Kvl.3多肽疫苗對多發(fā)性硬化的治療作用及其免疫藥理機制的研究
發(fā)布時間：2018-11-01 20:27
【摘要】：第一部分Kv1.3多肽性疫苗的制備及其誘導的抗體特異性的鑒定目的Kv1.3離子通道為電壓門控鉀通道,主要表達于淋巴細胞、巨噬細胞等免疫相關細胞并調控細胞的活化、分化、增殖及遷移等多種生理功能。因此,Kv1.3作為研發(fā)自身免疫性疾病治療的新靶點受到廣泛關注。本部分以課題組前期實驗篩選出的hKv1.3關鍵肽段作為抗原決定表位,與PADRE序列連接制備特異性抑制Kv1.3通道的多肽性疫苗并鑒定其誘導抗體能否特異性結合于大鼠Kvl.3通道。方法將前期篩選出的Kv1.3肽段與PADRE序列耦聯合成為線性多肽性疫苗并主動免疫Lewis大鼠和BABL/c小鼠,酶聯免疫吸附法(ELISA)檢測大鼠和小鼠提取的血清中anti-Kv1.3抗體的滴度；收集大鼠所有抗血清并通過飽和硫酸銨沉淀和免疫親和層析法純化抗血清以獲得anti-Kv1.3抗體。通過質粒轉染建立穩(wěn)定表達rKv1.3通道蛋白的HEK293細胞系(HEK/rKv1.3),采用細胞免疫熒光法和Western Blot法檢測純化的anti-Kv1.3抗體能否特異性識別結合rKv1.3蛋白。結果PADRE-hKv1.3多肽性疫苗免疫大鼠和小鼠后均誘導出anti-Kv1.3抗體,大鼠抗體滴度高于小鼠抗體滴度；飽和硫酸銨沉淀和免疫親和層析法提純獲得純化的anti-Kv1.3抗體,免疫熒光法證實anti-Kvl.3抗體能在細胞水平上與rKvl.3蛋白特異性結合,Western Blot法驗證anti-KV1.3抗體能在蛋白水平上與rKv1.3蛋白特異性結合。結論篩選出的hKv1.3關鍵肽段與PADRE序列構建出的多肽性疫苗能夠成功誘導Kv1.3特異性抗體的產生,并能特異性識別rKv1.3通道蛋白。第二部分Kv1.3多肽性疫苗生物安全性的評價目的明確PADRE-hKv1.3多肽性疫苗是否對免疫大鼠各臟器及其功能造成損傷,對其潛在的毒副作用進行檢測。方法PADRE-hKv1.3多肽性疫苗免疫Lewis大鼠3次、每次間隔2周,免疫過程進行大鼠體重和一般情況的監(jiān)測；最后一次免疫后2周進行大鼠心電檢測及胸部X線放射檢測；取血檢測大鼠血常規(guī)和血生化：處死大鼠將各相關臟器分離取出并肉眼觀察臟器形態(tài)特征并記錄各臟器重量系數,之后多聚甲醛固定石蠟包埋進行HE染色病理檢測。結果各組大鼠(正常組、假免疫組、PADRE-hKv1.3免疫組)在整個免疫期間一般情況良好,體重增加相當,體表及肺部未出現感染病灶。PADRE-hKv1.3多肽性疫苗免疫未對大鼠心臟大體形態(tài)、超微結構及心電功能產生不利影響。PADRE-hKv1.3多肽性疫苗注射未對大鼠肝、脾、腎和大腦等臟器造成損害,而各組大鼠肺臟均出現輕微充血表現。PADRE-hKv1.3多肽性疫苗免疫對大鼠血細胞及血生化指標無不良影響。結論PADRE-hKv1.3多肽性疫苗對免疫動物各臟器及其功能沒有顯著毒副作用。第三部分Kv1.3多肽性疫苗對實驗性自身免疫性腦脊髓炎(EAE)的治療作用及其免疫藥理機制研究目的PADRE-hKv1.3多肽治療性疫苗通過阻斷Kv1.3通道選擇性作用于致病性免疫細胞,本部分實驗通過構建MS動物病理模型EAE來明確該疫苗對自身免疫性疾病的治療作用及其免疫藥理機制。方法Lewis大鼠完成PADRE-hKv1.3多肽治療性疫苗3次免疫后,以豚鼠勻漿液作為免疫抗原構建EAE模型,記錄整個病程每天的臨床評分、體重、EAE發(fā)病時間、發(fā)病率及死亡率等。免疫后第14天留取脊髓組織HE染色觀察炎癥浸潤程度；固藍髓鞘染色(LFB)觀察脊髓脫髓鞘面積；組織免疫熒光檢測脊髓T淋巴細胞和小膠質細胞／巨噬細胞浸潤活化的情況,CD68和iNOS或Arg-1熒光雙染檢測小膠質細胞/巨噬細胞的分型。第14天處死大鼠提取脾臟和CNS中淋巴細胞,T淋巴細胞分型采用流式細胞術進行檢測；脾淋巴細胞和CNS組織相關細胞因子的mRNA表達量采用RT-PCR法進行檢測。免疫后第28天,采用MBP和NeuN蛋白熒光染色檢測各組恢復期MBP降解和神經元存活情況并進行Bielschowsky氏軸突銀染觀察軸突缺失面積。結果采用豚鼠脊髓勻漿液作為免疫抗原成功構建了EAE動物模型,各組EAE發(fā)病率均為100%。與單純EAE和假免疫+EAE組相比,PADRE-hKv1.3疫苗預處理改善了EAE臨床癥狀,顯著地減輕脊髓組織的炎癥浸潤和脫髓鞘；減少脊髓炎癥區(qū)域T細胞的浸潤,使脾臟和CNS中的淋巴細胞向CD4+IL-10+Treg細胞偏移分化并抑制Thl和Th17細胞的分化。EAE組和假免疫+EAE組促炎細胞因子IL-17A和IL-1p的mRNA在脾臟淋巴細胞和CNS組織中表達均增高,而給予PADRE-hKvl.3多肽疫苗預處理后這些細胞因子mRNA表達均下降；相反,PADRE-hKv1.3多肽疫苗預處理使脾臟淋巴細胞中抑炎因子IL-10 mRNA表達升高,CNS中IL-10和FoxP3基因表達量均增高。PADRE-hKv1.3多肽疫苗預處理使脊髓炎癥區(qū)域活化的小膠質細胞/巨噬細胞數量減少并使小膠質細胞/巨噬細胞向M2型偏移分化�；謴推诓±頇z測顯示PADRE-hKvl.3多肽疫苗預處理顯著改善MBP降解、軸突缺失和神經元死亡等EAE特征性病理變化。結論PADRE-hKv1.3多肽性疫苗影響炎癥細胞的浸潤及其分型,改善脊髓組織炎癥浸潤微環(huán)境并顯著減輕脊髓病理改變,進而發(fā)揮其對EAE/MS病變的治療作用改善其臨床癥狀。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R744.51
本文編號：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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R744.51
【相似文獻】
相關期刊論文 前10條
1 趙小寧,惠宏襄,徐德忠;多肽疫苗研究進展[J];國外醫(yī)學.預防.診斷.治療用生物制品分冊;1997年06期
2 深白色;;信使[J];中國處方藥;2007年03期
3 王曉勤;抗寄生蟲多肽疫苗的研制策略[J];國外醫(yī)學(寄生蟲病分冊);1994年01期
4 劉慶良;多肽疫苗的設計[J];國外醫(yī)學.預防.診斷.治療用生物制品分冊;1998年01期
5 江南,戴保民,李勝富,方芝茂;賴型鉤體基因重組多肽疫苗動物免疫保護試驗[J];中華微生物學和免疫學雜志;1999年06期
6 呂鳳林,何鳳慈;利用納米材料制作多肽疫苗佐劑的思考[J];生物化學與生物物理進展;2001年06期
7 張劍軍;徐惠綿;鄭志超;趙巖;張濤;王振寧;閻影;;熱休克蛋白90α多肽疫苗的抗腫瘤效應研究[J];山東醫(yī)藥;2010年20期
8 趙蓮君;李琳;鄒征云;;惡性腫瘤個體化多肽疫苗的原理與臨床研究[J];現代腫瘤醫(yī)學;2014年03期
9 蔣常生;;微膠粒乙型肝炎多肽疫苗的制備[J];國外醫(yī)學.生物制品分冊;1982年03期
10 劉寧,劉紅軍,宋磊;腫瘤的多肽疫苗[J];齊魯藥事;2005年04期
相關會議論文 前5條
1 王芳;才子斌;王毅;徐煌;侯立中;顏煒群;;乳腺癌HER-2/neu肽疫苗的實驗研究[A];增強自主創(chuàng)新能力 促進吉林經濟發(fā)展——啟明杯·吉林省第四屆科學技術學術年會論文集（下冊）[C];2006年
2 劉征;馬翠卿;張玲;姚智燕;馮惠東;魏林;;A族鏈球菌多價疫苗的構建及表達[A];河北省免疫學會第六次免疫學大會資料匯編[C];2010年
3 尚小云;;表位改造在腫瘤治療性多肽疫苗研究中的進展[A];第六屆全國免疫學學術大會論文集[C];2008年
4 呂鳳林;;基于HPV-6、11之L1蛋白的多肽疫苗研究[A];2004年中國科學院新生病毒性疾病控制學術討論會摘要集[C];2004年
5 王立新;Walter J．Urba;;清除CD122~+細胞增強DC／多肽疫苗在免疫重建荷瘤小鼠體內誘導的治療性抗腫瘤應答[A];中國免疫學會第五屆全國代表大會暨學術會議論文摘要[C];2006年
相關重要報紙文章 前1條
1 記者 白毅;腫瘤抗原NY-ESO-1b多肽疫苗完成臨床前研究[N];中國醫(yī)藥報;2006年
相關博士學位論文 前3條
1 龍蕊;Kvl.3多肽疫苗對多發(fā)性硬化的治療作用及其免疫藥理機制的研究[D];華中科技大學;2016年
2 王芳;高特異性HER2/neu手性多肽疫苗實驗研究[D];吉林大學;2004年
3 曹勝利;血吸蟲病多肽疫苗的設計與合成研究[D];北京醫(yī)科大學;1997年
相關碩士學位論文 前3條
1 郭沛;納米磷酸鈣佐劑的制備及其輔助多肽疫苗免疫效果的觀察[D];南京農業(yè)大學;2012年
2 吳梅芝;基于EGFR二聚化靶向策略的多肽疫苗構建[D];廣東藥學院;2013年
3 吳孔田;CCR5自體多肽疫苗的構建及其免疫原性的研究[D];第四軍醫(yī)大學;2006年
，本文編號：2305014
本文鏈接：http://sikaile.net/yixuelunwen/shenjingyixue/2305014.html
最近更新
教材專著
