MUC4多表位嵌合疫苗誘導特異性細胞毒T淋巴細胞（CTL）體外殺傷功能的實驗研究
發(fā)布時間：2018-09-17 12:08
【摘要】： 背景: 粘蛋白(mucin,MUC)為高度糖基化的大分子糖蛋白,覆蓋在粘膜上皮表面,作用包括保護粘膜上皮、參與上皮細胞的更新分化以及調節(jié)細胞粘附、免疫反應和細胞信號傳導等。目前共發(fā)現(xiàn)20余種粘蛋白,包括:MUC1-2、MUC3/17、MUC3A、MUC3B、MUC4、MUC5AC、MUC5B、MUC6-16、MUC18-20等。 1991年Porchet等首先從人類支氣管cDNA文庫中獲得MUC4cDNA,定位于3q29的候選癌基因,與鼠唾液酸粘蛋白復合物SMC具有60%同源性。MUC4基因可以通過不同的轉錄后剪接方式編碼三種不同的MUC4蛋白亞型:膜結合型,分泌型和無特征結構型,在人體內分布最為廣泛的是膜結合型,其分子結構包括一個分子量為850KD的胞外部分MUC4α和分子量為80KD的跨膜部分MUC4β。目前研究發(fā)現(xiàn)MUC4蛋白具有重要的生物學功能,包括抑制細胞與細胞、細胞與基質之間的連接,幫助腫瘤細胞逃避免疫細胞攻擊,促進腫瘤細胞生長和轉移,抑制細胞凋亡等,在腫瘤的發(fā)生發(fā)展過程中起著重要作用。 腫瘤疫苗是目前抗腫瘤研究的一個活躍領域,可分為細胞疫苗、蛋白疫苗、肽疫苗和核酸疫苗等幾大類,而表位疫苗是目前核酸疫苗的一個研究方向,其中多表位嵌合疫苗是指同時攜帶多個抗原相關表位以及輔助性表位的疫苗,這類疫苗在誘導細胞免疫方面有獨特的優(yōu)勢,包括:1、內源性的表位呈遞可以避免表位肽被降解,而且有研究顯示DNA疫苗能為APC提供長時期的內源性抗原表達,誘導CD4細胞產生Th1細胞因子,通過“交叉呈遞和交叉激活”而活化CTL;2、去除非免疫相關成分,減少免疫耐受和自身免疫的發(fā)生,引入各種免疫輔助成分,增強免疫應答效果;3、設計和制備方便,可以有不同HLA限制性表位,有廣譜性。因此多表位嵌合疫苗成為目前疫苗研究的一個熱點。 樹突狀細胞(Dendritic cells)是已知體內功能最強的專職抗原呈遞細胞(Antigen presenting cell,APC),其抗原呈遞能力是巨噬細胞的10-100倍,在啟動抗腫瘤的細胞免疫中起著關鍵作用,目前在腫瘤的免疫治療研究中廣為使用,成為研究的熱點和重點。以腫瘤相關抗原誘導特異性CTL產生而發(fā)揮抗腫瘤的作用已經有報道,并取得一定的研究成果。MUC4特異性表達于各種腫瘤組織和腫瘤細胞系,特別在胰腺腫瘤中,由于MUC4在慢性胰腺炎和正常胰腺中不表達,在胰腺癌組織中高表達,被視為胰腺癌相關抗原而成為胰腺癌基因治療和免疫治療的一個新靶標。已有研究證明在MUC4表達的腫瘤組織中,MUC4蛋白可以激發(fā)機體產生體液免疫和細胞免疫。高表達MUC4的HPAF細胞是具有強轉移特性的胰腺癌細胞系,在轉染反義MUC4 mRNA后,MUC4蛋白表達下調,細胞增殖克隆能力變弱,接種小鼠后表現(xiàn)腫瘤生長緩陵,轉移能力降低。上述研究證明對于表達MUC4蛋白的腫瘤,MUC4有作為基因治療靶點的潛力。 本研究中,我們首先利用生物信息學分析MUC4蛋白序列,獲得HLA-A1和HLA-A2限制性表位,這些表位和通用Th輔助表位PADRE直接串接成多表位嵌合基因并構建腺病毒載體,SDS-page凝膠電泳檢測其在真核細胞中的表達,并利用重組腺病毒感染DC細胞,TNF-α誘導成熟后體外刺激自身來源的PBLs,獲得特異性CTL。標準Cr~(51)釋放實驗和Elispot檢測CTL對靶細胞的殺傷作用。 結果: 重組多表位嵌合基因腺病毒轉染真核細胞COS-7,可以表達目的蛋白。多表位嵌合基因轉染DC,TNF-α刺激成熟并外照射后,體外與自身來源的PBLs共同作用刺激特異性CTL產生,Cr~(51)實驗結果顯示實驗組rAd-CMV-MUC4/PADRE誘導CTL殺傷MUC4和HLA-A2雙陽性靶細胞HCT-116與單陽性靶細胞BXPC-3,MCF-7有顯著統(tǒng)計學差異,而對照組rAd-CMV-GFP和IL-2組沒有差異,顯示CTL殺傷有特異性。Elispot檢測rAd-CMV-MUC4/PADRE、rAd-CMV-GFP、空白組分別和HCT-116作用后IFN-γ表達,當效靶比為40/1時,rAd-CMV-MUC4/PADRE組IFN-γ分泌量和其余兩組有顯著差別。 結論: 本研究成功構建了含有腫瘤抗原MUC4的多表位嵌合基因腺病毒顆粒,在真核細胞可以表達多表位嵌合蛋白。重組病毒顆粒體外能高效感染DC細胞,且不影響DC成熟。重組MUC4多表位嵌合基因腺病毒轉染的DC可以有效刺激針對MUC4的HLA限制性CTL產生,體外對表達MUC4和HLA-A2雙陽性細胞有殺傷作用。這些資料為我們將來臨床應用含MUC4/PADRE的腺病毒載體治療MUC4相關腫瘤打下基礎。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392
本文編號：2245871
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392
【共引文獻】
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