本文選題：4-1BBL　切入點(diǎn)：CEACAM6　出處：《蘇州大學(xué)》2015年博士論文

【摘要】：第一部分:含p IRES-CEACAM6-4-1BBL質(zhì)粒減毒沙門氏菌對(duì)大鼠大腸癌生長(zhǎng)的影響目的:研究減毒沙門氏菌(SL3261)為載體的CEACAM6聯(lián)合4-1BBL基因疫苗對(duì)實(shí)驗(yàn)性大腸癌的抑制作用。方法:實(shí)驗(yàn)大鼠隨機(jī)分四組,每組6只,連續(xù)18周皮下注射1,2-二甲肼以建立大鼠大腸癌模型,同時(shí)于第8、10、12及14周每組分別灌服p IRES/SL3261、p IRES-CEACAM6/SL3261、p IRES-4-1BBL/SL3261、p IRES-CEACAM6-4-1BBL/SL3261減毒沙門氏菌疫苗,濃度為109/ml的細(xì)菌2ml,第18周處死荷瘤大鼠,解剖并觀察大鼠大腸成瘤數(shù)量,收集大鼠大腸及脾臟組織,病理HE染色并評(píng)判腸道腫瘤Dukes分期,RT-PCR檢測(cè)大鼠脾臟細(xì)胞CMV基因。結(jié)果:p IRES/SL3261空質(zhì)粒組平均成瘤數(shù)為11.7±2.1個(gè),明顯多于p IRES-4-1BBL/SL3261組(5.7±1.2個(gè)),p IRES-CEACAM6//SL3261組(5.0±1.4個(gè)),p IRES-CEACAM64-1BBL/SL3261組(4.3±1.4個(gè));Dukes分期分析,p IRES-CEACAM6-4-1BBL/SL3261組5只為A期,1只為B-C期;p IRES-4-1BBL/SL3261組中3只為A期,3只為B-C期;p IRES-CEACAM6/SL3261中1只為A期,5只為B-C期;p IRES/SL3261組中4只為B-C期,2只為D期。RT-PCR檢測(cè)各組脾臟均有CMV基因表達(dá)。結(jié)論:各組疫苗菌可以將外源基因有效轉(zhuǎn)錄至大鼠體內(nèi)并表達(dá)相關(guān)蛋白,含外源基因的疫苗菌均能抑制實(shí)驗(yàn)性大腸癌的生長(zhǎng),以含雙基因疫苗菌的抑癌作用最強(qiáng)。第二部分:含p IRES-CEACAM6-4-1BBL質(zhì)粒減毒沙門氏菌抑制大鼠大腸癌的機(jī)制研究——腫瘤局部免疫反應(yīng)目的:通過(guò)研究腫瘤組織內(nèi)局部免疫反應(yīng)分析雙基因疫苗抑制腫瘤生長(zhǎng)機(jī)制。方法:采用免疫組織化學(xué)SP法檢測(cè)四組大腸腫瘤組織的CD3、CD4、CD8、CD56、CD45RO、Foxp3、CEACAM6、IL-4、IL-17并分別評(píng)測(cè)各組指標(biāo)表達(dá)分值。結(jié)果:與p IRES/SL3261組相比,p IRES-4-1BBL/SL3261組與p IRES-CEACAM6-4-1BBL/SL3261組CD3、CD8+、CD56+、CD45RO表達(dá)明顯增高(P0.05),而IL-4、IL-17和FOXP3表達(dá)明顯下調(diào)(P0.05),另外,p IRES-CEACAM6/SL3261組CD45RO表達(dá)較p IRES/SL3261組明顯增高(P0.05)。結(jié)論:含有CEACAM6和4-1BBL雙基因的重組減毒沙門氏菌可能通過(guò)提高CD3+CD8+T細(xì)胞和NK細(xì)胞的浸潤(rùn),減少Foxp3細(xì)胞表達(dá),促進(jìn)Th1極化,抑制Th2及Th17極化,誘導(dǎo)特異與非特異性免疫,并打破免疫抑制微環(huán)境的機(jī)制來(lái)抑制腸道腫瘤。第三部分:含p IRES-CEACAM6-4-1BBL質(zhì)粒減毒沙門氏菌抑制大鼠大腸癌的機(jī)制研究——全身免疫反應(yīng)目的:通過(guò)研究脾臟組織免疫學(xué)指標(biāo)分析雙基因疫苗抑制腫瘤生長(zhǎng)的機(jī)制。方法:采用免疫組織化學(xué)SP法檢測(cè)四組大腸腫瘤組織的IFN-γ、CD3、CD4、CD8、CD56、Foxp3、IL-4、IL-17并分別評(píng)測(cè)各組指標(biāo)表達(dá)分值。結(jié)果:與p IRES/SL3261組相比,p IRES-4-1BBL/SL3261組與p IRES-CEACAM6-4-1BBL/SL3261組IFN-γ、CD3、CD8+、CD56+表達(dá)明顯增高(P0.05),而IL-4、IL-17和FOXP3表達(dá)明顯下調(diào)(P0.05),而各組CD4表達(dá)無(wú)明顯差別(P0.05)。結(jié)論:通過(guò)研究雙基因疫苗的全身免疫反應(yīng),含有CEACAM6和4-1BBL雙基因的重組減毒沙門氏菌可以刺激機(jī)體產(chǎn)生特異與非特異性的抗腫瘤免疫。
[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 鹵2.1. Analysis of 4. 3 鹵1. 4 Dukes staging in p IRES-4-1BBL/SL3261 group (5. 7 鹵1. 2) IRES-CEACAM64-1BBL/SL3261 group (4. 3 鹵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- 緯 CD _ 3 ~ (+) ~ (-) ~ (-) CD _ (3) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (-) ~ (. In comparison with p IRES-CEACAM6-4-1BBL/SL3261 group, the expression of CD56 in IFN- 緯 ~ (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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.34

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本文編號(hào)：1683923