Kvl.3多肽疫苗对多发性硬化的治疗作用及其免疫药理机制的研究
发布时间：2018-11-01 20:27
【摘要】：第一部分Kv1.3多肽性疫苗的制备及其诱导的抗体特异性的鉴定目的Kv1.3离子通道为电压门控钾通道,主要表达于淋巴细胞、巨噬细胞等免疫相关细胞并调控细胞的活化、分化、增殖及迁移等多种生理功能。因此,Kv1.3作为研发自身免疫性疾病治疗的新靶点受到广泛关注。本部分以课题组前期实验筛选出的hKv1.3关键肽段作为抗原决定表位,与PADRE序列连接制备特异性抑制Kv1.3通道的多肽性疫苗并鉴定其诱导抗体能否特异性结合于大鼠Kvl.3通道。方法将前期筛选出的Kv1.3肽段与PADRE序列耦联合成为线性多肽性疫苗并主动免疫Lewis大鼠和BABL/c小鼠,酶联免疫吸附法(ELISA)检测大鼠和小鼠提取的血清中anti-Kv1.3抗体的滴度；收集大鼠所有抗血清并通过饱和硫酸铵沉淀和免疫亲和层析法纯化抗血清以获得anti-Kv1.3抗体。通过质粒转染建立稳定表达rKv1.3通道蛋白的HEK293细胞系(HEK/rKv1.3),采用细胞免疫荧光法和Western Blot法检测纯化的anti-Kv1.3抗体能否特异性识别结合rKv1.3蛋白。结果PADRE-hKv1.3多肽性疫苗免疫大鼠和小鼠后均诱导出anti-Kv1.3抗体,大鼠抗体滴度高于小鼠抗体滴度；饱和硫酸铵沉淀和免疫亲和层析法提纯获得纯化的anti-Kv1.3抗体,免疫荧光法证实anti-Kvl.3抗体能在细胞水平上与rKvl.3蛋白特异性结合,Western Blot法验证anti-KV1.3抗体能在蛋白水平上与rKv1.3蛋白特异性结合。结论筛选出的hKv1.3关键肽段与PADRE序列构建出的多肽性疫苗能够成功诱导Kv1.3特异性抗体的产生,并能特异性识别rKv1.3通道蛋白。第二部分Kv1.3多肽性疫苗生物安全性的评价目的明确PADRE-hKv1.3多肽性疫苗是否对免疫大鼠各脏器及其功能造成损伤,对其潜在的毒副作用进行检测。方法PADRE-hKv1.3多肽性疫苗免疫Lewis大鼠3次、每次间隔2周,免疫过程进行大鼠体重和一般情况的监测；最后一次免疫后2周进行大鼠心电检测及胸部X线放射检测；取血检测大鼠血常规和血生化：处死大鼠将各相关脏器分离取出并肉眼观察脏器形态特征并记录各脏器重量系数,之后多聚甲醛固定石蜡包埋进行HE染色病理检测。结果各组大鼠(正常组、假免疫组、PADRE-hKv1.3免疫组)在整个免疫期间一般情况良好,体重增加相当,体表及肺部未出现感染病灶。PADRE-hKv1.3多肽性疫苗免疫未对大鼠心脏大体形态、超微结构及心电功能产生不利影响。PADRE-hKv1.3多肽性疫苗注射未对大鼠肝、脾、肾和大脑等脏器造成损害,而各组大鼠肺脏均出现轻微充血表现。PADRE-hKv1.3多肽性疫苗免疫对大鼠血细胞及血生化指标无不良影响。结论PADRE-hKv1.3多肽性疫苗对免疫动物各脏器及其功能没有显著毒副作用。第三部分Kv1.3多肽性疫苗对实验性自身免疫性脑脊髓炎(EAE)的治疗作用及其免疫药理机制研究目的PADRE-hKv1.3多肽治疗性疫苗通过阻断Kv1.3通道选择性作用于致病性免疫细胞,本部分实验通过构建MS动物病理模型EAE来明确该疫苗对自身免疫性疾病的治疗作用及其免疫药理机制。方法Lewis大鼠完成PADRE-hKv1.3多肽治疗性疫苗3次免疫后,以豚鼠匀浆液作为免疫抗原构建EAE模型,记录整个病程每天的临床评分、体重、EAE发病时间、发病率及死亡率等。免疫后第14天留取脊髓组织HE染色观察炎症浸润程度；固蓝髓鞘染色(LFB)观察脊髓脱髓鞘面积；组织免疫荧光检测脊髓T淋巴细胞和小胶质细胞／巨噬细胞浸润活化的情况,CD68和iNOS或Arg-1荧光双染检测小胶质细胞/巨噬细胞的分型。第14天处死大鼠提取脾脏和CNS中淋巴细胞,T淋巴细胞分型采用流式细胞术进行检测；脾淋巴细胞和CNS组织相关细胞因子的mRNA表达量采用RT-PCR法进行检测。免疫后第28天,采用MBP和NeuN蛋白荧光染色检测各组恢复期MBP降解和神经元存活情况并进行Bielschowsky氏轴突银染观察轴突缺失面积。结果采用豚鼠脊髓匀浆液作为免疫抗原成功构建了EAE动物模型,各组EAE发病率均为100%。与单纯EAE和假免疫+EAE组相比,PADRE-hKv1.3疫苗预处理改善了EAE临床症状,显著地减轻脊髓组织的炎症浸润和脱髓鞘；减少脊髓炎症区域T细胞的浸润,使脾脏和CNS中的淋巴细胞向CD4+IL-10+Treg细胞偏移分化并抑制Thl和Th17细胞的分化。EAE组和假免疫+EAE组促炎细胞因子IL-17A和IL-1p的mRNA在脾脏淋巴细胞和CNS组织中表达均增高,而给予PADRE-hKvl.3多肽疫苗预处理后这些细胞因子mRNA表达均下降；相反,PADRE-hKv1.3多肽疫苗预处理使脾脏淋巴细胞中抑炎因子IL-10 mRNA表达升高,CNS中IL-10和FoxP3基因表达量均增高。PADRE-hKv1.3多肽疫苗预处理使脊髓炎症区域活化的小胶质细胞/巨噬细胞数量减少并使小胶质细胞/巨噬细胞向M2型偏移分化。恢复期病理检测显示PADRE-hKvl.3多肽疫苗预处理显著改善MBP降解、轴突缺失和神经元死亡等EAE特征性病理变化。结论PADRE-hKv1.3多肽性疫苗影响炎症细胞的浸润及其分型,改善脊髓组织炎症浸润微环境并显著减轻脊髓病理改变,进而发挥其对EAE/MS病变的治疗作用改善其临床症状。
[Abstract]:The preparation of the first partial Kv1.3 polypeptide vaccine and its induced antibody specificity are Kv1.3 ion channels as voltage gated potassium channels, which are mainly expressed in immune related cells such as lymphocytes, macrophages and the like, and regulate the activation, differentiation, proliferation and migration of the cells. Therefore, Kv1.3 is widely concerned as a new target for developing autoimmune diseases. In this part, the key peptide fragment of hKv1.3, which was screened out by the previous experiment group, was used as the epitope of antigen to prepare the polypeptide vaccine with specific inhibition of Kv1.3 channel with PADRE sequence and to identify whether the induced antibody can specifically bind to the Kvl. 3 channel in the rat. Methods The anti-Kv1.3 antibody in serum extracted from rats and mice was detected by enzyme-linked immunosorbent assay (ELISA). All antiserum was collected and the antiserum was purified by saturated calmodulin precipitation and immunoaffinity chromatography to obtain anti-Kv1.3 antibody. A stable expression of rKv1.3 channel protein was established by plasmid transfection (HEK/ rKv1.3), and the purified anti-Kv1.3 antibody was detected by cell immunofluorescence and Western blot. Results Anti-Kv1.3 antibody was induced in mice and mice immunized with PADRE-hKv1.3 polypeptide vaccine. Anti-Kv1.3 antibody was purified by saturated salt precipitation and immunoaffinity chromatography, and purified anti-Kv1.3 antibody was purified. Immunofluorescence assay confirmed that anti-Kvl. 3 antibody could be specifically combined with rKvl. 3 protein on the cell level, and the anti-KV1.3 antibody could be specifically combined with rKv1.3 protein on the protein level. Conclusion The screened hKv1.3 critical peptide segment and PADRE sequence can successfully induce the generation of Kv1.3 specific antibody, and can specifically recognize the rKv1.3 channel protein. The purpose of the evaluation of the biological safety of the second part Kv1.3 polypeptide vaccine is to determine whether the PADRE-hKv1.3 polypeptide vaccine has damage to the organs and functions of the immune rats and detect the potential toxic and side effects. Methods PADRE-hKv1.3 polypeptide vaccine was used to immunize Lewis rats for 3 times, and the body weight and general condition were monitored every 2 weeks. Rats were killed, the organs were separated and removed with naked eyes, the organ weight coefficient was recorded, and then HE stained pathological examination was carried out on paraffin embedded paraffin. Results All groups of rats (normal group, sham-immunized group, PADRE-hKv1.3 immune group) had good general condition during the whole immunization period, and the body surface and lung were not infected with infection. The immune function of PADRE-hKv1.3 polypeptide vaccine did not adversely affect the general morphology, ultrastructure and ECG function of rat heart. The injection of PADRE-hKv1.3 polypeptide vaccine did not cause damage to liver, spleen, kidney, brain and other organs of rats, and slight hyperemia was observed in the lungs of each group. PADRE-hKv1.3 polypeptide vaccine has no adverse effect on blood cell and blood biochemical indexes of rats. Conclusion PADRE-hKv1.3 polypeptide vaccine has no obvious toxic and side effects on the organs and functions of immune animals. The third part of Kv1.3 polypeptide vaccine plays a role in the treatment of experimental autoimmune encephalomyelitis (EAE) and its immune pharmacological mechanism, and the PADRE-hKv1.3 polypeptide therapeutic vaccine can selectively act on pathogenic immune cells by blocking Kv1.3 channels. In this part, the therapeutic effect of the vaccine on autoimmune diseases and its immune pharmacological mechanism were determined by constructing the pathological model of MS animal. Methods After three immunizations of PADRE-hKv1.3 polypeptide therapeutic vaccine, Lewis rats were immunized with the homogenate of guinea pig as immune antigen, and the clinical scores, body weight, morbidity, morbidity and mortality of the whole course were recorded. HE staining of spinal cord tissue was observed on the 14th day after immunization to observe the degree of inflammation and infiltration. LFB was used to observe the surface area of spinal cord, and immunofluorescence was used to detect the infiltration and activation of T lymphocytes and microglial cells/ macrophages. CD68 and iNOS or Arg-1 fluorescent double staining examined the typing of microglial cells/ macrophages. Lymphocytes in spleen and CNS were extracted on Day 14, and T lymphocyte subsets were detected by flow cytometry; the mRNA expression of cytokines in spleen lymphocytes and CNS tissues was detected by RT-PCR. In the 28th day after immunization, the degradation and neuronal survival of each group were detected by means of fluorescent staining and TUNEL staining, and the missing area of axons was observed by Bielschowsky's neurite staining. Results The animal model was successfully constructed using guinea pig spinal cord homogenate as an immune antigen, and the incidence rate of each group was 100%. The PADRE-hKv1.3 vaccine pretreatment improves the clinical symptoms of the spinal cord, obviously reduces inflammation and infiltration of the spinal cord tissue, and reduces the infiltration of T cells in the inflammatory area of the spinal cord, Lymphocytes in the spleen and CNS were differentiated from CD4 + IL-10 + Treg cells and inhibited the differentiation of Thl and Th17 cells. The mRNA expression of IL-17A and IL-1p increased in spleen lymphocytes and CNS tissues, while the expression of these cytokines was decreased after pretreatment with PADRE-hKvl. 3 polypeptide vaccine. The expression of IL-10 and FoxP3 in the spleen lymphocytes increased with the pretreatment of PADRE-hKv1.3 polypeptide vaccine. The pretreatment of the PADRE-hKv1.3 polypeptide vaccine reduced the number of microglial cells/ macrophages activated in the spinal inflammatory region and differentiated the microglial cells/ macrophages towards the M2 type. The pathological examination of recovery period showed that PADRE-hKvl. 3 polypeptide vaccine pretreatment significantly improved the pathological changes of nerve cell degradation, loss of axons and neuronal death. Conclusion PADRE-hKv1.3 polypeptide vaccine affects the infiltration and classification of inflammatory cells, improves the microenvironment of inflammatory infiltration of spinal cord tissue and significantly reduces the pathological changes of spinal cord, and then plays its role in improving the therapeutic effect of PADRE-hKv1.3 polypeptide vaccine to improve its clinical symptoms.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.51
本文编号：2305014
[Abstract]:The preparation of the first partial Kv1.3 polypeptide vaccine and its induced antibody specificity are Kv1.3 ion channels as voltage gated potassium channels, which are mainly expressed in immune related cells such as lymphocytes, macrophages and the like, and regulate the activation, differentiation, proliferation and migration of the cells. Therefore, Kv1.3 is widely concerned as a new target for developing autoimmune diseases. In this part, the key peptide fragment of hKv1.3, which was screened out by the previous experiment group, was used as the epitope of antigen to prepare the polypeptide vaccine with specific inhibition of Kv1.3 channel with PADRE sequence and to identify whether the induced antibody can specifically bind to the Kvl. 3 channel in the rat. Methods The anti-Kv1.3 antibody in serum extracted from rats and mice was detected by enzyme-linked immunosorbent assay (ELISA). All antiserum was collected and the antiserum was purified by saturated calmodulin precipitation and immunoaffinity chromatography to obtain anti-Kv1.3 antibody. A stable expression of rKv1.3 channel protein was established by plasmid transfection (HEK/ rKv1.3), and the purified anti-Kv1.3 antibody was detected by cell immunofluorescence and Western blot. Results Anti-Kv1.3 antibody was induced in mice and mice immunized with PADRE-hKv1.3 polypeptide vaccine. Anti-Kv1.3 antibody was purified by saturated salt precipitation and immunoaffinity chromatography, and purified anti-Kv1.3 antibody was purified. Immunofluorescence assay confirmed that anti-Kvl. 3 antibody could be specifically combined with rKvl. 3 protein on the cell level, and the anti-KV1.3 antibody could be specifically combined with rKv1.3 protein on the protein level. Conclusion The screened hKv1.3 critical peptide segment and PADRE sequence can successfully induce the generation of Kv1.3 specific antibody, and can specifically recognize the rKv1.3 channel protein. The purpose of the evaluation of the biological safety of the second part Kv1.3 polypeptide vaccine is to determine whether the PADRE-hKv1.3 polypeptide vaccine has damage to the organs and functions of the immune rats and detect the potential toxic and side effects. Methods PADRE-hKv1.3 polypeptide vaccine was used to immunize Lewis rats for 3 times, and the body weight and general condition were monitored every 2 weeks. Rats were killed, the organs were separated and removed with naked eyes, the organ weight coefficient was recorded, and then HE stained pathological examination was carried out on paraffin embedded paraffin. Results All groups of rats (normal group, sham-immunized group, PADRE-hKv1.3 immune group) had good general condition during the whole immunization period, and the body surface and lung were not infected with infection. The immune function of PADRE-hKv1.3 polypeptide vaccine did not adversely affect the general morphology, ultrastructure and ECG function of rat heart. The injection of PADRE-hKv1.3 polypeptide vaccine did not cause damage to liver, spleen, kidney, brain and other organs of rats, and slight hyperemia was observed in the lungs of each group. PADRE-hKv1.3 polypeptide vaccine has no adverse effect on blood cell and blood biochemical indexes of rats. Conclusion PADRE-hKv1.3 polypeptide vaccine has no obvious toxic and side effects on the organs and functions of immune animals. The third part of Kv1.3 polypeptide vaccine plays a role in the treatment of experimental autoimmune encephalomyelitis (EAE) and its immune pharmacological mechanism, and the PADRE-hKv1.3 polypeptide therapeutic vaccine can selectively act on pathogenic immune cells by blocking Kv1.3 channels. In this part, the therapeutic effect of the vaccine on autoimmune diseases and its immune pharmacological mechanism were determined by constructing the pathological model of MS animal. Methods After three immunizations of PADRE-hKv1.3 polypeptide therapeutic vaccine, Lewis rats were immunized with the homogenate of guinea pig as immune antigen, and the clinical scores, body weight, morbidity, morbidity and mortality of the whole course were recorded. HE staining of spinal cord tissue was observed on the 14th day after immunization to observe the degree of inflammation and infiltration. LFB was used to observe the surface area of spinal cord, and immunofluorescence was used to detect the infiltration and activation of T lymphocytes and microglial cells/ macrophages. CD68 and iNOS or Arg-1 fluorescent double staining examined the typing of microglial cells/ macrophages. Lymphocytes in spleen and CNS were extracted on Day 14, and T lymphocyte subsets were detected by flow cytometry; the mRNA expression of cytokines in spleen lymphocytes and CNS tissues was detected by RT-PCR. In the 28th day after immunization, the degradation and neuronal survival of each group were detected by means of fluorescent staining and TUNEL staining, and the missing area of axons was observed by Bielschowsky's neurite staining. Results The animal model was successfully constructed using guinea pig spinal cord homogenate as an immune antigen, and the incidence rate of each group was 100%. The PADRE-hKv1.3 vaccine pretreatment improves the clinical symptoms of the spinal cord, obviously reduces inflammation and infiltration of the spinal cord tissue, and reduces the infiltration of T cells in the inflammatory area of the spinal cord, Lymphocytes in the spleen and CNS were differentiated from CD4 + IL-10 + Treg cells and inhibited the differentiation of Thl and Th17 cells. The mRNA expression of IL-17A and IL-1p increased in spleen lymphocytes and CNS tissues, while the expression of these cytokines was decreased after pretreatment with PADRE-hKvl. 3 polypeptide vaccine. The expression of IL-10 and FoxP3 in the spleen lymphocytes increased with the pretreatment of PADRE-hKv1.3 polypeptide vaccine. The pretreatment of the PADRE-hKv1.3 polypeptide vaccine reduced the number of microglial cells/ macrophages activated in the spinal inflammatory region and differentiated the microglial cells/ macrophages towards the M2 type. The pathological examination of recovery period showed that PADRE-hKvl. 3 polypeptide vaccine pretreatment significantly improved the pathological changes of nerve cell degradation, loss of axons and neuronal death. Conclusion PADRE-hKv1.3 polypeptide vaccine affects the infiltration and classification of inflammatory cells, improves the microenvironment of inflammatory infiltration of spinal cord tissue and significantly reduces the pathological changes of spinal cord, and then plays its role in improving the therapeutic effect of PADRE-hKv1.3 polypeptide vaccine to improve its clinical symptoms.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.51
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