, an average of 3.9 times, 55 cases of positive cerebrospinal fluid oligoclonal bands (positive rate 52.88%), 104 patients had new positive signs / symptoms, and the head MRI had new lesions. The first part 1, the design route was divided into three groups: ms (a) group (edss:4), MS (b) group (edss: > 4-6) according to the extended disability scale (EDSS). The MS (c) group (edss: > 6-9), 20 cases of non inflammatory disease (nind) of the nervous system as the control group. The ELISA method was used to detect the changes in serum IL-23 and IL-17A levels before and after 1 weeks of RRMS patients in the nind group and the large dose methylprednisolone impact treatment. The changes of IL-23 and IL-17A levels in the serum and cerebrospinal fluid after January were treated and their correlation with EDSS .2 was included in the patients: 66 patients with RRMS were divided into three groups according to EDSS: 27 cases in group MS (a), of which 8 were male and 19 in women (35.20 + 11.71) years, and 28 in group B (b), including 11 men, 17 women, average age (33.40 + 9.18) years and MS (c), including male 3, women,.3, methods and specimen collection: All the patients were taken 5ml of the elbow vein blood before treatment, and the cerebrospinal fluid (3ml) was extracted from the lumbar puncture, then the supernatant was removed in the separation tube immediately after the centrifugation. The patients were treated with methylprednisolone needle (1.0givggtqd-3, 0.5givgttqd-4) after the cryopreservation in.Rrms group. The same method was preserved for 1 weeks after 1 weeks. After taking oral prednisone tablets 60mg/d, gradually decreasing in January, taking oral protection of gastric mucosa, calcium supplementation, potassium supplement, oral administration of prednisone for 3 weeks (using hormone therapy in January), 5ml of elbow vein blood, 3ml of cerebrospinal fluid in the lumbar puncture, and 20 cases of non inflammatory (nind) patients. Control group. Second part 1, design route: test the changes of IL-23, IL-17A, IL-21, IL-22, tgf- beta, IL-6 and other Th17 cell related cytokines in serum and cerebrospinal fluid before and after the treatment of methylprednisolone in RRMS patients, and the correlation between the cytokines and.2, including 38 cases in the RRMS group, including 13 males and 25 women in the RRMS group. Age (31.4 + 9.5) years old.3, method and specimen collection: all the patients were taken 5ml of the elbow vein blood before treatment, and the cerebrospinal fluid (3ml) was extracted from the lumbar puncture, then the supernatant was removed in the separation tube immediately after 5 centrifugation, and the patients in group.Rrms were given the 1.0givggtqd-3, 0.5givgttqd-4, and then oral prednisone 60mg/d, gradually decreasing in January. At the same time, combined with protective stomach medicine, calcium supplement and potassium supplement, 3 weeks after oral administration of prednisone tablets (using hormone therapy in January), the 5ml of the elbow vein was recovered, 3ml of cerebrospinal fluid was extracted from the waist, and the same method was preserved. 20 cases of non inflammatory disease (nind) of the nervous system were used as the control group. The third part: 1, the design was involved in the first or second. In some studies, the.Elisa method of RRMS patients in the remission outpatient follow-up period was used to detect the serum Th17 cell related cytokines and tau protein, the level of S100 protein, 14-3-3 protein, neurofilament protein, and EDSS associated.2 in the serum of nind and RRMS patients before and after the treatment and remission period. The patients were included in the case of RRMS follow up. There were 41 patients, including 14 male and 27 female, average age (33.6 + 10.1) years, 41 patients had no new symptoms and signs, and there were no lesions in the skull MRI. The patients were divided into three groups according to EDSS: ms (a), MS (b) group (edss: > 4-7), MS (c) group (edss: > 6-9), including 17 cases of MS (5, 12, average). Age (33.12 + 10.28) years, MS (b) group 18 cases, including 7 men, 11 women, average age (36.27 + 11.29) years, MS (c) group 6 cases, including 2 men, 4 cases, average age (35.44 + 15.70) years of.3, method and specimen collection: all the patients were collected before the treatment of the elbow vein blood 5ml, immediately after centrifugation and the supernatant in the separation tube, marked, frozen to be measured. Results: 1, the level of IL-23 and IL-17A in serum of rrms1 and January decreased, and the level of il-23,17a in cerebrospinal fluid was also lower than that before treatment, but both of them were still higher than that in group nind. The level of IL-17A in the cerebrospinal fluid of RRMS patients was higher than that in serum; 2, after the methylprednisolone shock treatment RRMS, the patients were all decreased in MS (a) group and MS group. 3, IL-17A level in the cerebrospinal fluid of MS group was positively correlated with EDSS; 4, the level of IL-17A, IL-23, IL-21, IL-22 in the serum and cerebrospinal fluid after RRMS was lower than that before the treatment. The level of tgf- beta was higher than that before the treatment, and the level of tgf- beta was higher than that before the treatment. 5, RRMS patients had lower levels of tgf- beta in the blood and cerebrospinal fluid before treatment and the cerebrospinal fluid after treatment. Gf- beta level was significantly higher than that in nind group; 6, correlation analysis: IL-23 and IL-17A were positive in serum before RRMS treatment; IL-23, IL-17A, IL-21 22 in cerebrospinal fluid were positively correlated; 7, RRMS recovery period, IL-23, IL-17A, IL-21, and IL-21 in serum and cerebrospinal fluid were still higher than those in the group. IL-17A was positively correlated with IL-23 in the patient's serum and cerebrospinal fluid, IL-17A and IL-23 in cerebrospinal fluid were positively correlated with IL-22, suggesting that the 3 inflammatory cytokines have the same coordination relationship in the pathogenesis of RRMS; 9, the serum IL-23, IL-17A, IL-21, IL-22 levels of RRMS patients in the remission stage were lower than those in the acute relapse period and after treatment, but still higher than those in the nind group; 10. The serum S100 protein level decreased after the shock treatment, but the recovery period was still higher than that in the nind group, and there was no significant change in serum tau protein, 14-3-3 protein and neurofilament level. 11, the serum IL-17A, IL-23, IL-21, IL-22, tgf- beta were positively related to S100 protein in the serum of RRMS patients, and the level of S100 protein was positively correlated. Conclusion: 1, methylprednisolone was treated for the week. And after January IL-23, IL-17A level and EDSS score declined, suggesting that il-23,17a was associated with RRMS; 2, methylprednisolone could play an anti-inflammatory effect by reducing IL-23, IL-17A level in patients with RRMS; 3, IL-17A levels in RRMS patients were associated with the severity of the disease; 4 By inhibiting inflammatory cytokines IL-23, IL-17A, IL-21, IL-22, and up regulation of tgf- beta, the anti-inflammatory effect is played. 6, there are still high levels of inflammatory cytokines in the recovery phase RRMS, which may be associated with the recurrence of MS; 7, IL-17A, IL-23 and IL-22 in RRMS patients are in the same direction in the pathogenesis of RRMS. 8, the high level of IL-23, IL-17A, IL-21, IL-22 in the sera of RRMS patients in the remission period may be associated with the recurrence of RRMS; 9, the level of S100 protein in the serum of RRMS patients is still higher, suggesting that the RRMS patients continue to have the proliferation of glial cells; 10, S100 protein levels in the serum of the RRMS patients can predict the clinical manifestation of the RRMS patients. Severity, 11, IL-23, IL-17A, IL-21, IL-22 and other cytokines were correlated with inflammatory demyelination and non demyelination in RRMS patients.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.51
，

本文编号：1919387