UL144基因修饰的树突状细胞诱导免疫耐受
发布时间：2018-09-14 17:52
【摘要】： 淋巴细胞的活化不仅需要来自于抗原受体[包括T细胞受体(T cell receptor,TCR)与B细胞受体(B cell receptor,BCR)]的信号,还需要来自于共刺激或共抑制性分子的第二信号,这些第二信号共同调控着淋巴细胞活化的程度、质量及持续性。对T细胞而言,其表面的共刺激和共抑制分子不仅包括了CD28、CTLA-4(cytotoxicT-lymphocyte antigen-4)等免疫球蛋白(immunoglobulin,Ig)超家族成员,还包括了CD27、CD30、4-1BB和Fas等肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)-TNF家族成员。过去一直认为免疫球蛋白超家族成员主要接受来自抗原递呈细胞(antigen-presenting cell,APC)和外周组织上表达的B7家族蛋白的信号,而TNFR-TNF超家族成员除极个别的受体,如神经生长因子受体结合神经妥乐平、HVEM(herpesvius entry mediator)可与Ⅰ型单纯疱疹病毒(herpes simplex virus typeⅠ,HSVI)糖蛋白D(glycoprotein D)结合外,几乎均只与本家族成员发生联系。这些表面分子调节T细胞活化的一般规律是Ig超家族成员在T细胞活化早期决定其活化或抑制;而TNFR-TNF超家族成员则在T细胞活化晚期维持活化状态或诱导调亡,该超家族成员的这一功能特点主要是因为其大部分成员都是在T细胞活化后诱导表达。 BTLA(B and T lymphocyte attenuator)是新近发现的一Ig超家族抑制性受体,其虽与CTLA-4具有相似的结构,但其配体却不是经典的B7家族成员,而是TNFR-TNF超家族的HVEM。这是迄今为止发现的唯一一对Ig超家族成员与TNFR-TNF超家族成员相互作用的受配体。除BTLA外,HVEM还有另一个重要的膜结合分子——LIGHT,传递的信号维持T细胞的活化。通过和BTLA和LIGHT的结合,HVEM扮演一个双相调节T细胞激活的角色。对BTLA-HVEM复合物的晶体结构进行分析,提示HVEM有可能能同时结合LIGHT和BTLA,体外以纯化蛋白进行实验,也的确发现三者可形成复合物。UL144是人巨细胞病毒上一段和HVEM具有同源性的长独特型序列,UL144结合BTLA,但不结合LIGHT,并且抑制T细胞的增殖,选择性的模拟了HVEM的共抑制功能。 UL144基因产物与BTLA结合,在体外能抑制T细胞增殖,并且效果强于HVEM。由于BTLA表达于分化阶段的CD_4~+T细胞,并在Th1型细胞获得永久表达。在本研究中,我们考虑拟通过转基因的方式诱导DC表达UL144,观察其是否可能成为一种耐受型DC,或表现出某些耐受型特征,这种DC能否介导免疫抑制作用。在本课题中,我们拟选用实验性自身免疫性心肌炎为模型,研究通过调控UL144-BTLA通路重建自身免疫耐受,实现对Th1型反应为主的自身免疫性疾病进行治疗。 在各种用于细胞转染的载体中,重组腺病毒对DC具有良好的感染效率,且易于构建、纯化并获得较高滴度,因而成为本研究首选的载体。本研究设计以hCMVDNA为模板,通过PCR扩增出UL144全长片段;用常规分子生物学方法构建了UL144全长片段的腺病毒表达载体(pAdEasy-1系统);腺病毒载体经HEK293细胞包装产生含UL144片段的重组腺病毒Ad-CMV-UL144,用氯化铯密度梯度离心法获得纯化的高滴度腺病毒;采用TCID_(50)方法对重组腺病毒滴度进行检测;运用AD-CMV-UL144感染DC,研究其表型变化以及细胞因子分泌情况;通过感染DC,与T细胞进行混合淋巴反应研究其抑制活性;用纯化的猪心肌肌凝蛋白免疫Balb/c小鼠,诱导实验性自身免疫性心肌炎动物模型的产生,并分别于再次免疫后0～2天与14～16天经尾静脉注射重组腺病毒感染的DC,观察Ad-CMV-UL144对实验性自身免疫性心肌炎的治疗效果,研究其在体内的抑制作用。 第一部分携带UL144基因片段的重组腺病毒载体的构建与纯化 本部分采用常规分子生物学方法,先从CMV-DNA阳性的病人外周血提取cDNA,根据Primer3软件设计的引物,PCR扩增获得UL144目的片段;经pMD18-T载体获得多克隆位点,装载进入重组穿梭质粒pAdTrack-CMV。以PmeⅠ酶切带目的片段的穿梭质粒pAdTrack-CMV后,与重组腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌DH-5α,发生同源重组获得携带UL144基因的重组腺病毒骨架,经鉴定后纯化该骨架质粒,以内切酶PacⅠ线性化骨架,转染HEK293细胞获得完整的重组腺病毒颗粒UL144。重组的腺病毒转入HeLa细胞中,经RT-PCR鉴定UL144基因成功表达。重组腺病毒经过大量扩增,收取细胞,PBS重悬,反复冻融3次后,经CsCl双密度梯度超速离心纯化。通过有限稀释法测定半数组织细胞感染量(TCID_(50)),判定病毒滴度(Reed-Muenech法),AD-CMV-UL144的滴度为3×10~(10)/mL,对照AD-GFP的滴度为1×10~(10)/mL。 第二部分重组腺病毒介导表达UL144蛋白对小鼠骨髓来源的树突状细胞功能的影响 本部分实验以重组腺病毒介导UL144转染小鼠DC,观察其对DC免疫表型和分泌细胞因子的影响;观察UL144基因修饰的DC抗原提呈能力的改变及对效应性T细胞刺激能力的变化。我们采用GM-CSF、IL-4体外扩增Balb/c小鼠骨髓来源的DC,培养过程中将其感染携带UL144编码序列的重组腺病毒载体,设正常DC及GFP对照病毒处理DC为对照。培养6天的未成熟DC,加入脂多糖(LPS)继续培养24小时,诱导为成熟DC。结果发现:DC细胞表面CD40、CD80(B7.1)、CD86(B7.2)、Ia~d(MHCⅡ)的表达水平随LPS诱导成熟而明显上调,而UL144基因修饰的DC细胞CD40、CD80、CD86、Ia~d的表达水平低于对照组,并对LPS诱导的上述分子的表达水平上调有一定抑制作用,说明其能抑制DC成熟。UL144基因修饰的DC分泌TNF-α、IL-6和IL-1β的能力减弱,该DC与OVA_(323-339)肽特异反应性淋巴细胞的共培养实验表明,UL144基因修饰的DC能直接抑制OVA_(323-339)肽特异性淋巴细胞的增殖,提示UL144基因修饰的DC可抑制效应性,该作用部分可能是通过TNF-α和IL-6分泌减少而引起。我们的实验从免疫表型、细胞因子分泌、抗原提呈功能等方面证明,UL144基因修饰的DC具有维持相对未成熟状态的能力,并减弱了对抗原特异性T效应细胞的刺激功能。 第三部分UL144蛋白修饰的树突状细胞对实验性自身免疫性心肌炎的防治 选择6周龄的Balb/c小鼠,分别于0天和第7天皮下注射200μg纯化的猪心肌肌凝蛋白,制备实验性自身免疫性心肌炎动物模型。分别于初次免疫后的两周内,按每周1次经小鼠尾静脉注射5×10~6个重组腺病毒介导表达UL144蛋白的DC,并同时以空载病毒Ad-GFP转染的DC作为其对照组,再设立单纯免疫组与正常组对照。小鼠于初次免疫后第21天眼球取血后,脱颈处死,解剖获取心脏与脾脏。心肌病理检查与血浆cTnI浓度判断心肌病损程度,测定小鼠血浆抗心肌肌凝蛋白抗体的滴度与总IgG的量判断小鼠的体液免疫功能,测定小鼠脾脏单个核细胞对Con A和心肌肌凝蛋白刺激的增殖能力及细胞因子的分泌能力反映小鼠细胞免疫功能,流式细胞术测定脾及淋巴结的淋巴细胞亚群的活化状况。 虽然Ad-CMV-UL144处理组和Ad-CMV-GFP对照组有几只小鼠因不明原因死亡,但结果显示,UL144基因修饰的DC能够减轻小鼠心肌病损程度,使肌凝蛋白自身抗体水平均明显下降、脾单个核细胞对心肌肌凝蛋白的刺激明显受抑、引流淋巴结与脾脏中活化的T、B细胞明显下降。这些结果表明UL144基因修饰的DC能够减轻心肌炎的病损程度,对肌凝蛋白诱导的实验性自身免疫性心肌炎具有一定的治疗效果。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2243439
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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1 何蓉;人类巨细胞病毒UL144、UL146和UL147基因多态性研究[D];中国医科大学;2006年
2 毛志芹;人类巨细胞病毒UL144、UL139、UL149基因多态性与其致病性关系的研究[D];中国医科大学;2005年
3 蔡刚;炎症因子介导树突状细胞过度表达HVEM重建特异性自身免疫耐受[D];第二军医大学;2007年
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