异种抗原α-gal联合免疫细胞治疗结直肠癌的实验研究

发布时间：2018-05-23 20:00

本文选题：α-gal表位 + anti-Gal抗体　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：研究背景：α-gal表位是广泛存在于非灵长类哺乳动物细胞表面的碳水化合物结构,由GGTA1基因编码的α1,3GT酶催化形成,能够与anti-Gal抗体特异性结合。由于物种进化,人类细胞不表达α-gal表位,但血清中存在天然anti-Gal抗体。α-gal/anti-Gal在物种间的表达差异为抗肿瘤治疗开拓了思路,其介导的抗肿瘤免疫治疗疗效已在黑色素瘤、胰腺癌等动物模型中得到验证,也为结直肠癌免疫治疗提供了新策略。然而,已被应用于临床试验的α-gal糖脂的治疗效果良莠不齐,原因在于该疗法主要依赖患者体内潜在的免疫系统功能,但肿瘤患者体内DC细胞和T细胞功能不足,不能激发有效的抗肿瘤免疫应答。近年,免疫细胞过继转移治疗取得长足进展。笔者认为α-gal可联合细胞免疫治疗提高抗肿瘤疗效。一方面,可利用α-gal表位提高肿瘤免疫原性,诱导新型特异性DC-CTL疫苗,重建患者免疫功能;另一方面,可通过“病毒-基因”疗法诱导自体肿瘤组织表达α-gal表位,转变为自体瘤苗,为DC-CTL疫苗提供体内治疗的靶点,在识别、提呈和效应多个环节增强抗肿瘤免疫应答效果。研究目的：1)通过诱导肿瘤细胞或组织表达α-gal表位来解决肿瘤抗原免疫原性低的难题,为启动抗肿瘤免疫应答奠定基础。2)研发新型特异性DC-CTL疫苗,为增强细胞过继转移治疗的疗效奠定基础。3)初步探讨工程化α-gal瘤苗联合新型DC-CTL疫苗治疗结直肠癌的可行性和治疗策略,为进一步完成临床前研究和临床应用奠定基础。研究方法：1)通过质粒重组、病毒包装构建调控GGTA1基因表达的实验慢病毒Lenti-GGTAl和对照空转病毒Lenti-control,体外感染人结肠癌细胞SW620和人胃癌细胞BGC823,建立稳定表达α-gal表位的细胞瘤苗,Western Blot鉴定基因表达产物,荧光显微镜观察α-gal表位的定位表达情况,流式细胞术检测阳性表达率。2)通过质粒重组、病毒包装构建E2F-1启动子调控GGTA1基因表达的AAV-GGTA1和对照空转病毒AAV-control,同时建立裸鼠荷人结肠癌皮下移植瘤模型,瘤内多点注射AAV-GGTA1,并以AAV-control和PBS作为对照,Western Blot鉴定肿瘤组织内融合蛋白表达,冰冻切片间接免疫荧光染色观察α-gal表位表达。3)取健康人外周血分离PBMC培养DC和CTL,分别用普通结肠癌细胞抗原(SW620-normal)、对照慢病毒感染后的结肠癌细胞抗原(SW620-control)和α-gal修饰的结肠癌瘤苗抗原(SW620-a-gal)致敏DC,进一步激活CTL,流式细胞术检测DC和CTL细胞表型,ELISA检测DC分泌的IL-12、IL-10以及CTL分泌的TNF-α、IL-4, LDH释放法检测各组CTL的体外杀伤能力。研究结果：1)经多次酶切、PCR和Western Blot鉴定,Lenti-GGTA1和Lenti-control构建成功,符合实验用滴度。以MOI 200感染SW620细胞、MOI 40感染BGC823细胞,成功建立人结肠癌细胞瘤苗SW620-α-gal和人胃癌细胞瘤苗BGC823-α-galc通过荧光显微镜可以观察到α-gal表位表达于细胞膜上,实验组阳性表达率高达97%。2)经多次酶切、PCR和测序鉴定,AAV-GGTA1和AAV-control构建成功,滴度符合实验需要。建立裸鼠荷人结肠癌皮下移植瘤模型成功率100%。Western Blot检测实验组肿瘤组织内有融合蛋白表达,间接免疫荧光染色可以观察到肿瘤组织中广泛表达α-gal表位。α-gal修饰的结肠癌抗原致敏的DC疫苗CD80和HLA-DR表达升高,CD86表达有升高趋势,CD83表达降低,分泌IL-12增加,分泌IL-10降低,以其激活的CD8+T细胞、NKT细胞比例增加,Treg细胞比例降低,分泌TNF-α增加,体外靶向杀伤能力增强。研究结论：1)体外实验中,所制备的慢病毒可以诱导人肿瘤细胞广泛稳定表达α-gal表位,提高肿瘤细胞免疫原性。2)体内实验中,所制备的AAV可以在体感染人结肠癌肿瘤组织,使其广泛表达α-gal表位,转变为自体瘤苗,提高肿瘤组织免疫原性。3)可以利用α-gal修饰的结肠癌细胞抗原制备DC疫苗,该方法可有效激活第二信号系统,使DC活化程度提高,分泌IL-12的功能增强,分泌IL-10的功能减低;该DC疫苗可以在体外诱导出强有力的CTL,其CD8表达增加,NKT细胞比例增加,Treg细胞比例降低,分泌TNF-α的能力增强。
[Abstract]:Background: alpha -gal epitope is a carbohydrate structure widely existed on the surface of non primate mammalian cells, catalyzed by alpha 1,3GT enzyme encoded by GGTA1 gene, and can specifically bind to anti-Gal antibody. Human cells do not express alpha -gal epitopes due to species evolution, but there are natural anti-Gal antibodies in serum. Alpha -gal/anti-Gal exists in serum. The difference in expression between species opens up a way of antitumor therapy, and its mediated antitumor immunotherapy has been verified in animal models such as melanoma, pancreatic cancer and other animal models. However, the therapeutic effect of alpha -gal glycolipid, which has been applied to clinical trials, is uncommon. This therapy mainly depends on the potential immune system function in the patient's body, but the function of DC and T cells in the tumor patients is not enough to stimulate the effective anti-tumor immune response. In recent years, the adoptive transfer therapy of immune cells has made great progress. I think that alpha -gal can improve the antitumor effect with cell immunotherapy. On the one hand, it can be used. The alpha -gal epitopes improve the immunogenicity of the tumor, induce a new specific DC-CTL vaccine, and reconstruct the immune function of the patient. On the other hand, it can be induced by the "virus gene" therapy to induce autologous tumor tissue to express the alpha -gal epitopes and turn into the autologous tumor vaccine. It provides the target for the treatment of the DC-CTL vaccine in vivo, and enhances the resistance in recognition, presentation and effect. The effect of tumor immune response. 1) 1) a new specific DC-CTL vaccine was developed by inducing tumor cell or tissue expression of alpha epitopes to solve the problem of low immunogenicity of tumor antigen, to establish a basic.2 for anti-tumor immune response), and to establish a basic.3 for enhancing the effect of cell adoptive transfer therapy. The feasibility and treatment strategy of gal vaccine combined with new DC-CTL vaccine in the treatment of colorectal cancer lay the foundation for further completion of pre clinical research and clinical application. Research methods: 1) the experimental lentivirus Lenti-GGTAl and control Lenti-control, which regulate the expression of GGTA1 gene by plasmid recombination, are constructed by recombinant virus packaging, and the human knot is infected in vitro. Colon cancer cell SW620 and human gastric cancer cell BGC823, establish a cell tumor vaccine that expresses the epitopes of alpha -gal stable, Western Blot identification of gene expression products, fluorescence microscope to observe the localization and expression of alpha -gal epitopes, positive expression rate.2 in flow cytometry, and construct E2F-1 promoter to regulate the GGTA1 gene expression by plasmid recombination and E2F-1 promoter to regulate the GGTA1 gene expression AAV -GGTA1 and control AAV-control were used to establish a model of subcutaneous transplantation of human colon cancer in nude mice. AAV-GGTA1 was injected into the tumor, and AAV-control and PBS were used as control. Western Blot was used to identify the expression of fusion protein in tumor tissue, and the expression of alpha -gal epitopes of frozen section indirect immunofluorescence staining was used to observe the peripheral blood of healthy people. In vitro, DC and CTL were cultured with common colon cancer cell antigen (SW620-normal), colon cancer cell antigen (SW620-control) after lentivirus infection and colon cancer vaccine antigen (SW620-a-gal) modified by alpha -gal (SW620-a-gal) sensitized DC, and CTL was further activated and the phenotype of DC and CTL cells was detected by flow cytometry. L secreted TNF- alpha, IL-4, LDH release assay in vitro to detect the killing ability of CTL in each group. Results: 1) after multiple enzyme digestion, PCR and Western Blot identified, Lenti-GGTA1 and Lenti-control were constructed successfully, conforming to the experimental titer. MOI 200 infected SW620 cells, 40 infected cells and successfully established human colon cancer cell tumor vaccine The BGC823- alpha -galc of gastric cancer cell tumor vaccine can be observed on the cell membrane by fluorescence microscope. The positive expression rate of the experimental group is up to 97%.2). The positive rate of the experimental group is up to 97%.2). After several enzyme cuts, PCR and sequencing are identified, AAV-GGTA1 and AAV-control are successfully constructed, and the titer conforms to the experimental needs. The success rate of the model of the subcutaneous transplantation tumor of the nude mice bearing human colon cancer is 100%.Wes Tern Blot detected the expression of fusion protein in the tumor tissue of the experimental group. Indirect immunofluorescence staining could observe the extensive expression of alpha -gal epitopes in the tumor tissues. The expression of CD80 and HLA-DR in the DC vaccine sensitized by alpha -gal modified colon cancer antigen was elevated, the expression of CD86 was elevated, the expression of CD83 decreased, the secretion of IL-12 increased, and the secretion IL-10 decreased. The proportion of activated CD8+T cells, the proportion of NKT cells increased, the proportion of Treg cells decreased, the secretion of TNF- alpha increased, and the target killing ability in vitro enhanced. 1) in vitro, the prepared lentivirus could induce the human tumor cells to express the alpha -gal epitopes widely and steadily, and improve the immunogenicity of the tumor cells in vivo. In vivo, the prepared AAV can be prepared. In vivo infection of human colon cancer tissue, making it widely expressed in the epitopes of alpha -gal, transforming into an autologous tumor vaccine and improving the tumor tissue immunogenic.3) can be used to prepare the DC vaccine by using the colon cancer cell antigen modified by alpha -gal. This method can effectively activate the second signal system, improve the activation range of DC, enhance the function of the secretory IL-12, and secrete the work of IL-10. It can be reduced; the DC vaccine can induce strong CTL in vitro, increase the expression of CD8, increase the proportion of NKT cells, decrease the proportion of Treg cells, and enhance the ability to secrete TNF- alpha.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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