Ag85A与Ag85BDNA疫苗联合应用对移植性鼠膀胱肿瘤的免疫调节效应

发布时间：2018-03-09 09:10

本文选题：膀胱肿瘤　切入点：BCG　出处：《山西医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:探讨联合应用Ag85A与Ag85B DNA疫苗抗膀胱肿瘤的免疫效应。方法:采用体外培养的小鼠膀胱肿瘤细胞株BTT739,皮下注射T739小鼠的左后肢内侧,成功构建可移植性小鼠膀胱肿瘤模型。取48只,随机数字表法随机分为6组,即生理盐水组、pcDNA3.1+组、卡介苗(BCG)组、pcDNA-Ag85A组、pcDNA-Ag85B组以及pcDNA—Ag85A+pcDNA—Ag85B组。荷瘤后第7天、14天和21天各肌注1次,末次肌注后7天处死小鼠,流式细胞仪检测各组小鼠脾脏的CD4~+和CD8~+ T细胞亚群数量及所占百分比并计算CD4~+/CD8~+比值;酶联免疫吸附测定(ELISA)法检测小鼠血清中的干扰素γ(γ-IFN)分泌水平;定期测量肿瘤大小,计算肿瘤体积;剥离肿瘤称重并作肿瘤组织病理学检查。结果:1.经双酶切鉴定,成功提取重组质粒pcDNA-Ag85A及pcDNA-Ag85B。 2.CD4~+和CD8~+ T细胞亚群流式细胞学检测结果:Ag85A与Ag85B DNA疫苗联合免疫组CD4~+和CD8~+ T细胞亚群的数量及CD4~+/CD8~+的比值明显高于DNA疫苗单独免疫组,差异有显著性(p＜0.05),但不及BCG组(p＜0.05)。 3.小鼠血清IFN-γ水平的ELISA检测结果:Ag85A与Ag85B DNA疫苗联合免疫组小鼠血清IFN-γ的水平明显高于DNA疫苗单独免疫组,差异有显著性(p＜0.05),但不及BCG组(p＜0.05)。 4.肿瘤称重:Ag85A与Ag85B DNA疫苗联合免疫组小鼠的肿瘤重量明显低于DNA疫苗单独免疫组,差异有显著性(p＜0.05),但高于BCG组(p＜0.05)。 5.肿瘤组织病理学变化:Ag85A与Ag85B DNA疫苗联合免疫组肿瘤坏死(核浓缩、核碎裂、核溶解,胞浆红染)较DNA疫苗单独免疫组明显,且瘤体周围及瘤体内炎细胞(中性粒细胞、淋巴细胞、单核细胞)浸润:但BCG组肿瘤坏死更为明显,瘤体周围及瘤体内有大量炎细胞浸润。结论:1.Ag85A与Ag85B DNA疫苗联合免疫在抗膀胱肿瘤过程中具有明显的免疫效应,且明显优于DNA疫苗单独免疫,二者联用具有协同增强免疫作用; 2.Ag85A与Ag85B DNA疫苗联合免疫抗膀胱肿瘤效应尚达不到BCG水平。
[Abstract]:Objective: to investigate the immunological effect of combined use of Ag85A and Ag85B DNA vaccine against bladder cancer. Methods: the mouse bladder tumor cell line BTT739 was cultured in vitro and injected subcutaneously into the medial left hind limb of T739 mouse. 48 mice were randomly divided into 6 groups. That is, normal saline group (pcDNA3.1), BCG (BCG) group, pcDNA-Ag85A group (pcDNA-Ag85B) and pcDNA-Ag85A pcDNA-Ag85B group. The number and percentage of CD4 ~ and CD8T lymphocyte subsets in spleen of each group were detected by flow cytometry and the ratio of CD4 ~ / / CD8 ~ ~ was calculated. The levels of interferon 纬 (纬 -IFNs) in serum of mice were detected by Elisa, and tumor size was measured regularly. Tumor volume was calculated, tumor weight was removed and histopathological examination was performed. Results 1. The recombinant plasmid pcDNA-Ag85A and pcDNA-Ag85B were successfully extracted by double enzyme digestion. 2. The number of CD4 ~ and CD8T subsets and the ratio of CD4 ~ / / CD8 ~ ~ ~ of CD4 ~ / CD8 ~ ~ were significantly higher in CD4 ~ and CD8- T cell subsets immunized with Ag85B DNA vaccine than those in DNA vaccine alone group, the difference was significant (P < 0.05), but less than that in BCG group (p < 0.05). 3. The serum IFN- 纬 level of mice immunized with Ag85B DNA vaccine was significantly higher than that of mice immunized with DNA vaccine alone (P < 0.05), but less than that of BCG group (P < 0.05). 3. The level of serum IFN- 纬 in mice immunized with Ag85B DNA vaccine was significantly higher than that in mice immunized with DNA vaccine alone (P < 0.05). 4. The tumor weight of mice immunized with Ag85B DNA vaccine was significantly lower than that of mice immunized with DNA vaccine alone (P < 0.05), but higher than that of BCG group (P < 0.05). 5. Histopathological changes of tumor tissues in the tumor necrosis group (nuclear concentration, nuclear fragmentation, nucleolysis, cytoplasmic red stain) in the combined immunization group with Ag85B DNA vaccine were significantly higher than those in the DNA vaccine alone group, and the inflammatory cells (neutrophils) around the tumor and in the tumor body. Lymphocyte, monocyte) infiltration: however, tumor necrosis was more obvious in BCG group, and a large number of inflammatory cells were infiltrated around the tumor and in the tumor body. Conclusion the combined immunization of Ag85B DNA vaccine with 1: 1 Ag85A vaccine has obvious immune effect in the process of anti-bladder tumor, and is obviously superior to that of DNA vaccine alone. The combination of the two vaccines can enhance the immunity synergistically. 2. The effect of Ag85A combined with Ag85B DNA vaccine on bladder tumor was not up to the level of BCG.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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