新型可复制型双质粒抗肾癌DNA疫苗的抑瘤活性及免疫学机制研究
发布时间：2018-10-14 08:16
【摘要】：研究背景： 肾细胞癌是成人肾脏肿瘤中最常见的一种类型，进展期肾癌预后相对较差，传统的治疗方式如化疗、放疗都不敏感，细胞因子治疗客观反应率仅为15%，仅对部分患者有效。近年来，DNA疫苗作为一种新的免疫治疗方式在恶性肿瘤治疗领域进行了广泛研究。本课题基于自主研发的可复制型DNA疫苗载体系统，成功构建了新型可复制型双质粒抗肾癌DNA疫苗，兼具抗肾癌与抗血管双重功效，希望能够为肾癌的免疫治疗提供一种新的思路。 研究目的： 本课题的目的是对新型可复制型双质粒抗肾癌DNA疫苗进行药效学评价，分析其可能的免疫机制，并为了进一步评价双质粒疫苗的抑瘤活性，建立同时稳定表达人G250抗原及荧光素酶报告基因的小鼠肾癌单克隆细胞系。 研究方法： 1. Renca-hG250/Luc单克隆细胞系的建立：将鉴定正确的pIRES-Luc-hyg质粒利用阳离子脂质体法转染Renca／hG250细胞系，之后利用潮霉素加压筛选和有限稀释法最终获得同时稳定表达人G250基因及荧光素酶报告基因Luc的小鼠肾癌单克隆细胞系Renca-hG250/Luc，并通过Western blot、流式细胞术及细胞体外活体成像检测hG250和Luc的表达情况。 2.双质粒疫苗动物实验研究：双质粒抗肾癌疫苗pSVK-tG250+CAVE及其他对照疫苗通过肌肉注射＋电穿孔的方式免疫Balb/c小鼠，间隔1周共免3次，并在末次免疫后1周皮下接种2×105个Renca-hG250/Luc细胞，建立荷瘤小鼠模型。首先观察肿瘤生长情况及荷瘤小鼠生存时间，评价抑瘤效果；其次研究双质粒疫苗在小鼠体内的免疫学机制：收集免疫后小鼠血清，检测hG250和mVEGFR2特异性IgG抗体水平，以及hG250特异性IgG1/IgG2的比值；处死小鼠并分离脾脏淋巴细胞，CCK-8法检测脾脏淋巴细胞的特异性增值活性，ELISPOT检测hG250及mVEGFR2特异性脾脏淋巴细胞IFN-γ分泌，ELISA法检测脾脏淋巴细胞Th1类细胞因子（IL-2和INF-γ）的分泌，最后LDH法检测脾脏淋巴细胞CTL杀伤活性。 结果： 1.成功建立了同时稳定表达hG250和荧光素酶报告基因的小鼠肾癌单克隆细胞系Renca-hG250/Luc，其hG250和luc的阳性表达率分别达到81.87%和81.03%，，能够有效地与荧光素酶底物发生反应放出荧光。 2.双质粒疫苗pSVK-tG250+CAVE免疫小鼠后，发现与PBS组、pSVK空载体及pSVK-tG250、CAVE单独应用组相比，成瘤时间最多延长10天（P＜0.05），小鼠皮下肿瘤生长明显受到抑制，小鼠的平均生存期延长；双质粒疫苗在肌肉注射加电穿孔免疫方式下能够诱导机体产生较高水平的hG250和mVEGFR2特异性抗体，并且小鼠血清中抗hG250IgG1/IgG2a的比值为0.76，低于其他各组；在hG250和mVEGFR2抗原的刺激下，双质粒疫苗组小鼠脾脏淋巴细胞特异性IFN-γ分泌数分别达到405±52个/5×105和453±61个/5×105脾脏淋巴细胞；此外，双质粒组小鼠脾脏淋巴细胞具有更高的增值活性，hG250抗原刺激后脾脏淋巴细胞培养上清中可检测到Th1类细胞因子IL-2和IFN-γ大量释放；最后，双质粒疫苗组对肾癌靶细胞的CTL杀伤活性得到了明显增强，在效靶比为40:1、20:1和10:1时分别达到51.12%、39.23%和25.88%。 结论： 新型可复制型抗肾癌双质粒疫苗能够在小鼠体内诱导产生高效的抗肾癌体液免疫应答和细胞免疫应答，具有抗肾癌和抗血管双重功效，有效地抑制肾癌的生长，为肾癌的免疫治疗提供了新的方法。
[Abstract]:Background of the study: Renal cell carcinoma is one of the most common types in adult kidney tumors. The prognosis of renal cell carcinoma is relatively poor. Traditional treatment methods such as chemotherapy and radiotherapy are not sensitive. The objective response rate of cytokine therapy is only 15%. In recent years, DNA vaccine has been widely used as a new way of immunotherapy in the treatment of malignant tumor This topic is based on the self-developed replicable DNA vaccine vector system, successfully constructed a novel replicable double plasmid anti-renal cell carcinoma DNA vaccine, has both anti-renal cell carcinoma and anti-blood vessel double efficacy, and hopes to provide a new immune therapy for renal cancer. Train of thought. The purpose of this study is to evaluate the pharmacodynamics of a novel replicable double plasmid anti-renal cell carcinoma DNA vaccine, analyze its possible immune mechanism, and to further evaluate the double quality. Tumor tumor suppressor activity of particle vaccine and establishment of mouse stably expressing human G250 antigen and luciferase reporter gene renal cell carcinoma monoclonal antibody Cell line. Study method: 1. Establishment of the Renca-hG250/ Luc monoclonal cell line: The correct pIRES-Luc-hyg plasmid was transfected with a cationic liposome method Renca/ hG250 cell line, after which the mouse renal cell carcinoma monoclonal cell line Renca-hG250/ Luc stably expressing human G250 gene and luciferase reporter gene Luc is finally obtained by using hygromycin pressurization screening and limited dilution method, Expression of hG250 and Luc. Two-plasmid vaccine animal experiment study: Double-plasmid anti-renal cell carcinoma vaccine pSVK-tG250 + CAVE and other control vaccines were immunized by intramuscular injection and electroporation. 50/ Luc cells were used to establish the model of tumor-bearing mice. The tumor growth and survival time of tumor-bearing mice were first observed, the tumor suppressor effect was evaluated; secondly, the immunological mechanism of the double-plasmid vaccine in mice was studied: the serum of the immunized mice was collected, the levels of hG250 and mVEGFR2-specific IgG antibodies were detected, and hG2 50-specific IgG1/ IgG2 ratio; killing mice and isolating spleen lymphocytes, CCK-8 method for detecting specific value-added activity of spleen lymphocytes, ELISPOT detection hG250 and mVEGFR2-specific spleen lymphocyte IFN-7721 secretion, and ELISA method for detecting Th1 cells of spleen lymphocytes secretion of factor (IL-2 and INF-inf), last LD H-method inspection Results: 1. Mouse renal cell carcinoma monoclonal cell line Renca-hG250/ Luc stably expressing hG250 and luciferase reporter gene was successfully established. The positive rates of hG250 and luc were 81.87% and 81.0 respectively. When pSVK-tG250 + CAVE mice immunized with pSVK-tG250 + CAVE were immunized with pSVK-tG250 + CAVE, it was found that the tumor time was more than 10 days (P <0.05) compared with PBS group, pSVK empty vector and pSVK-tG250 and CAVE alone group. and the ratio of anti-hG250IgG1/ IgG2a in the serum of the mice is 0. 76 and low. In other groups, in the stimulation of hG250 and mVEGFR2 antigen, the number of specific IFN-tau secretion in spleen lymphocytes of mice immunized with double plasmid vaccine was 405, 52/ 5, 105 and 453, 61/ 5, 105, respectively. In addition, the spleen lymphocytes of the double-plasmid group had higher value-added activity, and the spleen lymphocytes cultured in the hG250 group were able to detect the large release of the Th1 cytokines IL-2 and IFN-, and finally, the CTL killing activity of the double-plasmid vaccine group on the target cells of the renal cell carcinoma was significantly enhanced, and the effect target ratio was 40: 1, 20: 1 and 10: 1 hour Conclusion: A new type of replicable anti-RCC double plasmid vaccine can induce high-efficient humoral immune response in renal cell carcinoma in vivo. and cell immune response, and has the effects of resisting kidney cancer and resisting blood.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11
[Abstract]:Background of the study: Renal cell carcinoma is one of the most common types in adult kidney tumors. The prognosis of renal cell carcinoma is relatively poor. Traditional treatment methods such as chemotherapy and radiotherapy are not sensitive. The objective response rate of cytokine therapy is only 15%. In recent years, DNA vaccine has been widely used as a new way of immunotherapy in the treatment of malignant tumor This topic is based on the self-developed replicable DNA vaccine vector system, successfully constructed a novel replicable double plasmid anti-renal cell carcinoma DNA vaccine, has both anti-renal cell carcinoma and anti-blood vessel double efficacy, and hopes to provide a new immune therapy for renal cancer. Train of thought. The purpose of this study is to evaluate the pharmacodynamics of a novel replicable double plasmid anti-renal cell carcinoma DNA vaccine, analyze its possible immune mechanism, and to further evaluate the double quality. Tumor tumor suppressor activity of particle vaccine and establishment of mouse stably expressing human G250 antigen and luciferase reporter gene renal cell carcinoma monoclonal antibody Cell line. Study method: 1. Establishment of the Renca-hG250/ Luc monoclonal cell line: The correct pIRES-Luc-hyg plasmid was transfected with a cationic liposome method Renca/ hG250 cell line, after which the mouse renal cell carcinoma monoclonal cell line Renca-hG250/ Luc stably expressing human G250 gene and luciferase reporter gene Luc is finally obtained by using hygromycin pressurization screening and limited dilution method, Expression of hG250 and Luc. Two-plasmid vaccine animal experiment study: Double-plasmid anti-renal cell carcinoma vaccine pSVK-tG250 + CAVE and other control vaccines were immunized by intramuscular injection and electroporation. 50/ Luc cells were used to establish the model of tumor-bearing mice. The tumor growth and survival time of tumor-bearing mice were first observed, the tumor suppressor effect was evaluated; secondly, the immunological mechanism of the double-plasmid vaccine in mice was studied: the serum of the immunized mice was collected, the levels of hG250 and mVEGFR2-specific IgG antibodies were detected, and hG2 50-specific IgG1/ IgG2 ratio; killing mice and isolating spleen lymphocytes, CCK-8 method for detecting specific value-added activity of spleen lymphocytes, ELISPOT detection hG250 and mVEGFR2-specific spleen lymphocyte IFN-7721 secretion, and ELISA method for detecting Th1 cells of spleen lymphocytes secretion of factor (IL-2 and INF-inf), last LD H-method inspection Results: 1. Mouse renal cell carcinoma monoclonal cell line Renca-hG250/ Luc stably expressing hG250 and luciferase reporter gene was successfully established. The positive rates of hG250 and luc were 81.87% and 81.0 respectively. When pSVK-tG250 + CAVE mice immunized with pSVK-tG250 + CAVE were immunized with pSVK-tG250 + CAVE, it was found that the tumor time was more than 10 days (P <0.05) compared with PBS group, pSVK empty vector and pSVK-tG250 and CAVE alone group. and the ratio of anti-hG250IgG1/ IgG2a in the serum of the mice is 0. 76 and low. In other groups, in the stimulation of hG250 and mVEGFR2 antigen, the number of specific IFN-tau secretion in spleen lymphocytes of mice immunized with double plasmid vaccine was 405, 52/ 5, 105 and 453, 61/ 5, 105, respectively. In addition, the spleen lymphocytes of the double-plasmid group had higher value-added activity, and the spleen lymphocytes cultured in the hG250 group were able to detect the large release of the Th1 cytokines IL-2 and IFN-, and finally, the CTL killing activity of the double-plasmid vaccine group on the target cells of the renal cell carcinoma was significantly enhanced, and the effect target ratio was 40: 1, 20: 1 and 10: 1 hour Conclusion: A new type of replicable anti-RCC double plasmid vaccine can induce high-efficient humoral immune response in renal cell carcinoma in vivo. and cell immune response, and has the effects of resisting kidney cancer and resisting blood.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11
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