减毒沙门氏菌为载体的CEACAM6联合4-1BBL基因疫苗对大鼠肠癌生长的影响及机制研究

发布时间：2018-03-30 02:19

本文选题：4-1BBL　切入点：CEACAM6　出处：《苏州大学》2015年博士论文

【摘要】：第一部分:含p IRES-CEACAM6-4-1BBL质粒减毒沙门氏菌对大鼠大肠癌生长的影响目的:研究减毒沙门氏菌(SL3261)为载体的CEACAM6联合4-1BBL基因疫苗对实验性大肠癌的抑制作用。方法:实验大鼠随机分四组,每组6只,连续18周皮下注射1,2-二甲肼以建立大鼠大肠癌模型,同时于第8、10、12及14周每组分别灌服p IRES/SL3261、p IRES-CEACAM6/SL3261、p IRES-4-1BBL/SL3261、p IRES-CEACAM6-4-1BBL/SL3261减毒沙门氏菌疫苗,浓度为109/ml的细菌2ml,第18周处死荷瘤大鼠,解剖并观察大鼠大肠成瘤数量,收集大鼠大肠及脾脏组织,病理HE染色并评判肠道肿瘤Dukes分期,RT-PCR检测大鼠脾脏细胞CMV基因。结果:p IRES/SL3261空质粒组平均成瘤数为11.7±2.1个,明显多于p IRES-4-1BBL/SL3261组(5.7±1.2个),p IRES-CEACAM6//SL3261组(5.0±1.4个),p IRES-CEACAM64-1BBL/SL3261组(4.3±1.4个);Dukes分期分析,p IRES-CEACAM6-4-1BBL/SL3261组5只为A期,1只为B-C期;p IRES-4-1BBL/SL3261组中3只为A期,3只为B-C期;p IRES-CEACAM6/SL3261中1只为A期,5只为B-C期;p IRES/SL3261组中4只为B-C期,2只为D期。RT-PCR检测各组脾脏均有CMV基因表达。结论:各组疫苗菌可以将外源基因有效转录至大鼠体内并表达相关蛋白,含外源基因的疫苗菌均能抑制实验性大肠癌的生长,以含双基因疫苗菌的抑癌作用最强。第二部分:含p IRES-CEACAM6-4-1BBL质粒减毒沙门氏菌抑制大鼠大肠癌的机制研究——肿瘤局部免疫反应目的:通过研究肿瘤组织内局部免疫反应分析双基因疫苗抑制肿瘤生长机制。方法:采用免疫组织化学SP法检测四组大肠肿瘤组织的CD3、CD4、CD8、CD56、CD45RO、Foxp3、CEACAM6、IL-4、IL-17并分别评测各组指标表达分值。结果:与p IRES/SL3261组相比,p IRES-4-1BBL/SL3261组与p IRES-CEACAM6-4-1BBL/SL3261组CD3、CD8+、CD56+、CD45RO表达明显增高(P0.05),而IL-4、IL-17和FOXP3表达明显下调(P0.05),另外,p IRES-CEACAM6/SL3261组CD45RO表达较p IRES/SL3261组明显增高(P0.05)。结论:含有CEACAM6和4-1BBL双基因的重组减毒沙门氏菌可能通过提高CD3+CD8+T细胞和NK细胞的浸润,减少Foxp3细胞表达,促进Th1极化,抑制Th2及Th17极化,诱导特异与非特异性免疫,并打破免疫抑制微环境的机制来抑制肠道肿瘤。第三部分:含p IRES-CEACAM6-4-1BBL质粒减毒沙门氏菌抑制大鼠大肠癌的机制研究——全身免疫反应目的:通过研究脾脏组织免疫学指标分析双基因疫苗抑制肿瘤生长的机制。方法:采用免疫组织化学SP法检测四组大肠肿瘤组织的IFN-γ、CD3、CD4、CD8、CD56、Foxp3、IL-4、IL-17并分别评测各组指标表达分值。结果:与p IRES/SL3261组相比,p IRES-4-1BBL/SL3261组与p IRES-CEACAM6-4-1BBL/SL3261组IFN-γ、CD3、CD8+、CD56+表达明显增高(P0.05),而IL-4、IL-17和FOXP3表达明显下调(P0.05),而各组CD4表达无明显差别(P0.05)。结论:通过研究双基因疫苗的全身免疫反应,含有CEACAM6和4-1BBL双基因的重组减毒沙门氏菌可以刺激机体产生特异与非特异性的抗肿瘤免疫。
[Abstract]:Part one: effects of attenuated Salmonella containing p IRES-CEACAM6-4-1BBL plasmid on the growth of Colorectal carcinoma in Rats objective: to study the inhibitory effect of CEACAM6 combined with 4-1BBL gene vaccine on experimental colorectal cancer in rats with attenuated Salmonella sp. SL3261). Methods: experimental rats were randomly divided into four groups. Six rats in each group were injected subcutaneously with 1m2-dimethylhydrazine for 18 weeks to establish a rat model of colorectal cancer. At the same time, the rats were given pIRES / SL3261p IRES-CEACAM6 / SL3261p IRES-4-1BBL / SL3261p IRES-CEACAM6-4-1BBL/SL3261 attenuated Salmonella vaccine at the concentration of 109/ml in 2ml. the tumor bearing rats were killed at the 18th week. The number of large intestine tumorigenesis in rats was observed, and the tissues of large intestine and spleen were collected. The pathological HE staining and Dukes staging of intestinal tumors were used to detect the CMV gene of spleen cells in rats. Results the average number of tumors in the empty plasmid group was 11.7 卤2.1. Analysis of 4. 3 卤1. 4 Dukes staging in p IRES-4-1BBL/SL3261 group (5. 7 卤1. 2) IRES-CEACAM64-1BBL/SL3261 group (4. 3 卤1. 4) Dukes staging analysis of 5 patients with stage A in p IRES-CEACAM6-4-1BBL/SL3261 group and 3 patients with stage A stage A IRES-4-1BBL/SL3261 group with 3 patients with stage A stage A with 3 patients with stage A IRES-CEACAM6/SL3261 with 3 patients with stage A IRES-CEACAM6/SL3261 with 5 patients with stage A stage A with 4 patients with stage B C stage IRES/SL3261 group. In order to detect the expression of CMV gene in spleen of two B-C phase D mice by RT-PCR. Conclusion: the foreign genes can be effectively transcribed into the rat body and related proteins can be expressed by each group of vaccine bacteria. Vaccine bacteria containing exogenous genes could inhibit the growth of experimental colorectal cancer. The second part: the mechanism of attenuated Salmonella mutans containing p IRES-CEACAM6-4-1BBL plasmid to inhibit colorectal cancer in rats. Objective: to study the local immune response of tumor tissue by studying the local immunoreactivity of tumor tissue. The inhibition mechanism of tumor growth by double gene vaccine should be analyzed. Methods: immunohistochemical SP method was used to detect the expression of IL-17 in four groups of colorectal neoplasms. Results: compared with the p IRES/SL3261 group, the expression of IL-4 IL-17 was evaluated in the p IRES-4-1BBL/SL3261 group and the p IRES/SL3261 group. The expression of CD3, CD8, CD56, CD45RO, IL-4, IL-17 and FOXP3 in IRES-CEACAM6-4-1BBL/SL3261 group was significantly increased, while the expression of IL-17 and FOXP3 in IRES-CEACAM6/SL3261 group was significantly lower than that in p IRES/SL3261 group. Conclusion: the recombinant attenuated Salmonella containing both CEACAM6 and 4-1BBL genes may increase CD3 CD8 T fine. The infiltration of cells and NK cells, Reduce the expression of Foxp3 cells, promote Th1 polarization, inhibit Th2 and Th17 polarization, induce specific and nonspecific immunity. And breaking the mechanism of immunosuppressive microenvironment to inhibit intestinal tumours. Part 3: study on the mechanism of attenuated Salmonella containing p IRES-CEACAM6-4-1BBL plasmid in inhibiting colorectal cancer in rats-systemic immune response: to study the histology of spleen. Methods: immunohistochemical SP method was used to detect IFN- 纬 CD _ 3 ~ (+) ~ (-) ~ (-) CD _ (3) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (. In comparison with p IRES-CEACAM6-4-1BBL/SL3261 group, the expression of CD56 in IFN- 纬 ~ (3 +) CD _ (3) and CD _ (8) was significantly increased, while the expression of IL-4, IL-17 and FOXP3 was down-regulated, while the expression of CD4 in each group was not significantly different. Conclusion: by studying the systemic immune response of double gene vaccine, the expression of IL-17 and FOXP3 is significantly lower than that of p IRES-CEACAM6-4-1BBL/SL3261. Recombinant attenuated Salmonella containing CEACAM6 and 4-1BBL genes can stimulate specific and non-specific anti-tumor immunity.
【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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