口服抗原诱导免疫性血小板减少模型T细胞耐受的实验研究

发布时间：2018-05-23 08:49

本文选题：特发性血小板减少性紫癜 + 口服耐受　；参考：《山东大学》2008年博士论文

【摘要】： 特发性血小板减少性紫癜(idiopathic thrombocytopenic purpura,ITP),也称自身免疫性血小板减少性紫癜(autoimmune thrombocytopenic purpura,AITP),是临床最为常见的出血性疾病,约占出血性疾病总数的30%。本病的实质是一种自身免疫性疾病,其发病机制复杂,主要是由于人体自身免疫耐受机制被打破,体液免疫和细胞免疫紊乱,产生抗自身血小板抗体或激活细胞毒T细胞,导致血小板破坏增加和/或生成障碍。目前,国内外对于ITP的一、二线治疗以肾上腺皮质激素、静脉丙种球蛋白及脾切除为主,但仍有25%～30%的患者上述治疗无效或短期内复发,成为难治性ITP,迁延不愈,甚至危及生命,对这部分患者目前有名目繁多的非常规治疗比如免疫抑制剂、骨髓移植等,但是常常缺乏有效性和安全性。因此亟需探索更为有效和安全的治疗方法。自身免疫性疾病的发生主要与自身免疫耐受的破坏有关,因此去除导致耐受破坏的因素或再次诱导对自身抗原的耐受,有助于控制自身免疫性疾病的发生和发展。近年来研究人员从自身免疫性疾病发病机理的不同角度出发,开始探寻一些新型的免疫治疗方法,有些方法已逐步应用于临床试验。口服耐受(oral tolerance)是通过肠道粘膜接触抗原而诱导的一种免疫应答低下状态,是诱导机体产生免疫耐受的重要方法之一。口服耐受的确切机制迄今尚不清楚,目前认为其形成机制主要依赖于口服抗原的剂量:低剂量口服抗原可刺激调节性T细胞(包括CD4~+CD25~(high)Tr细胞、CD8~+CD28~-Ts细胞、NKT细胞、_γδT细胞等)的分化,引起主动性免疫抑制(active suppression),其机制可能是通过分泌抑制性细胞因子(女HTGF-β、IL-4、IL-10等),诱导以Th2和Th3应答为主的效应,即发生“免疫偏离”。而这种主动性抑制还能作用于非特异性免疫应答,即旁路抑制(bystander suppression);高剂量口服抗原可导致T细胞克隆缺失(clonal deletion)和/或克隆无能(clonal anergy),诱导耐受的产生。另外,何种机制占优势还与口服抗原的时间、形式及次数有关。口服耐受具有应用方便,无明显毒性,且不必明确特异性自身抗原的独特优点,因此为治疗自身免疫性疾病提供了新的途径和思路。口服耐受应用于T细胞介导的自身免疫性疾病动物模型的防治已有不少成功的报道,如自身免疫性脑脊髓炎、胶原或佐剂诱发的关节炎、1型糖尿病(NOD小鼠)、自身免疫性葡萄膜炎、自身免疫性重症肌无力等,且多发性硬化、类风湿性关节炎、1型糖尿病、自身免疫性葡萄膜炎等已有临床试验报道。基于ITP的自身免疫性发病机制,应用口服抗原诱导免疫耐受的研究具有可行性。已有前期研究应用口服自身血小板治疗ITP,虽然存在获取足够自身血小板的困难和血小板保存方法的局限,但是这种方法具备极大的潜在优势,如自身抗原的易接近性(血小板膜糖蛋白)、评价指标的客观性(血小板计数)等,而且除了自身血小板,已有专家指出还可以应用同种异体血小板、培养的自身血小板以及分离纯化的血小板糖蛋白如GPⅡb/Ⅲa。 ITP动物模型的建立有利于阐明ITP的发病机制,分析药物的治疗作用,并进一步探讨新的治疗方法。一个理想的ITP动物模型应该能模拟人类ITP的所有特点,如易感性、发病机制和临床过程,同时具备可操作性和稳定性。数十年来,已有众多的造模方法应用于ITP的研究,如抗血小板血清及多/单克隆抗体造模方法、(NZW×BXSB)F1小鼠自发模型等。我们参照国外有关文献加以改进,用主动免疫法建立理想的ITP动物模型,并通过喂食血小板抗原,研究口服耐受对ITP的防治作用,并探讨其可能机制。第一部分免疫性血小板减少(ITP)小鼠模型的建立目的:建立ITP小鼠模型,初步探讨其体液及细胞免疫机制方法:应用Wistar大鼠血小板(模型组)或PBS(对照组)腹腔免疫CBA/CaJ小鼠,每周取血检测血常规,监测血小板计数及平均血小板体积(MPV)水平的变化,并应用流式细胞术检测血小板相关抗体(PAIgG),应用Western blot技术检测血小板洗脱抗体特异性,应用CCK-8检测特异性抗血小板淋巴细胞增殖反应,应用ELISA试剂盒检测血浆IFN-γ、IL-2、IL-4、IL-10水平,同时观察骨髓细胞形态学,尤其是巨核细胞系的数量和发育情况。结果:与对照组相比,模型组血小板计数于初次免疫后第3周达第一个低谷,第6周达第二个低谷(P＜0.05),第8、9周降至最低(P＜0.01),第10周骤然回升至正常甚至更高,MPV相应地逐渐增大再逐渐恢复,而骨髓巨核细胞形态和数量均无显著变化;免疫机制研究显示PAIgG显著增高(P＜0.05),Westernblot显示血小板洗脱抗体可与与GPIbα(CD42b)分子量相当的蛋白结合,特异性抗血小板淋巴细胞增殖反应增强(P＜0.05);与免疫前相比,模型组血浆IL-2、IFN-γ、IL-4、IL-10水平均有所增高(P＜0.05),分别于免疫后第6周、第3和7周、第4周、第4周达峰值,第10周均降至正常水平。结论:基于抗原模拟原理成功建立了理想的、稳定的、最大程度模拟人体的ITP小鼠模型,不仅发病过程与人类ITP相似,而且发病机制亦与人类ITP相符,为ITP的实验研究提供了有力的工具。第二部分小鼠GPIbα(CD42b)GST融合蛋白的原核表达及其诱导ITP小鼠模型口服耐受的初步研究目的:构建小鼠GPIba(CD42b)原核表达载体,制备GPIba-GST融合蛋白,并探讨经口服途径GPIbα-GST融合蛋白对ITP小鼠模型的防治作用及可能机制。方法:将小鼠GPIbα的开放读码框分成四个片段,片段之间有10-15个氨基酸的重叠,经RT-PCR扩增目的基因,重组到原核表达质粒pGEX-6P-1中,用限制性内切酶酶切和DNA测序法进行鉴定,IPTG诱导重组质粒pGexGPIba转化的大肠杆菌BL21(DE3),通过SDS-PAGE、Western blot分析表达产物,大量表达后经谷胱甘肽Sepharose 4B亲和层析获得纯化产物。按不同剂量(高、中、低)将ITP小鼠模型分组,每组4-6只,以灌胃针喂食实验小鼠纯化GPIbα/GST融合蛋白(耐受组)或PBS(溶剂对照组),并设空白对照组(免疫PBS,口服PBS),观察血小板参数,检测PAIgG以及血浆IFN-γ、IL-2、IL-4、IL-10、TGF-β水平,同时应用流式细胞技术检测外周血、脾脏及肠系膜淋巴结中CD4~+CD25~(high)调节性T细胞占CD4~+T细胞的比例(%CD4~+CD25~(high)/CD4~+)。结果:限制性内切酶酶切和DNA测序分析证实成功获得小鼠GPIbα四个基因片段,并准确克隆入pGEX-6P-1,pGexGPIbα经诱导表达出分子量与理论值相符的GST融合蛋白,其中GST/GPIbα1-214和GST/GPIbα306-537为可溶性表达,GST/GPIbα198-315和GST/GPIbα527-734为包涵体表达,用变性复性方法获得目的蛋白,另外免疫印迹法证实GST/GPIbα306-537能与抗小鼠GPIbot(CD42b)单克隆抗体特异性结合,纯化后的目的蛋白纯度均达90%以上。口服耐受实验中,与溶剂对照组相比,高剂量耐受组血小板减少的幅度缓和,血小板降至最低的时间延迟至第10周,血小板减少的持续时间缩短至1～2周(P＜0.05),MPV变化不显著,PAIgG水平降低;调节性T细胞检测结果显示与空白对照组相比,溶剂对照组各组织的%CD4~+CD25~(high)/CD4~+明显减低(P＜0.05),而高剂量耐受组虽然亦有所减低,但未达显著水平;与溶剂对照组相比,高剂量耐受组各组织的%CD4~+CD25~(high)/CD4~+平均水平均有所增加,但无显著差异;血浆细胞因子检测结果显示与空白对照组相比,溶剂对照组IL-2、IFN-γ、IL-4、IL-10水平增高(P＜0.01),TGF-β水平降低(P＜0.01),而高剂量耐受组IL-4、IL-10水平增高(P＜0.01),IFN-γ水平有所增高,IL-2、TGF-β水平有所降低,但均未达显著水平;与溶剂对照组相比,高剂量耐受组IL-2、IFN-γ、IL-10水平降低(P＜0.05),TGF-β水平增高(P＜0.05),IL-4水平亦有所增高,但无显著差异。结论:成功构建pGexGPIbα原核表达载体,并高效表达、大量纯化GPIbα/GST融合蛋白,该融合蛋白经口服途径可改善ITP小鼠模型的病程和病情,可能是通过活化CD4~+CD25~(high)及其他调节性T细胞分泌抑制性细胞因子纠正Th1偏离,从而诱导以Th2和Th3应答为主的效应。
[Abstract]:Idiopathic thrombocytopenic purpura (idiopathic thrombocytopenic purpura, ITP), also known as autoimmune thrombocytopenic purpura (autoimmune thrombocytopenic purpura, AITP), is the most common clinical hemorrhagic disease. The essence of the total number of hemorrhagic diseases is an autoimmune disease, the pathogenesis of which is a autoimmune disease. Complex, mainly because the body's own immune tolerance mechanism is broken, humoral immunity and cell immune disorders, producing anti autoplatelet antibodies or activating cytotoxic T cells, resulting in increased platelet destruction and / or formation of obstacles.
At present, the first and second line treatment of ITP at home and abroad is adrenocortical hormone, intravenous gamma globulin and splenectomy, but there are still 25% to 30% of the patients who are invalid or relapsed in the short term. It becomes a refractory ITP, and it does not heal, even endangers life. There are many unconventional treatments such as immunosuppression, such as the immunosuppression in this part of the patients. Agents, bone marrow transplants, etc., but often lack effectiveness and safety. Therefore, it is urgent to explore more effective and safe treatment methods.
The occurrence of autoimmune diseases is mainly related to the destruction of autoimmune tolerance, so it is helpful to control the occurrence and development of autoimmune diseases. In recent years, the researchers began to explore from the different angles of the pathogenesis of autoimmune diseases. Some new immunotherapy methods have been gradually applied to clinical trials.
Oral tolerance (oral tolerance) is an immune response to immune tolerance induced by intestinal mucosal contact antigen. It is one of the important methods to induce immune tolerance. The exact mechanism of oral tolerance is not yet clear. It is believed that its formation mechanism is mainly dependent on the dose of oral antigen: low dose oral antigen can be stimulated. The differentiation of regulatory T cells (including CD4~+CD25~ (high) Tr cells, CD8~+CD28~-Ts cells, NKT cells, Tr Delta T cells, etc.) causes active immune suppression (active suppression), and its mechanism may be induced by secreting inhibitory cytokines (HTGF- beta, IL-4, IL-10, etc.) and inducing the effect of "immune deviation". This active inhibition can also affect the non specific immune response, namely, bystander suppression; high dose oral antigen can lead to T cell clone deletion (clonal deletion) and / or clone inability (clonal anergy), induced tolerance. In addition, what mechanism is dominant in time, form and times of oral antigen. Of
Oral tolerance has the advantages of convenient application, no obvious toxicity, and no specific advantages of specific autoantigens. Therefore, it provides new ways and ideas for the treatment of autoimmune diseases. The prevention and control of oral tolerance used in the prevention and control of autoimmune diseases in T cells has been successfully reported, such as autoimmune cerebrospinal meninges. Arthritis, collagen or adjuvant induced arthritis, type 1 diabetes (NOD mice), autoimmune uveitis, autoimmune myasthenia, and multiple sclerosis, rheumatoid arthritis, type 1 diabetes, and autoimmune uveitis have been reported in clinical trials.
Based on the autoimmune pathogenesis of ITP, it is feasible to use oral antigen to induce immune tolerance. Previous studies have used oral self platelets to treat ITP. Although there are difficulties in obtaining sufficient blood platelets and the limitations of platelet preservation methods, this method has a great potential advantage, such as self antigen. The accessibility (platelet membrane glycoprotein), the objectivity of the evaluation index (platelet count) and so on, and in addition to its own platelets, experts have pointed out that allogeneic platelets, the cultured self platelets and the purified platelet glycoproteins such as GP II B / III A. can also be used.
The establishment of the ITP animal model is beneficial to elucidate the pathogenesis of ITP, to analyze the therapeutic effect of drugs, and to further explore new treatments. An ideal ITP animal model should be able to simulate all the characteristics of human ITP, such as susceptibility, pathogenesis and clinical processes, as well as operational and stability. The modeling method was applied to the research of ITP, such as antiplatelet serum and multi / monoclonal antibody modeling method, (NZW * BXSB) F1 mouse spontaneous model.
We have improved the relevant foreign literature to establish an ideal ITP animal model with active immunization, and study the preventive and therapeutic effects of oral tolerance on ITP by feeding platelet antigens and explore the possible mechanism.
Part one establishment of immune thrombocytopenia (ITP) mouse model
Objective: to establish a ITP mouse model and to explore its humoral and cellular immune mechanisms.
Methods: the Wistar rat platelets (model group) or PBS (control group) were immunized with CBA/CaJ mice intraperitoneally, blood routine was detected every week, the changes of platelet count and average platelet volume (MPV) were monitored, and platelet related antibody (PAIgG) was detected by flow cytometry, and Western blot technique was used to detect the specificity of platelet elution antibody. The specific antiplatelet lymphocyte proliferation reaction was detected by CCK-8, and the level of plasma IFN- gamma, IL-2, IL-4, IL-10 was detected by ELISA kit. The number and development of megakaryocyte, especially the megakaryocyte, were observed.
Results: compared with the control group, the platelet count in the model group reached the first low valley at the first third weeks after the first immunization, reached second low valleys in sixth weeks (P < 0.05), and decreased to the lowest (P < 0.01) at week 8,9. The tenth weeks abruptly recovered to normal or even higher, and the MPV correspondingly increased and then gradually recovered, but the morphology and quantity of megakaryocyte were not significantly changed. The study of immune mechanism showed significant increase in PAIgG (P < 0.05). Westernblot showed that platelet elution antibody could be combined with the protein of GPIb alpha (CD42b), and the proliferation response of specific antiplatelet lymphocyte was enhanced (P < 0.05). Compared with pre immunization, the levels of plasma IL-2, IFN- gamma, IL-4 and IL-10 were increased (P < 0.05), respectively. After sixth weeks, third and seventh weeks, fourth weeks, fourth weeks, the peak reached tenth weeks after immunization.
Conclusion: Based on the principle of antigen simulation, an ideal, stable and maximum simulated ITP mouse model is established. The pathogenesis is similar to human ITP, and the pathogenesis is also consistent with human ITP, which provides a powerful tool for the experimental research of ITP.
Prokaryotic expression of mouse GPIb alpha (CD42b) GST fusion protein in second parts and preliminary study on oral tolerance induced by ITP mouse model
Objective: to construct a mouse GPIba (CD42b) prokaryotic expression vector, to prepare GPIba-GST fusion protein, and to explore the preventive effect and possible mechanism of GPIb alpha -GST fusion protein on ITP mouse model by oral route.
Methods: the open reading frame of GPIb alpha in mice was divided into four segments, and 10-15 amino acids were overlapped between the fragments. The target gene was amplified by RT-PCR and reorganized into the prokaryotic expression plasmid pGEX-6P-1. The recombinant plasmid was identified by restriction endonuclease digestion and DNA sequencing, and IPTG induced the recombinant Escherichia coli BL21 (DE3) transformed by recombinant plasmid pGexGPIba through SDS-PAG. E and Western blot were used to analyze the expression products, and then the purified products were obtained by glutathione Sepharose 4B affinity chromatography. The ITP mice model was grouped in different doses (high, medium, low), and each group of 4-6 mice was treated with gavage needle feeding experimental mice to purify GPIb alpha /GST fusion protein (tolerance group) or PBS (solvent control group), and set up a blank control group (immune PBS,) The platelet parameters were observed and the levels of PAIgG and plasma IFN- gamma, IL-2, IL-4, IL-10, TGF- beta were measured, and the proportion of CD4~+CD25~ (high) regulatory T cells in the spleen and mesenteric lymph nodes in the peripheral blood was detected by flow cytometry, and the proportion of CD4~+CD25~ (high) regulatory T cells in the spleen and mesenteric lymph nodes was proportional to the proportion of CD4~+T cells (%CD4~+ PBS).
Results: four gene fragments of GPIb alpha in mice were successfully obtained by restriction endonuclease digestion and DNA sequencing, and pGexGPIb alpha was accurately cloned into pGEX-6P-1, and pGexGPIb alpha was induced to express the GST fusion protein which was in accordance with the theoretical value, in which GST/GPIb alpha 1-214 and GST/GPIb alpha 306-537 were soluble, GST/GPIb a 198-315 and GST/GPIb alpha 527-734. For inclusion body expression, the target protein was obtained by denaturing refolding method. In addition, the immunoblot assay showed that GST/GPIb alpha 306-537 could be specifically combined with anti mouse GPIbot (CD42b) monoclonal antibody. The purity of the purified target protein was more than 90%. The extent of thrombocytopenia in high dose tolerance group was compared with the solvent tolerance test. The duration of thrombocytopenia was delayed to tenth weeks, the duration of thrombocytopenia was shortened to 1~2 weeks (P < 0.05), the change of MPV was not significant, and the level of PAIgG decreased. The results of regulatory T cell detection showed that the% CD4~+CD25~ (high) /CD4~+ of each tissue in the control group decreased significantly (P < 0.05) compared with the blank control group (P < 0.05), and the high dose was resistant to the control group. Compared with the solvent control group, the average level of%CD4~+CD25~ (high) /CD4~+ in the high dose tolerance group was increased, but there was no significant difference compared with the solvent control group. The results of plasma cytokine detection showed that the level of IL-2, IFN- gamma, IL-4, IL-10 increased (P < 0.01), TGF compared with the blank control group, TGF (P < 0.01), TGF Beta level decreased (P < 0.01), while high dose tolerance group IL-4, IL-10 level increased (P < 0.01), IFN- gamma level increased, IL-2, TGF- beta level decreased, but not significant level. Compared with the solvent control group, the level of IL-2, IFN- gamma and IL-10 decreased (P < 0.05), TGF- beta level increased (0.05), and the level also increased, compared with the solvent control group. But there is no significant difference.
Conclusion: pGexGPIb alpha prokaryotic expression vector is successfully constructed and highly expressed and GPIb alpha /GST fusion protein is purified. The fusion protein can improve the course and condition of the ITP mouse model by oral pathway. It may be induced by activating CD4~+CD25~ (high) and other regulatory T cells to secrete the inhibitory cytokine to correct Th1 deviation, thus inducing Th2 and T. The effect of H3 response.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;R554.6

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