【摘要】：研究背景： 腎細(xì)胞癌是成人腎臟腫瘤中最常見的一種類型，進(jìn)展期腎癌預(yù)后相對較差，傳統(tǒng)的治療方式如化療、放療都不敏感，細(xì)胞因子治療客觀反應(yīng)率僅為15%，僅對部分患者有效。近年來，DNA疫苗作為一種新的免疫治療方式在惡性腫瘤治療領(lǐng)域進(jìn)行了廣泛研究。本課題基于自主研發(fā)的可復(fù)制型DNA疫苗載體系統(tǒng)，成功構(gòu)建了新型可復(fù)制型雙質(zhì)粒抗腎癌DNA疫苗，兼具抗腎癌與抗血管雙重功效，希望能夠為腎癌的免疫治療提供一種新的思路。 研究目的： 本課題的目的是對新型可復(fù)制型雙質(zhì)粒抗腎癌DNA疫苗進(jìn)行藥效學(xué)評價，分析其可能的免疫機(jī)制，并為了進(jìn)一步評價雙質(zhì)粒疫苗的抑瘤活性，建立同時穩(wěn)定表達(dá)人G250抗原及熒光素酶報告基因的小鼠腎癌單克隆細(xì)胞系。 研究方法： 1. Renca-hG250/Luc單克隆細(xì)胞系的建立：將鑒定正確的pIRES-Luc-hyg質(zhì)粒利用陽離子脂質(zhì)體法轉(zhuǎn)染Renca／hG250細(xì)胞系，之后利用潮霉素加壓篩選和有限稀釋法最終獲得同時穩(wěn)定表達(dá)人G250基因及熒光素酶報告基因Luc的小鼠腎癌單克隆細(xì)胞系Renca-hG250/Luc，并通過Western blot、流式細(xì)胞術(shù)及細(xì)胞體外活體成像檢測hG250和Luc的表達(dá)情況。 2.雙質(zhì)粒疫苗動物實驗研究：雙質(zhì)粒抗腎癌疫苗pSVK-tG250+CAVE及其他對照疫苗通過肌肉注射＋電穿孔的方式免疫Balb/c小鼠，間隔1周共免3次，并在末次免疫后1周皮下接種2×105個Renca-hG250/Luc細(xì)胞，建立荷瘤小鼠模型。首先觀察腫瘤生長情況及荷瘤小鼠生存時間，評價抑瘤效果；其次研究雙質(zhì)粒疫苗在小鼠體內(nèi)的免疫學(xué)機(jī)制：收集免疫后小鼠血清，檢測hG250和mVEGFR2特異性IgG抗體水平，以及hG250特異性IgG1/IgG2的比值；處死小鼠并分離脾臟淋巴細(xì)胞，CCK-8法檢測脾臟淋巴細(xì)胞的特異性增值活性，ELISPOT檢測hG250及mVEGFR2特異性脾臟淋巴細(xì)胞IFN-γ分泌，ELISA法檢測脾臟淋巴細(xì)胞Th1類細(xì)胞因子（IL-2和INF-γ）的分泌，最后LDH法檢測脾臟淋巴細(xì)胞CTL殺傷活性。 結(jié)果： 1.成功建立了同時穩(wěn)定表達(dá)hG250和熒光素酶報告基因的小鼠腎癌單克隆細(xì)胞系Renca-hG250/Luc，其hG250和luc的陽性表達(dá)率分別達(dá)到81.87%和81.03%，，能夠有效地與熒光素酶底物發(fā)生反應(yīng)放出熒光。 2.雙質(zhì)粒疫苗pSVK-tG250+CAVE免疫小鼠后，發(fā)現(xiàn)與PBS組、pSVK空載體及pSVK-tG250、CAVE單獨應(yīng)用組相比，成瘤時間最多延長10天（P＜0.05），小鼠皮下腫瘤生長明顯受到抑制，小鼠的平均生存期延長；雙質(zhì)粒疫苗在肌肉注射加電穿孔免疫方式下能夠誘導(dǎo)機(jī)體產(chǎn)生較高水平的hG250和mVEGFR2特異性抗體，并且小鼠血清中抗hG250IgG1/IgG2a的比值為0.76，低于其他各組；在hG250和mVEGFR2抗原的刺激下，雙質(zhì)粒疫苗組小鼠脾臟淋巴細(xì)胞特異性IFN-γ分泌數(shù)分別達(dá)到405±52個/5×105和453±61個/5×105脾臟淋巴細(xì)胞；此外，雙質(zhì)粒組小鼠脾臟淋巴細(xì)胞具有更高的增值活性，hG250抗原刺激后脾臟淋巴細(xì)胞培養(yǎng)上清中可檢測到Th1類細(xì)胞因子IL-2和IFN-γ大量釋放；最后，雙質(zhì)粒疫苗組對腎癌靶細(xì)胞的CTL殺傷活性得到了明顯增強(qiáng)，在效靶比為40:1、20:1和10:1時分別達(dá)到51.12%、39.23%和25.88%。 結(jié)論： 新型可復(fù)制型抗腎癌雙質(zhì)粒疫苗能夠在小鼠體內(nèi)誘導(dǎo)產(chǎn)生高效的抗腎癌體液免疫應(yīng)答和細(xì)胞免疫應(yīng)答，具有抗腎癌和抗血管雙重功效，有效地抑制腎癌的生長，為腎癌的免疫治療提供了新的方法。
[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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.11

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