【作者】 罗进；

【导师】 阎小君；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 随着免疫识别理论、分子生物学知识和电子显微技术的不断进展与突破,大量研究证实发现T细胞对于抗原的识别是与MHC分子结合递呈的抗原短肽。有效的抗原表位肽可以通过树突细胞呈递到MHC-Ⅰ/MHC-Ⅱ途径引起机体的免疫反应。近年来又有科学家提出复合表位或改构表位可以增强单表位的功能。随着DC疫苗在临床的应用及其相关研究的进一步深入,寻找有效的T细胞表位以增强DC疫苗在抗肿瘤及治疗感染性疾病的作用受到越来越多的重视。如何使外来抗原通过MHC-Ⅰ途径诱发CD8+CTL反应是DC在临床应用中的关键。该课题的设计主要针对病因明确的病毒性疾病,拟选择以乙型肝炎病毒源性肝炎为疾病模型,以细胞免疫所攻击的乙型肝炎病毒核心蛋白为靶蛋白,通过软件预测的方法分析该蛋白的生物学特性及T细胞表位肽,将设计得到的表位修饰并合成。建立可标准化的DC培养平台;通过实验探讨表位肽对DC功能的影响以及其作用机理,为DC在临床的应用奠定基础。实验分为四部分,具体如下:第一部分T细胞表位肽的设计与筛选目的预测HBV核心蛋白的一般生物学特性及该蛋白片段上有效的HLA-2限制性CTL表位。方法从Genebank中选择B型(adw) HBV的核心蛋白,用软件CLC Protein Workbench3版本和SignalP3.0软件(http://www.cbs.dtu.dk/services/SignalP-2.0/)对HBV核心抗原的编码蛋白质进行预测的一般生物学特性;采用SYFPEITHI远程预测数据库、PREDEPP数据库、基序法和多项式法共同对HBV核心抗原编码蛋白进行HLA-A2限制性CTL表位预测。结果通过软件对HBV核心蛋白进行分析,明确了该蛋白质的跨膜区,信号肽,亚细胞定位,抗原性位点。筛选了5条T细胞表位肽。结论预测方案的联合使用可以提高T细胞表位肽筛选的准确率。第二部分T细胞表位肽的鉴定、修饰与合成目的鉴定筛选出的5条肽生物学特性,修饰并合成与抗原结合性能最好的肽,为进一步探讨外源性小分子表位肽进入细胞内激发MHC1类反应奠定基础。方法用抗原性能结合实验分别从亲和力、结合稳定性和MHC-肽复合体稳定性三方面鉴定5条表位肽与抗原的结合性能,筛选出结合性能最好的两条肽,并用辅助性T细胞表位、B细胞表位和棕榈酰基修饰,Fmoc方案合成。结果筛选出2条T细胞表位肽,合成9条肽,构成11组表位肽。结论利用抗原结合性能实验和Fmoc合成方案可以完成抗原肽的鉴定与合成。第三部分人外周血单核细胞来源的树突细胞转化平台的搭建目的建立一种可标准化、规模操作DC培养、转化及鉴定平台。方法将人外周血的PBMC用CD14+磁珠标记,在磁场中分离出CD14+PBMC;将分选得到的细胞在DC培养袋中加入IL-4,GM-CSF共培养,第10天,收集细胞计数,Typanlan染色观察活性,流式细胞仪检测细胞表型,扫描电镜观察细胞形态。结果光学显微镜下,树突状细胞悬浮生长,大小不等,形态极不规则,向四周伸出不同数量的粗细不一、形态迥异的胞质突起。CD14+PBMC在初始血液中的比例为14%,而经过分离纯化后的CD14+细胞的浓度达到94.8%。在培养10天后的细胞悬液中,由于大部分细胞转化为DC,所以分子表面标志发生改变,CD14+细胞的浓度仅为0.65%;在培养出的DC细胞表面高表达HLA-I(70.43%),HLA-Dr(0.65%),CD80(89.45% ),CD86(86.32%),部分表达CD40(29.61%)。这些细胞表面分子标志都是人PBMC转化为DC后的特征性分子。扫描电镜可见被固定的树突状细胞仍保持其特征性的突起,使细胞呈星状、多角形或极不规则形。突起长短不一,分支形成1-4级的亚突起。结论从PBMC中分离纯化的CD14+细胞通过加入细胞因子可以在细胞培养袋中非贴壁式培养出高纯度的DC。第四部分DC介导的靶向性杀伤功能及机制的初步探讨目的对比负载不同表位肽的DC的生物学功能筛选出杀伤功能最佳的表位肽组合,从微分子水平探讨组合肽的功能性差异。方法用四噻唑蓝法实验检测细胞毒功能;ELISPOT实验检测负载不同表位的DC刺激T细胞分泌γ-IFN的功能;原子力显微镜分析DC经小分子肽作用后表面精细结构改变;激光共聚焦显微镜分析小分子肽进入细胞后不同时间的表现;免疫荧光显微镜观察DC摄取肽的过程。结果细胞经第T1,T1+T2,T1+Th,T1-AAA-Th,pal-KSST1,pal-KSST1AAATh,T1+B,pal-KSST1Th,T1+Th,B组短肽处理后,细胞活性较对照组有显著性升高(p<0.05),其中第T1+B组肽则可使细胞活性呈现极显著升高(p<0.01),而经T2,Th,T1Th短肽处理后,细胞活性较对照组有显著性降低(p<0.05),其中第T2组肽使细胞活性呈现极显著降低。筛选出的表位肽T1可以上调DC对T细胞刺激能力(P<0.01),而经过棕榈酰丝氨酸修饰的表位或B细胞、Th协同的T细胞表位负载DC刺激能力也有同样表现(P<0.01),无论辅助性T细胞表位还是B细胞表位对于T1均有协同作用。将辅助性T细胞表位直接偶联到T1表位上,破坏了T1刺激DC的能力。即使在T1表位和Th内部通过AAA连接,并以棕榈酰丝氨酸修饰也不会表现出比单独的T1表位明显的功能增强。激光共聚焦的结果显示小分子肽可以在30分钟被DC快速摄取,免疫荧光显微镜发现肽的摄取是在5min内完成的;小分子肽的摄取由胞体完成后向树突传递,而且树突的肽浓度相对胞体较低。小分子肽被细胞摄取后的12h内,始终不曾进入细胞核。在透射电镜下,负载肽的DC线粒体增大,增多,核糖体和内质网丰富。而MTT活性检测实验显示这些被肽激活的DC的功能上调。结论DC经不同短肽处理后,细胞活性会产生一定程度的改变。短肽使T淋巴细胞的杀伤活性增高的原因主要是通过DC激活了细胞免疫途径,发挥了CTL效应。棕榈酰基修饰后可以上调T细胞表位的功能;B细胞表位和Th可以通过表位间的协同作用增强T细胞表位肽激活CTL的效应。AFM观察到细胞表面形貌变化受外源性小分子肽一级结构的影响,而且直接影响细胞的活性。小分子肽不作为遗传物质参与细胞的信息传递,这一现象为表位肽作为疫苗,与DC共培养后回输机体的安全性提供了理论基础。研究发现软件预测是一种可行的T细胞表位肽筛选方案,极大的缩减了实验的工作量,不同类别的表位肽之间的协同作用对于上调DC的生物学功能大于表位间的偶联效应。棕榈酰化表位肽也可以上调DC对于T细胞的刺激能力。不同的小分子肽对DC表面的精细结构的影响不一样,产生的CTL杀伤效应也不同。更多还原

【Abstract】 With the development of immunology, molecular biology, and micro-electronics technology, insights regarding peptide binding with major histocompatability complex (MHC) molecules have revealed a vital role in recognition between T cell and antigen. Antigen epitope can be presented on MHC-Ⅰor II molecules by antigen presenting cells and promote an immune response. Recently, the theory that both was considered that both compound-epitopes and modified epitopes can be more effective at enhancing the function of cytotoxic lymphocytes (CTL) was proposed. Therefore, attention has focused on finding efficient epitopes that upregulate the function of dendritic cells (DC) for treating cancer and infectious diseases. It is key for the application of DC therapy in clinic to determine how to best induce CD8+CTL through MHC-I by exogenous peptides.Our research is focused on the treatment of hepatitis B virus (HBV). The HBV core protein was selected as the antigen source for presentation by DC. The HBV core protein was analysed by specialized software. Biological characteristics of HBV core protein were acquired. T epitopes were analyzed, modified and synthesized. A DC culturing platform was established and standardized. Experiments were designed to elucidate the function and mechanism of peptide-pulsed-DC.Specific Aim I: Screening of T Epitopes Objective. To predict the characteristics of efficient T epitopes restricted by HLA-A2 of the HBV core protein. Methods. B type(adw subtype) HBV core protein from Genebank was selected and predicted normal characteristics by CLC Protein Workbench3 version and SignalP3.0 software(http://www.cbs.dtu.dk/services/SignalP-2.0/)and T epitopes restricted by HLA-A2 by SYFPEITHI databank, PREDEEP databank, matrix. Results. The cross-membrane region, signal peptides, and antigenic sites of HBV core protein were identified and five T epitopes were selected for analysis. Conclusion. Combining several prediction methods can boost the prediction accuracy of T epitopes.Specific Aim II: Identifying, Modifying, and Synthesizing T EpitopesObjective. To Identify, modify, and synthesize five peptides for elucidating the mechanism of exogenous peptides entering DC by the MHC I pathway. Methods. Antigen binding experiments were performed to identify affinities, binding affinities, and MHC-peptide complex stability of five epitopes. Two peptides with the best performance were selected, modified with Th, B, and palmitoyl, then synthesized. Results. Two peptides were acquired, and nine peptides were synthesized and eleven epitopes were obtained. Conclusion. Antigen binding experiments with the Fmoc synthesis method can be used to identify and synthesize epitopes.Specific Aim III: Establishing a Platform of DC GenerationObjective. To establish a reproducible, large-scale platform to generate DC. Methods. Human peripheral blood mononuclear cells (PBMC) were labeled with CD14+ magnetic beads. CD14+ cells were isolated and cultured with IL-4 and GM-CSF in a culture bag for ten days, then harvested. The viability of DC was determined by Typanlan staining. DC phenotype was determined by FACS. The morphology of these DC was observed under a scanning electron microscope. Results. DC suspended in culture medium result in variable cell size and irregular morphology. Cytoplasmic dendrites were extended. 14% of PBMC are CD14+. Magnetic bead purification of PBMC resulted in 94.8% purity of CD14+ cells. After ten days of culture, the majority of PBMC expressed a DC phenotype with only 0.65% of cells expressing CD14. Certain cell surface molecules were expressed at ten days including HLA-I (70.43%),HLA-Dr (0.65%),CD80 (89.45%),CD86 (86.32%), and CD40 (29.61%). By electron microscopy, DC retained their specific morphology. Conclusion. CD14+ cells isolated from PBMC can be non-adherently cultured and transformed into pure DC in culture bags.Specific Aim IV: The Function and Mechanism of Peptide-Pulsed DCObjective. To compare the biological functions of DC loaded with different epitopes and select the peptide that results in the most effective CTL response. To investigate functional differences in DC pulsed with different peptides. Methods. The cytotoxity of T cells was tested by MTT assay and the amount of secretion ofγ-IFN was detected by ELISPOT. Fine structures on the DC surface loaded with different peptides were observed under atomic force microscope(AFM). The behavior of peptides in the DC at different time points were analyzed by laser scanning confocal microscope (LSCM). Results. The function of DC are upregulated when they were pulsed by peptide through MTT assay. Cell viability was significantly higher(p<0.05)when they were pulsed with T1,T1+T2,T1+Th,T1-AAA-Th,pal-KSST1,pal-KSST1AAATh,pal-KSST1Th,T1+B,T1+Th,B epitopes and especially by T1+B epitope(p<0.01). However, cell viability was significantly lower when treated with T2,Th,T1Th, and was at its lowest when treated with T2 epitope. T1 can upregulate DC function (p<0.01). T epitopes all show higher activity when they are modified by B epitope, helper T cell, and palmitoyl(p<0.01=. The results of the laser scanning confocal microscope demonstrated that small peptides can be taken up within 30 minutes by DC and transfer to branches from the cytoplasm. However, the concentration of peptide is lower in the branches than in the cell body. Peptide never was able to enter the nuclear within 12 hours in our experiment. Under TEM, peptide-pulsed DC display larger and a greater number of mitochondria in the cytoplasm. Ribosomes and endoplasmic reticulum are abundant. Conclusion. Viability of DC can change when they are treated with peptides. The reason that the epitope increased the CTL response is that exogenous peptide can induce a cellular response through the cross-presenting pathway of MHC I and promote the CTL reaction. DC function can be upregulated by epitope modified by palmitol B epitope. Both Th epitope and B epitope all have synergistic effect on T epitopes. Even if three alanine were linked between them and palmitoyl were joined to the N-terminal, the epitope did not induce an antigen-specific T cell reaction differently compared to T1 epitope. However, the way peptides entered into dendritic cell were changed. Cell-surface DC morphology changes when DC were loaded with different protein sequence under AFM. Fine structures on the surface of DC have a strong relationship with viability. Small peptides can upregulate DC function but cannot alter genetic information transmission. Small peptide still enter the cell through the endosome/lysosome pathway and are presented by MHC I on the cell surface.In our investigation, it is feasible to predict T epitopes by software, which greatly reduces conventional workload. Small peptides are taken up by DC through endocytosis. Different epitopes have different effects on DC in a CTL reaction, fine structures on the cell surface, and the function of DC. This finding suggests that small peptides do not directly participate in genetic information transmission when DC are loaded with peptides. Our research provides evidence that peptide-pulsed DC may be employed therapeutically in clinic.更多还原