本文選題：特發(fā)性血小板減少性紫癜 + 口服耐受��； 參考：《山東大學(xué)》2008年博士論文

【摘要】： 特發(fā)性血小板減少性紫癜(idiopathic thrombocytopenic purpura,ITP),也稱自身免疫性血小板減少性紫癜(autoimmune thrombocytopenic purpura,AITP),是臨床最為常見的出血性疾病,約占出血性疾病總數(shù)的30%。本病的實質(zhì)是一種自身免疫性疾病,其發(fā)病機制復(fù)雜,主要是由于人體自身免疫耐受機制被打破,體液免疫和細(xì)胞免疫紊亂,產(chǎn)生抗自身血小板抗體或激活細(xì)胞毒T細(xì)胞,導(dǎo)致血小板破壞增加和/或生成障礙。 目前,國內(nèi)外對于ITP的一、二線治療以腎上腺皮質(zhì)激素、靜脈丙種球蛋白及脾切除為主,但仍有25%～30%的患者上述治療無效或短期內(nèi)復(fù)發(fā),成為難治性ITP,遷延不愈,甚至危及生命,對這部分患者目前有名目繁多的非常規(guī)治療比如免疫抑制劑、骨髓移植等,但是常常缺乏有效性和安全性。因此亟需探索更為有效和安全的治療方法。 自身免疫性疾病的發(fā)生主要與自身免疫耐受的破壞有關(guān),因此去除導(dǎo)致耐受破壞的因素或再次誘導(dǎo)對自身抗原的耐受,有助于控制自身免疫性疾病的發(fā)生和發(fā)展。近年來研究人員從自身免疫性疾病發(fā)病機理的不同角度出發(fā),開始探尋一些新型的免疫治療方法,有些方法已逐步應(yīng)用于臨床試驗。 口服耐受(oral tolerance)是通過腸道粘膜接觸抗原而誘導(dǎo)的一種免疫應(yīng)答低下狀態(tài),是誘導(dǎo)機體產(chǎn)生免疫耐受的重要方法之一�？诜褪艿拇_切機制迄今尚不清楚,目前認(rèn)為其形成機制主要依賴于口服抗原的劑量:低劑量口服抗原可刺激調(diào)節(jié)性T細(xì)胞(包括CD4~+CD25~(high)Tr細(xì)胞、CD8~+CD28~-Ts細(xì)胞、NKT細(xì)胞、_γδT細(xì)胞等)的分化,引起主動性免疫抑制(active suppression),其機制可能是通過分泌抑制性細(xì)胞因子(女HTGF-β、IL-4、IL-10等),誘導(dǎo)以Th2和Th3應(yīng)答為主的效應(yīng),即發(fā)生“免疫偏離”。而這種主動性抑制還能作用于非特異性免疫應(yīng)答,即旁路抑制(bystander suppression);高劑量口服抗原可導(dǎo)致T細(xì)胞克隆缺失(clonal deletion)和/或克隆無能(clonal anergy),誘導(dǎo)耐受的產(chǎn)生。另外,何種機制占優(yōu)勢還與口服抗原的時間、形式及次數(shù)有關(guān)。 口服耐受具有應(yīng)用方便,無明顯毒性,且不必明確特異性自身抗原的獨特優(yōu)點,因此為治療自身免疫性疾病提供了新的途徑和思路�？诜褪軕�(yīng)用于T細(xì)胞介導(dǎo)的自身免疫性疾病動物模型的防治已有不少成功的報道,如自身免疫性腦脊髓炎、膠原或佐劑誘發(fā)的關(guān)節(jié)炎、1型糖尿病(NOD小鼠)、自身免疫性葡萄膜炎、自身免疫性重癥肌無力等,且多發(fā)性硬化、類風(fēng)濕性關(guān)節(jié)炎、1型糖尿病、自身免疫性葡萄膜炎等已有臨床試驗報道。 基于ITP的自身免疫性發(fā)病機制,應(yīng)用口服抗原誘導(dǎo)免疫耐受的研究具有可行性。已有前期研究應(yīng)用口服自身血小板治療ITP,雖然存在獲取足夠自身血小板的困難和血小板保存方法的局限,但是這種方法具備極大的潛在優(yōu)勢,如自身抗原的易接近性(血小板膜糖蛋白)、評價指標(biāo)的客觀性(血小板計數(shù))等,而且除了自身血小板,已有專家指出還可以應(yīng)用同種異體血小板、培養(yǎng)的自身血小板以及分離純化的血小板糖蛋白如GPⅡb/Ⅲa。 ITP動物模型的建立有利于闡明ITP的發(fā)病機制,分析藥物的治療作用,并進(jìn)一步探討新的治療方法。一個理想的ITP動物模型應(yīng)該能模擬人類ITP的所有特點,如易感性、發(fā)病機制和臨床過程,同時具備可操作性和穩(wěn)定性。數(shù)十年來,已有眾多的造模方法應(yīng)用于ITP的研究,如抗血小板血清及多/單克隆抗體造模方法、(NZW×BXSB)F1小鼠自發(fā)模型等。 我們參照國外有關(guān)文獻(xiàn)加以改進(jìn),用主動免疫法建立理想的ITP動物模型,并通過喂食血小板抗原,研究口服耐受對ITP的防治作用,并探討其可能機制。 第一部分免疫性血小板減少(ITP)小鼠模型的建立 目的:建立ITP小鼠模型,初步探討其體液及細(xì)胞免疫機制 方法:應(yīng)用Wistar大鼠血小板(模型組)或PBS(對照組)腹腔免疫CBA/CaJ小鼠,每周取血檢測血常規(guī),監(jiān)測血小板計數(shù)及平均血小板體積(MPV)水平的變化,并應(yīng)用流式細(xì)胞術(shù)檢測血小板相關(guān)抗體(PAIgG),應(yīng)用Western blot技術(shù)檢測血小板洗脫抗體特異性,應(yīng)用CCK-8檢測特異性抗血小板淋巴細(xì)胞增殖反應(yīng),應(yīng)用ELISA試劑盒檢測血漿IFN-γ、IL-2、IL-4、IL-10水平,同時觀察骨髓細(xì)胞形態(tài)學(xué),尤其是巨核細(xì)胞系的數(shù)量和發(fā)育情況。 結(jié)果:與對照組相比,模型組血小板計數(shù)于初次免疫后第3周達(dá)第一個低谷,第6周達(dá)第二個低谷(P＜0.05),第8、9周降至最低(P＜0.01),第10周驟然回升至正常甚至更高,MPV相應(yīng)地逐漸增大再逐漸恢復(fù),而骨髓巨核細(xì)胞形態(tài)和數(shù)量均無顯著變化;免疫機制研究顯示PAIgG顯著增高(P＜0.05),Westernblot顯示血小板洗脫抗體可與與GPIbα(CD42b)分子量相當(dāng)?shù)牡鞍捉Y(jié)合,特異性抗血小板淋巴細(xì)胞增殖反應(yīng)增強(P＜0.05);與免疫前相比,模型組血漿IL-2、IFN-γ、IL-4、IL-10水平均有所增高(P＜0.05),分別于免疫后第6周、第3和7周、第4周、第4周達(dá)峰值,第10周均降至正常水平。 結(jié)論:基于抗原模擬原理成功建立了理想的、穩(wěn)定的、最大程度模擬人體的ITP小鼠模型,不僅發(fā)病過程與人類ITP相似,而且發(fā)病機制亦與人類ITP相符,為ITP的實驗研究提供了有力的工具。 第二部分小鼠GPIbα(CD42b)GST融合蛋白的原核表達(dá)及其誘導(dǎo)ITP小鼠模型口服耐受的初步研究 目的:構(gòu)建小鼠GPIba(CD42b)原核表達(dá)載體,制備GPIba-GST融合蛋白,并探討經(jīng)口服途徑GPIbα-GST融合蛋白對ITP小鼠模型的防治作用及可能機制。 方法:將小鼠GPIbα的開放讀碼框分成四個片段,片段之間有10-15個氨基酸的重疊,經(jīng)RT-PCR擴增目的基因,重組到原核表達(dá)質(zhì)粒pGEX-6P-1中,用限制性內(nèi)切酶酶切和DNA測序法進(jìn)行鑒定,IPTG誘導(dǎo)重組質(zhì)粒pGexGPIba轉(zhuǎn)化的大腸桿菌BL21(DE3),通過SDS-PAGE、Western blot分析表達(dá)產(chǎn)物,大量表達(dá)后經(jīng)谷胱甘肽Sepharose 4B親和層析獲得純化產(chǎn)物。按不同劑量(高、中、低)將ITP小鼠模型分組,每組4-6只,以灌胃針喂食實驗小鼠純化GPIbα/GST融合蛋白(耐受組)或PBS(溶劑對照組),并設(shè)空白對照組(免疫PBS,口服PBS),觀察血小板參數(shù),檢測PAIgG以及血漿IFN-γ、IL-2、IL-4、IL-10、TGF-β水平,同時應(yīng)用流式細(xì)胞技術(shù)檢測外周血、脾臟及腸系膜淋巴結(jié)中CD4~+CD25~(high)調(diào)節(jié)性T細(xì)胞占CD4~+T細(xì)胞的比例(%CD4~+CD25~(high)/CD4~+)。 結(jié)果:限制性內(nèi)切酶酶切和DNA測序分析證實成功獲得小鼠GPIbα四個基因片段,并準(zhǔn)確克隆入pGEX-6P-1,pGexGPIbα經(jīng)誘導(dǎo)表達(dá)出分子量與理論值相符的GST融合蛋白,其中GST/GPIbα1-214和GST/GPIbα306-537為可溶性表達(dá),GST/GPIbα198-315和GST/GPIbα527-734為包涵體表達(dá),用變性復(fù)性方法獲得目的蛋白,另外免疫印跡法證實GST/GPIbα306-537能與抗小鼠GPIbot(CD42b)單克隆抗體特異性結(jié)合,純化后的目的蛋白純度均達(dá)90%以上�？诜褪軐嶒炛�,與溶劑對照組相比,高劑量耐受組血小板減少的幅度緩和,血小板降至最低的時間延遲至第10周,血小板減少的持續(xù)時間縮短至1～2周(P＜0.05),MPV變化不顯著,PAIgG水平降低;調(diào)節(jié)性T細(xì)胞檢測結(jié)果顯示與空白對照組相比,溶劑對照組各組織的%CD4~+CD25~(high)/CD4~+明顯減低(P＜0.05),而高劑量耐受組雖然亦有所減低,但未達(dá)顯著水平;與溶劑對照組相比,高劑量耐受組各組織的%CD4~+CD25~(high)/CD4~+平均水平均有所增加,但無顯著差異;血漿細(xì)胞因子檢測結(jié)果顯示與空白對照組相比,溶劑對照組IL-2、IFN-γ、IL-4、IL-10水平增高(P＜0.01),TGF-β水平降低(P＜0.01),而高劑量耐受組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,但均未達(dá)顯著水平;與溶劑對照組相比,高劑量耐受組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,但無顯著差異。 結(jié)論:成功構(gòu)建pGexGPIbα原核表達(dá)載體,并高效表達(dá)、大量純化GPIbα/GST融合蛋白,該融合蛋白經(jīng)口服途徑可改善ITP小鼠模型的病程和病情,可能是通過活化CD4~+CD25~(high)及其他調(diào)節(jié)性T細(xì)胞分泌抑制性細(xì)胞因子糾正Th1偏離,從而誘導(dǎo)以Th2和Th3應(yīng)答為主的效應(yīng)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392;R554.6

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相關(guān)期刊論文 前2條

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