减毒沙门氏菌为载体的CD40基因疫苗对实验性大肠癌生长的影响

发布时间：2018-02-15 03:50

本文关键词： CD40基因大肠癌共刺激分子减毒沙门氏菌基因疫苗　出处：《苏州大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分:重组大鼠CD40基因真核表达载体的构建及功能鉴定目的:构建含pIRES2-EGFP-CD40基因的真核表达质粒。方法:从大鼠的脾脏组织中完整克隆出大鼠CD40基因全长,成功构建真核表达质粒pIRES2-EGFP-CD40,通过脂质体介导转染COS7细胞,免疫双荧光法检测CD40的表达。结果:成功构建真核表达质粒pIRES2-EGFP-CD40并转染COS7细胞,证明真核表达质粒pIRES2-EGFP-CD40在COS7细胞中能够稳定表达目的基因。结论:成功构建了含pIRES2-EGFP-CD40基因的真核表达质粒。第二部分:含pIRES2-EGFP-CD40质粒减毒沙门氏菌的构建及功能鉴定目的:构建含pIRES2-EGFP-CD40质粒的减毒沙门氏菌菌疫苗。方法:采用电转化法将pIRES2-EGFP-CD40质粒成功转入鼠伤寒沙门氏菌(LB5000),经过甲基化修饰后的pIRES2-EGFP-CD40质粒通过电转化转入终宿主菌SL3261,通过抗生素筛选法挑取单个菌落进行扩增,最终抽提质粒并进行PCR鉴定。结果:终宿主菌SL3261在电转化转入pIRES2-EGFP-CD40质粒后,通过质粒抽提和PCR鉴定证实,其所获得的目的条带与原转化质粒条带大小相同。结论:成功构建了能携带大鼠CD40基因真核表达质粒pIRES2-EGFP-CD40的SL3261疫苗菌。第三部分:含pIRES2-EGFP-CD40质粒减毒沙门氏菌对大鼠大肠癌生长的影响目的:探讨减毒沙门氏菌为载体的CD40基因疫苗对荷瘤大鼠免疫系统的影响及治疗大肠癌可行性。方法:雄性6-8周龄SD大鼠18只随机分为3组,分别为SL3261组、空质粒(pIRES2-EGFP)组以及CD40(pIRES2-EGFP-CD40)组。对全部大鼠应用1,2-二甲肼连续皮下注射18周以构建大鼠大肠癌实验模型,分别于第8周、第10周、第12周及第16周根据所分组别不同共四次灌服SL3261、pIRES2-EGFP/SL3261和pIRES2-EGFP-CD40/SL3261,于18周后处死全部实验大鼠,将大鼠瘤体解剖后观察并记录瘤体大小、瘤体数量、有无转移灶等,根据观察指标进行Dukes分期;流式细胞仪检测外周血和脾脏细胞表型,PHA刺激脾脏细胞72小时后培养上清中IFN-γ的含量通过ELISA法检测,脾脏细胞中GFP的转录表达通过RT-PCR进行检测。结果:CD40组平均成瘤数为5.8±2.2个,明显低于SL3261组的12.7±3.1和空质粒组的11.6±2.4(P0.05),空质粒组和SL3261组两组的成瘤数相比较无明显统计学意义(P0.05);脾脏细胞和外周血表型检测显示CD40组的活化T细胞CD3+CD8+数量较其它荷瘤组明显增高(P0.05),PHA刺激脾细胞试验结果证实空质粒组和SL3261组分泌IFN-γ的能力也明显低于CD40组(P0.05)。结论:携带重组质粒pIRES2-EGFP-CD40的减毒沙门氏菌可以在荷瘤大鼠体内实现转染CD40基因并有效高表达,能显著提高荷瘤大鼠的细胞免疫功能,最终对实验性大鼠大肠癌具有明显的抑制作用。
[Abstract]:Part one: construction and functional Identification of Recombinant Rat CD40 Gene Eukaryotic expression Vector objective: to construct an eukaryotic expression plasmid containing pIRES2-EGFP-CD40 gene. Methods: the whole length of rat CD40 gene was cloned from rat spleen tissue. The eukaryotic expression plasmid pIRES2-EGFP-CD40 was successfully constructed and transfected into COS7 cells by liposome. The expression of CD40 was detected by double immunofluorescence assay. Results: the eukaryotic expression plasmid pIRES2-EGFP-CD40 was successfully constructed and transfected into COS7 cells. It is proved that the eukaryotic expression plasmid pIRES2-EGFP-CD40 can stably express the target gene in COS7 cells. Conclusion: the eukaryotic expression plasmid containing pIRES2-EGFP-CD40 gene was successfully constructed. Part 2: construction and functional identification of attenuated Salmonella mutans containing pIRES2-EGFP-CD40 plasmid. A attenuated Salmonella vaccine containing pIRES2-EGFP-CD40 plasmids was constructed. Methods: the pIRES2-EGFP-CD40 plasmid was successfully transferred to LB5000 of Salmonella typhimurium by electroporation. The methylated pIRES2-EGFP-CD40 plasmid was transformed into the terminal host strain SL3261 by electrotransformation. A single colony was selected for amplification by biotin screening method. The final plasmid was extracted and identified by PCR. Results: the final host strain SL3261 was transformed into pIRES2-EGFP-CD40 plasmid and confirmed by plasmid extraction and PCR identification. The obtained target bands were the same as the original transformed plasmid bands. Conclusion: the SL3261 vaccine bacteria carrying rat CD40 gene eukaryotic expression plasmid pIRES2-EGFP-CD40 were successfully constructed. Part 3: Salmonella mutans containing pIRES2-EGFP-CD40 plasmids were used to reduce the toxicity of Salmonella to rats. Objective: to investigate the effect of attenuated Salmonella CD40 gene vaccine on immune system of tumor-bearing rats and the feasibility of treating colorectal cancer. Methods: eighteen male SD rats aged 6-8 weeks were randomly divided into 3 groups. SL3261 group, pIRES2-EGFP group and CD40 pIRES2-EGFP-CD40 group were injected subcutaneously for 18 weeks to establish the experimental model of colorectal cancer in rats. At week 12 and week 16, SL3261pIRES2-EGFP / SL3261 and pIRES2-EGFP-CD40 / SL3261 were administered four times according to different groups. After 18 weeks, all the experimental rats were killed. The tumor size, the number of tumor, the number of tumor and the metastasis were observed and recorded. The content of IFN- 纬 in the culture supernatant of peripheral blood and spleen cells stimulated by phenotype of peripheral blood and spleen cells for 72 hours was detected by ELISA method. The expression of GFP in spleen cells was detected by RT-PCR. Results the average number of tumorigenesis was 5.8 卤2.2 in the CD 40 group. It was significantly lower than that in SL3261 group (12.7 卤3.1) and empty plasmid group (11.6 卤2.4 P0.05). There was no significant difference in the number of tumorigenesis between empty plasmid group and SL3261 group, and the phenotypic tests of spleen cells and peripheral blood showed that the number of CD3 CD8 in activated T cells in CD40 group was higher than that in other groups. The results of splenocyte stimulation test showed that the ability of secreting IFN- 纬 in empty plasmid group and SL3261 group was significantly lower than that in CD40 group. Conclusion: attenuated Salmonella mutans carrying recombinant plasmid pIRES2-EGFP-CD40 can transfect CD40 in tumor-bearing rats. The gene is effectively overexpressed, It can significantly improve the cellular immune function of tumor-bearing rats, and finally has a significant inhibitory effect on experimental colorectal cancer in rats.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34

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