【摘要】： 淋巴細(xì)胞的活化不僅需要來自于抗原受體[包括T細(xì)胞受體(T cell receptor,TCR)與B細(xì)胞受體(B cell receptor,BCR)]的信號(hào),還需要來自于共刺激或共抑制性分子的第二信號(hào),這些第二信號(hào)共同調(diào)控著淋巴細(xì)胞活化的程度、質(zhì)量及持續(xù)性。對(duì)T細(xì)胞而言,其表面的共刺激和共抑制分子不僅包括了CD28、CTLA-4(cytotoxicT-lymphocyte antigen-4)等免疫球蛋白(immunoglobulin,Ig)超家族成員,還包括了CD27、CD30、4-1BB和Fas等腫瘤壞死因子受體(tumor necrosis factor receptor,TNFR)-TNF家族成員。過去一直認(rèn)為免疫球蛋白超家族成員主要接受來自抗原遞呈細(xì)胞(antigen-presenting cell,APC)和外周組織上表達(dá)的B7家族蛋白的信號(hào),而TNFR-TNF超家族成員除極個(gè)別的受體,如神經(jīng)生長因子受體結(jié)合神經(jīng)妥樂平、HVEM(herpesvius entry mediator)可與Ⅰ型單純皰疹病毒(herpes simplex virus typeⅠ,HSVI)糖蛋白D(glycoprotein D)結(jié)合外,幾乎均只與本家族成員發(fā)生聯(lián)系。這些表面分子調(diào)節(jié)T細(xì)胞活化的一般規(guī)律是Ig超家族成員在T細(xì)胞活化早期決定其活化或抑制;而TNFR-TNF超家族成員則在T細(xì)胞活化晚期維持活化狀態(tài)或誘導(dǎo)調(diào)亡,該超家族成員的這一功能特點(diǎn)主要是因?yàn)槠浯蟛糠殖蓡T都是在T細(xì)胞活化后誘導(dǎo)表達(dá)。 BTLA(B and T lymphocyte attenuator)是新近發(fā)現(xiàn)的一Ig超家族抑制性受體,其雖與CTLA-4具有相似的結(jié)構(gòu),但其配體卻不是經(jīng)典的B7家族成員,而是TNFR-TNF超家族的HVEM。這是迄今為止發(fā)現(xiàn)的唯一一對(duì)Ig超家族成員與TNFR-TNF超家族成員相互作用的受配體。除BTLA外,HVEM還有另一個(gè)重要的膜結(jié)合分子——LIGHT,傳遞的信號(hào)維持T細(xì)胞的活化。通過和BTLA和LIGHT的結(jié)合,HVEM扮演一個(gè)雙相調(diào)節(jié)T細(xì)胞激活的角色。對(duì)BTLA-HVEM復(fù)合物的晶體結(jié)構(gòu)進(jìn)行分析,提示HVEM有可能能同時(shí)結(jié)合LIGHT和BTLA,體外以純化蛋白進(jìn)行實(shí)驗(yàn),也的確發(fā)現(xiàn)三者可形成復(fù)合物。UL144是人巨細(xì)胞病毒上一段和HVEM具有同源性的長獨(dú)特型序列,UL144結(jié)合BTLA,但不結(jié)合LIGHT,并且抑制T細(xì)胞的增殖,選擇性的模擬了HVEM的共抑制功能。 UL144基因產(chǎn)物與BTLA結(jié)合,在體外能抑制T細(xì)胞增殖,并且效果強(qiáng)于HVEM。由于BTLA表達(dá)于分化階段的CD_4~+T細(xì)胞,并在Th1型細(xì)胞獲得永久表達(dá)。在本研究中,我們考慮擬通過轉(zhuǎn)基因的方式誘導(dǎo)DC表達(dá)UL144,觀察其是否可能成為一種耐受型DC,或表現(xiàn)出某些耐受型特征,這種DC能否介導(dǎo)免疫抑制作用。在本課題中,我們擬選用實(shí)驗(yàn)性自身免疫性心肌炎為模型,研究通過調(diào)控UL144-BTLA通路重建自身免疫耐受,實(shí)現(xiàn)對(duì)Th1型反應(yīng)為主的自身免疫性疾病進(jìn)行治療。 在各種用于細(xì)胞轉(zhuǎn)染的載體中,重組腺病毒對(duì)DC具有良好的感染效率,且易于構(gòu)建、純化并獲得較高滴度,因而成為本研究首選的載體。本研究設(shè)計(jì)以hCMVDNA為模板,通過PCR擴(kuò)增出UL144全長片段;用常規(guī)分子生物學(xué)方法構(gòu)建了UL144全長片段的腺病毒表達(dá)載體(pAdEasy-1系統(tǒng));腺病毒載體經(jīng)HEK293細(xì)胞包裝產(chǎn)生含UL144片段的重組腺病毒Ad-CMV-UL144,用氯化銫密度梯度離心法獲得純化的高滴度腺病毒;采用TCID_(50)方法對(duì)重組腺病毒滴度進(jìn)行檢測(cè);運(yùn)用AD-CMV-UL144感染DC,研究其表型變化以及細(xì)胞因子分泌情況;通過感染DC,與T細(xì)胞進(jìn)行混合淋巴反應(yīng)研究其抑制活性;用純化的豬心肌肌凝蛋白免疫Balb/c小鼠,誘導(dǎo)實(shí)驗(yàn)性自身免疫性心肌炎動(dòng)物模型的產(chǎn)生,并分別于再次免疫后0～2天與14～16天經(jīng)尾靜脈注射重組腺病毒感染的DC,觀察Ad-CMV-UL144對(duì)實(shí)驗(yàn)性自身免疫性心肌炎的治療效果,研究其在體內(nèi)的抑制作用。 第一部分?jǐn)y帶UL144基因片段的重組腺病毒載體的構(gòu)建與純化 本部分采用常規(guī)分子生物學(xué)方法,先從CMV-DNA陽性的病人外周血提取cDNA,根據(jù)Primer3軟件設(shè)計(jì)的引物,PCR擴(kuò)增獲得UL144目的片段;經(jīng)pMD18-T載體獲得多克隆位點(diǎn),裝載進(jìn)入重組穿梭質(zhì)粒pAdTrack-CMV。以PmeⅠ酶切帶目的片段的穿梭質(zhì)粒pAdTrack-CMV后,與重組腺病毒骨架質(zhì)粒pAdEasy-1共轉(zhuǎn)化感受態(tài)大腸桿菌DH-5α,發(fā)生同源重組獲得攜帶UL144基因的重組腺病毒骨架,經(jīng)鑒定后純化該骨架質(zhì)粒,以內(nèi)切酶PacⅠ線性化骨架,轉(zhuǎn)染HEK293細(xì)胞獲得完整的重組腺病毒顆粒UL144。重組的腺病毒轉(zhuǎn)入HeLa細(xì)胞中,經(jīng)RT-PCR鑒定UL144基因成功表達(dá)。重組腺病毒經(jīng)過大量擴(kuò)增,收取細(xì)胞,PBS重懸,反復(fù)凍融3次后,經(jīng)CsCl雙密度梯度超速離心純化。通過有限稀釋法測(cè)定半數(shù)組織細(xì)胞感染量(TCID_(50)),判定病毒滴度(Reed-Muenech法),AD-CMV-UL144的滴度為3×10~(10)/mL,對(duì)照AD-GFP的滴度為1×10~(10)/mL。 第二部分重組腺病毒介導(dǎo)表達(dá)UL144蛋白對(duì)小鼠骨髓來源的樹突狀細(xì)胞功能的影響 本部分實(shí)驗(yàn)以重組腺病毒介導(dǎo)UL144轉(zhuǎn)染小鼠DC,觀察其對(duì)DC免疫表型和分泌細(xì)胞因子的影響;觀察UL144基因修飾的DC抗原提呈能力的改變及對(duì)效應(yīng)性T細(xì)胞刺激能力的變化。我們采用GM-CSF、IL-4體外擴(kuò)增Balb/c小鼠骨髓來源的DC,培養(yǎng)過程中將其感染攜帶UL144編碼序列的重組腺病毒載體,設(shè)正常DC及GFP對(duì)照病毒處理DC為對(duì)照。培養(yǎng)6天的未成熟DC,加入脂多糖(LPS)繼續(xù)培養(yǎng)24小時(shí),誘導(dǎo)為成熟DC。結(jié)果發(fā)現(xiàn):DC細(xì)胞表面CD40、CD80(B7.1)、CD86(B7.2)、Ia~d(MHCⅡ)的表達(dá)水平隨LPS誘導(dǎo)成熟而明顯上調(diào),而UL144基因修飾的DC細(xì)胞CD40、CD80、CD86、Ia~d的表達(dá)水平低于對(duì)照組,并對(duì)LPS誘導(dǎo)的上述分子的表達(dá)水平上調(diào)有一定抑制作用,說明其能抑制DC成熟。UL144基因修飾的DC分泌TNF-α、IL-6和IL-1β的能力減弱,該DC與OVA_(323-339)肽特異反應(yīng)性淋巴細(xì)胞的共培養(yǎng)實(shí)驗(yàn)表明,UL144基因修飾的DC能直接抑制OVA_(323-339)肽特異性淋巴細(xì)胞的增殖,提示UL144基因修飾的DC可抑制效應(yīng)性,該作用部分可能是通過TNF-α和IL-6分泌減少而引起。我們的實(shí)驗(yàn)從免疫表型、細(xì)胞因子分泌、抗原提呈功能等方面證明,UL144基因修飾的DC具有維持相對(duì)未成熟狀態(tài)的能力,并減弱了對(duì)抗原特異性T效應(yīng)細(xì)胞的刺激功能。 第三部分UL144蛋白修飾的樹突狀細(xì)胞對(duì)實(shí)驗(yàn)性自身免疫性心肌炎的防治 選擇6周齡的Balb/c小鼠,分別于0天和第7天皮下注射200μg純化的豬心肌肌凝蛋白,制備實(shí)驗(yàn)性自身免疫性心肌炎動(dòng)物模型。分別于初次免疫后的兩周內(nèi),按每周1次經(jīng)小鼠尾靜脈注射5×10~6個(gè)重組腺病毒介導(dǎo)表達(dá)UL144蛋白的DC,并同時(shí)以空載病毒Ad-GFP轉(zhuǎn)染的DC作為其對(duì)照組,再設(shè)立單純免疫組與正常組對(duì)照。小鼠于初次免疫后第21天眼球取血后,脫頸處死,解剖獲取心臟與脾臟。心肌病理檢查與血漿cTnI濃度判斷心肌病損程度,測(cè)定小鼠血漿抗心肌肌凝蛋白抗體的滴度與總IgG的量判斷小鼠的體液免疫功能,測(cè)定小鼠脾臟單個(gè)核細(xì)胞對(duì)Con A和心肌肌凝蛋白刺激的增殖能力及細(xì)胞因子的分泌能力反映小鼠細(xì)胞免疫功能,流式細(xì)胞術(shù)測(cè)定脾及淋巴結(jié)的淋巴細(xì)胞亞群的活化狀況。 雖然Ad-CMV-UL144處理組和Ad-CMV-GFP對(duì)照組有幾只小鼠因不明原因死亡,但結(jié)果顯示,UL144基因修飾的DC能夠減輕小鼠心肌病損程度,使肌凝蛋白自身抗體水平均明顯下降、脾單個(gè)核細(xì)胞對(duì)心肌肌凝蛋白的刺激明顯受抑、引流淋巴結(jié)與脾臟中活化的T、B細(xì)胞明顯下降。這些結(jié)果表明UL144基因修飾的DC能夠減輕心肌炎的病損程度,對(duì)肌凝蛋白誘導(dǎo)的實(shí)驗(yàn)性自身免疫性心肌炎具有一定的治療效果。
[Abstract]:Lymphocyte activation requires not only signals from antigen receptors (including T cell receptors (TCR) and B cell receptors (BCR), but also second signals from costimulatory or co-inhibitory molecules, which together regulate the degree, quality and persistence of lymphocyte activation. The co-stimulatory and co-inhibitory molecules on the surface include not only immunoglobulin (Ig) superfamily members such as CD28 and CTLA-4, but also tumor necrosis factor receptor (TNFR) TNF family members such as CD27, CD30, 4-1BB and Fas. Members of the protein superfamily mainly receive signals from antigen-presenting cell (APC) and B7 family proteins expressed in peripheral tissues, while members of the TNFR-TNF superfamily can bind to neurotrophin by a few receptors, such as nerve growth factor receptor, and HVEM (herpes vius entry mediator) can bind to herpes simplex virus type I (her). In addition to the binding of glycoprotein D (HSVI) to PES simplex virus type I, almost all of them are associated with members of this family. Or induction of apoptosis, this functional feature of the superfamily members is mainly due to the fact that most of its members are induced to express after T cell activation.
BTLA (B and T lymphocyte attenuator) is a recently discovered inhibitory receptor of the Ig superfamily. Although it has a similar structure to CTLA-4, its ligand is not a member of the classical B7 family, but the HEVM of the TNFR-TNF superfamily. This is the only pair of Ig superfamily members that has been found so far to interact with the members of the TNFR-TNF superfamily. In addition to BTLA, HVEM also has another important membrane binding molecule, LIGHT, which transmits signals to maintain T cell activation. By binding to BTLA and LIGHT, HVEM plays a biphasic role in regulating T cell activation. The analysis of the crystal structure of BTLA-HVEM complex suggests that HVEM may be able to bind both LIGHT and BTLA in vitro and be pure. UL144 is a long idiotypic sequence homologous to HVEM. UL144 binds to BTLA but does not bind to LIGHT and inhibits T cell proliferation. It selectively mimics the co-inhibition function of HVEM.
UL144 gene products bind to BTLA and inhibit T cell proliferation in vitro, and the effect is stronger than that of HVEM. Because BTLA is expressed in differentiated CD 4~+ T cells and is permanently expressed in Th1 cells, in this study, we considered the possibility of inducing DC to express UL144 by transgene to become a tolerant DC, or an epitope. In this study, we will choose experimental autoimmune myocarditis as a model to study the reconstruction of autoimmune tolerance by regulating the UL144-BTLA pathway to achieve the treatment of autoimmune diseases with Th1-type response.
In this study, the recombinant adenovirus was designed to amplify the full-length fragment of UL144 by PCR using hCMV DNA as template, and constructed the full-length fragment of UL144 by conventional molecular biological methods. Adenovirus expression vector (pAdEasy-1 system); adenovirus vector was packaged by HEK293 cells to produce recombinant adenovirus Ad-CMV-UL144 containing UL144 fragment, and purified adenovirus with high titer was obtained by cesium chloride density gradient centrifugation; the titer of recombinant adenovirus was detected by TCID_ (50) method; DC was infected by AD-CMV-UL144 to study the phenotype changes. And the secretion of cytokines. Through DC infection, mixed lymphocyte reaction with T cells was used to study the inhibitory activity. * Balb/c mice were immunized with purified porcine cardiac myosin, and induced the production of experimental autoimmune myocarditis animal models. The recombinant adenovirus was injected into the caudal vein 0~2 days and 14~16 days after re immunization respectively. To observe the therapeutic effect of Ad-CMV-UL144 on experimental autoimmune myocarditis and study its inhibitory effect in vivo.
Part one construction and purification of recombinant adenovirus vector carrying UL144 gene fragment
In this part, the conventional molecular biology method was used to extract the cDNA from peripheral blood of CMV-DNA positive patients. According to primers designed by Primer 3 software, UL144 target fragments were amplified by PCR; polyclonal sites were obtained by pMD18-T vector and loaded into the recombinant shuttle plasmid pAdTrack-CMV. The recombinant adenovirus plasmid carrying UL144 gene was obtained by homologous recombination with the recombinant adenovirus cytoskeleton plasmid pAdEasy-1. After identification, the recombinant adenovirus plasmid was purified and linearized with endonuclease Pac I. The recombinant adenovirus granule UL144 was transfected into HEK293 cells. UL144 gene was successfully expressed in eLa cells by RT-PCR. The recombinant adenovirus was amplified, collected, suspended with PBS and purified by CsCl double density gradient centrifugation after repeated freezing and thawing for three times. The titer of 10~ (10) /mL and control AD-GFP was 1 x 10~ (10) /mL.
Part 2 Effect of recombinant adenovirus-mediated expression of UL144 protein on the function of bone marrow-derived dendritic cells in mice
In this part, the effects of recombinant adenovirus-mediated UL144 transfection on DC immunophenotype and cytokine secretion were observed, and the changes of antigen presenting ability and stimulating ability of effector T cells were observed. The recombinant adenovirus vector carrying UL144 coding sequence was infected and treated with normal DC and GFP as control. Immature DC cultured for 6 days and added lipopolysaccharide (LPS) for 24 hours were induced to mature DC. The results showed that the expression levels of CD40, CD80 (B7.1), CD86 (B7.2), Ia~d (MHC II) on the surface of DC cells were significantly higher with LPS-induced maturation. The expression levels of CD40, CD80, CD86 and Ia~d in UL144-modified DC cells were lower than those in the control group, and the up-regulation of the above-mentioned molecules induced by LPS was inhibited to some extent, indicating that UL144-modified DC could inhibit the maturation of DC. The ability of secreting TNF-a, IL-6 and IL-1 beta in UL144-modified DC cells was weakened, and the specific reactive lymph nodes between the DC and OVA_ (323-339) peptide were fine. Co-culture experiments showed that UL144 gene-modified DC could directly inhibit the proliferation of OVA_ (323-339) peptide-specific lymphocytes, suggesting that UL144 gene-modified DC could inhibit the proliferation of OVA_ (323-339) peptide-specific lymphocytes, which may be partly due to the reduction of TNF-a and IL-6 secretion. It has been proved that UL144 gene modified DC has the ability to maintain relatively immature state and weaken the stimulation function to antigen-specific T-effector cells.
The third part is the prevention and treatment of experimental autoimmune myocarditis by UL144 protein modified dendritic cells.
6 week old Balb/c mice were injected subcutaneously with 200 * g purified porcine cardiac myosin at 0 days and seventh days respectively. The experimental autoimmune myocarditis models were prepared. After two weeks of the first immunization, the UL144 protein DC was expressed by 5 x 10~6 recombinant adenoviral vector 1 times a week, and at the same time, no load was observed. The DC transfected with virus Ad-GFP was used as the control group, and the simple immunization group was set up as the control group. The mice were sacrificed on the 21st day after the first immunization. The heart and spleen were dissected. Myocardial pathological examination and plasma cTnI concentration were used to judge the degree of myocardial lesion, and the titer of anti-myocardial coagulation protein antibody and total IgG were determined. The humoral immune function of mice was assessed by measuring the proliferation and cytokine secretion of splenic mononuclear cells stimulated by ConA and cardiac myosin. The activation of lymphocyte subsets in spleen and lymph nodes was measured by flow cytometry.
Although several mice in the Ad-CMV-UL144 treatment group and the AD-CMV-GFP control group died of unknown causes, the results showed that UL144 gene-modified DC could alleviate the degree of cardiomyopathy, reduce the level of autoantibodies to myosin, inhibit the stimulation of splenic mononuclear cells to myosin, and drain lymph nodes and spleens. These results indicate that UL144 gene modified DC can reduce the degree of myocarditis and has a certain therapeutic effect on experimental autoimmune myocarditis induced by myosin.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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