MUC4多表位嵌合疫苗诱导特异性细胞毒T淋巴细胞（CTL）体外杀伤功能的实验研究

发布时间：2018-09-17 12:08

【摘要】： 背景: 粘蛋白(mucin,MUC)为高度糖基化的大分子糖蛋白,覆盖在粘膜上皮表面,作用包括保护粘膜上皮、参与上皮细胞的更新分化以及调节细胞粘附、免疫反应和细胞信号传导等。目前共发现20余种粘蛋白,包括:MUC1-2、MUC3/17、MUC3A、MUC3B、MUC4、MUC5AC、MUC5B、MUC6-16、MUC18-20等。 1991年Porchet等首先从人类支气管cDNA文库中获得MUC4cDNA,定位于3q29的候选癌基因,与鼠唾液酸粘蛋白复合物SMC具有60%同源性。MUC4基因可以通过不同的转录后剪接方式编码三种不同的MUC4蛋白亚型:膜结合型,分泌型和无特征结构型,在人体内分布最为广泛的是膜结合型,其分子结构包括一个分子量为850KD的胞外部分MUC4α和分子量为80KD的跨膜部分MUC4β。目前研究发现MUC4蛋白具有重要的生物学功能,包括抑制细胞与细胞、细胞与基质之间的连接,帮助肿瘤细胞逃避免疫细胞攻击,促进肿瘤细胞生长和转移,抑制细胞凋亡等,在肿瘤的发生发展过程中起着重要作用。肿瘤疫苗是目前抗肿瘤研究的一个活跃领域,可分为细胞疫苗、蛋白疫苗、肽疫苗和核酸疫苗等几大类,而表位疫苗是目前核酸疫苗的一个研究方向,其中多表位嵌合疫苗是指同时携带多个抗原相关表位以及辅助性表位的疫苗,这类疫苗在诱导细胞免疫方面有独特的优势,包括:1、内源性的表位呈递可以避免表位肽被降解,而且有研究显示DNA疫苗能为APC提供长时期的内源性抗原表达,诱导CD4细胞产生Th1细胞因子,通过“交叉呈递和交叉激活”而活化CTL;2、去除非免疫相关成分,减少免疫耐受和自身免疫的发生,引入各种免疫辅助成分,增强免疫应答效果;3、设计和制备方便,可以有不同HLA限制性表位,有广谱性。因此多表位嵌合疫苗成为目前疫苗研究的一个热点。树突状细胞(Dendritic cells)是已知体内功能最强的专职抗原呈递细胞(Antigen presenting cell,APC),其抗原呈递能力是巨噬细胞的10-100倍,在启动抗肿瘤的细胞免疫中起着关键作用,目前在肿瘤的免疫治疗研究中广为使用,成为研究的热点和重点。以肿瘤相关抗原诱导特异性CTL产生而发挥抗肿瘤的作用已经有报道,并取得一定的研究成果。MUC4特异性表达于各种肿瘤组织和肿瘤细胞系,特别在胰腺肿瘤中,由于MUC4在慢性胰腺炎和正常胰腺中不表达,在胰腺癌组织中高表达,被视为胰腺癌相关抗原而成为胰腺癌基因治疗和免疫治疗的一个新靶标。已有研究证明在MUC4表达的肿瘤组织中,MUC4蛋白可以激发机体产生体液免疫和细胞免疫。高表达MUC4的HPAF细胞是具有强转移特性的胰腺癌细胞系,在转染反义MUC4 mRNA后,MUC4蛋白表达下调,细胞增殖克隆能力变弱,接种小鼠后表现肿瘤生长缓陵,转移能力降低。上述研究证明对于表达MUC4蛋白的肿瘤,MUC4有作为基因治疗靶点的潜力。本研究中,我们首先利用生物信息学分析MUC4蛋白序列,获得HLA-A1和HLA-A2限制性表位,这些表位和通用Th辅助表位PADRE直接串接成多表位嵌合基因并构建腺病毒载体,SDS-page凝胶电泳检测其在真核细胞中的表达,并利用重组腺病毒感染DC细胞,TNF-α诱导成熟后体外刺激自身来源的PBLs,获得特异性CTL。标准Cr~(51)释放实验和Elispot检测CTL对靶细胞的杀伤作用。结果: 重组多表位嵌合基因腺病毒转染真核细胞COS-7,可以表达目的蛋白。多表位嵌合基因转染DC,TNF-α刺激成熟并外照射后,体外与自身来源的PBLs共同作用刺激特异性CTL产生,Cr~(51)实验结果显示实验组rAd-CMV-MUC4/PADRE诱导CTL杀伤MUC4和HLA-A2双阳性靶细胞HCT-116与单阳性靶细胞BXPC-3,MCF-7有显著统计学差异,而对照组rAd-CMV-GFP和IL-2组没有差异,显示CTL杀伤有特异性。Elispot检测rAd-CMV-MUC4/PADRE、rAd-CMV-GFP、空白组分别和HCT-116作用后IFN-γ表达,当效靶比为40/1时,rAd-CMV-MUC4/PADRE组IFN-γ分泌量和其余两组有显著差别。结论: 本研究成功构建了含有肿瘤抗原MUC4的多表位嵌合基因腺病毒颗粒,在真核细胞可以表达多表位嵌合蛋白。重组病毒颗粒体外能高效感染DC细胞,且不影响DC成熟。重组MUC4多表位嵌合基因腺病毒转染的DC可以有效刺激针对MUC4的HLA限制性CTL产生,体外对表达MUC4和HLA-A2双阳性细胞有杀伤作用。这些资料为我们将来临床应用含MUC4/PADRE的腺病毒载体治疗MUC4相关肿瘤打下基础。
[Abstract]:Background:
Mucin (MUC) is a highly glycosylated macromolecule glycoprotein that covers the surface of mucosal epithelium. Its functions include protecting mucosal epithelium, participating in the regeneration and differentiation of epithelial cells and regulating cell adhesion, immune response and signal transduction. MUC6-16, MUC18-20 and so on.
In 1991, Porchet et et et et et et et al. first obtained MUC4 cDNA from human bronchial cDNA library, which was located in 3q29 candidate oncogene. MuC4 gene has 60% homology with SMC. MUC4 gene can encode three different MUC4 protein subtypes by different post-transcriptional splicing methods: membrane-bound, secretory and non-characteristic structural. Membrane-bound proteins are most widely distributed within the tumor cell membrane. Their molecular structure includes an extracellular molecule of 850 KD, and a transmembrane molecule of 80 KD. Current studies have found that MUC4 proteins have important biological functions, including inhibiting cell-to-cell, cell-to-matrix connections, and helping tumor cells escape epidemic details. Cell attack, promoting tumor cell growth and metastasis, inhibiting cell apoptosis and so on, plays an important role in the occurrence and development of tumor.
Nowadays, tumor vaccine is an active field of anti-tumor research, which can be divided into cell vaccine, protein vaccine, peptide vaccine and nucleic acid vaccine. Epitope vaccine is one of the research directions of nucleic acid vaccine. Multi-epitope chimeric vaccine is a kind of vaccine which carries many antigen-related epitopes and auxiliary epitopes at the same time. Vaccines have unique advantages in inducing cellular immunity, including: 1. Endogenous epitope presentation can prevent epitope peptides from being degraded, and studies have shown that DNA vaccines can provide long-term endogenous antigen expression for APC, induce CD4 cells to produce Th1 cytokines, and activate CTL through "cross-presentation and cross-activation"; 2. Relevant components can reduce the occurrence of immune tolerance and autoimmunity, introduce various immune adjuvant components to enhance the immune response effect; 3. The design and preparation are convenient and can have different HLA restriction epitopes with broad spectrum.
Dendritic cells are the most powerful professional antigen presenting cells (APCs) known in vivo. Their antigen presenting ability is 10-100 times that of macrophages. Dendritic cells play a key role in initiating anti-tumor cellular immunity. They are widely used in tumor immunotherapy research and become the focus of research. MuC4 is specifically expressed in various tumor tissues and tumor cell lines, especially in pancreatic tumors. Because MUC4 is not expressed in chronic pancreatitis and normal pancreas, it is highly expressed in pancreatic cancer tissues. MUC4 protein has been shown to stimulate humoral and cellular immunity in cancer tissues expressed in MUC4. HPAF cells with high MUC4 expression are pancreatic cancer cell lines with strong metastatic properties and are transfected with antisense MUC4 mRNA. After inoculation, the expression of MUC4 protein was down-regulated and the ability of cell proliferation and cloning was weakened. After inoculation, the tumor growth and metastasis were slowed down.
In this study, we first used bioinformatics to analyze MUC4 protein sequences and obtained HLA-A1 and HLA-A2 restricted epitopes. These epitopes were directly linked to Th epitope chimeric genes and constructed adenovirus vectors. SDS-page gel electrophoresis was used to detect its expression in eukaryotic cells, and the recombinant adenovirus was used to infect DC. The specific CTL was obtained by stimulating self-derived PBLs in vitro after TNF-a induced maturation. Cr 51 release assay and Elispot assay were used to detect the killing effect of CTL on target cells.
Result:
The recombinant adenovirus transfected Eukaryotic cells COS-7 could express the target protein. After transfection of multiepitope chimeric gene into DC, TNF-a stimulated maturation and external irradiation, specific CTL production was stimulated in vitro in combination with PBLs of its own origin. The results of Cr~ (51) showed that rAd-CMV-MUC4/PADRE induced CTL to kill both MUC4 and HLA-A2. HCT-116 was significantly different from BXPC-3 and MCF-7, but there was no difference between rAd-CMV-GFP and IL-2. CTL killing was specific. Elispot detected IFN-gamma expression in rAd-CMV-MUC4/PADRE, rAd-CMV-GFP, blank group and HCT-116, respectively. When the effective target ratio was 40/1, IFN-gamma score in rAd-CMV-MUC4/PADRE group was detected. There was a significant difference between the two groups.
Conclusion:
In this study, a multiepitope chimeric adenovirus containing tumor antigen MUC4 was successfully constructed, which could express multiepitope chimeric protein in eukaryotic cells. The recombinant adenovirus particles could infect DC cells efficiently without affecting DC maturation. The recombinant MUC4 multiepitope chimeric gene adenovirus could effectively stimulate HLA-restricted C against MUC4. TL is produced and can kill both MUC4 and HLA-A2 positive cells in vitro. These data will lay a foundation for the clinical application of adenovirus vector containing MUC4/PADRE in the treatment of MUC4-related tumors.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【共引文献】