本文關(guān)鍵詞： 佐劑 CpG-ODN Th1型免疫應(yīng)答 細(xì)胞毒性T淋巴細(xì)胞 細(xì)胞免疫　出處：《西北農(nóng)林科技大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：CD8~+ CTL反應(yīng)在腫瘤和慢性感染等疾病的治療中具有重要作用,然而在研究中多使用DNA疫苗和載體活疫苗等通過(guò)內(nèi)源性抗原加工提呈途徑誘生細(xì)胞免疫,往往具有安全性差、免疫效果不理想和抗載體效應(yīng)等缺點(diǎn)。蛋白和多肽等外源性抗原能夠通過(guò)交叉提呈和交叉致敏誘生CD8~+ CTL反應(yīng),但需要有效的疫苗佐劑來(lái)增強(qiáng)抗原提呈和共刺激信號(hào)并提供Th1類細(xì)胞因子環(huán)境。本論文初步評(píng)價(jià)了新型疫苗佐劑CpG-ODN與Al(OH)3或Montanide ISA 720等組成的復(fù)合佐劑在小鼠體內(nèi)促進(jìn)蛋白抗原通過(guò)交叉提呈和交叉致敏誘生CD8~+ CTL反應(yīng)的能力,從而為設(shè)計(jì)基于蛋白和多肽抗原的新型治療性疫苗提供理論基礎(chǔ)。 在本研究中,首先以雞卵清蛋白(OVA)為模型抗原,分別以CpG-ODN、Al(OH)3、Montanide ISA 720、CpG-ODN + Al(OH)3和CpG-ODN + Montanide ISA 720為疫苗佐劑,肌肉注射免疫C57BL/6小鼠,以胞內(nèi)細(xì)胞因子染色和體內(nèi)CTL殺傷等方法評(píng)價(jià)細(xì)胞免疫效果。在此基礎(chǔ)上,進(jìn)一步通過(guò)ELISPOT、胞內(nèi)細(xì)胞因子染色、體內(nèi)CTL殺傷和李斯特菌攻擊等方法評(píng)價(jià)了A、B和C三種具有不同化學(xué)結(jié)構(gòu)和生物學(xué)活性的CpG-ODN與Al(OH)3組成的復(fù)合佐劑對(duì)OVA抗原的細(xì)胞免疫佐劑效應(yīng)。 兩針免疫后結(jié)果表明,與無(wú)佐劑對(duì)照組相比,Al(OH)3本身并不能有效誘生細(xì)胞免疫,CpG-ODN或Montanide ISA 720單獨(dú)使用能夠在一定程度上增強(qiáng)抗原特異性CD8~+ T細(xì)胞的IFN-γ分泌和CTL活性,但不增強(qiáng)抗原特異性CD4~+ T細(xì)胞反應(yīng)。兩種復(fù)合佐劑具有比單佐劑更強(qiáng)的細(xì)胞免疫佐劑效應(yīng),其中CpG-ODN + Montanide ISA 720只能增強(qiáng)抗原特異性CD4~+和CD8~+ T細(xì)胞的IFN-γ分泌,而CpG-ODN + Al(OH)3不但能夠增強(qiáng)抗原特異性CD4~+和CD8~+ T細(xì)胞的IFN-γ反應(yīng),還能夠增強(qiáng)CD8~+ CTL反應(yīng)。三種不同類型CpG-ODN的比較研究結(jié)果表明,B和C型具有相近的細(xì)胞免疫佐劑效應(yīng),不但能夠促進(jìn)抗原特異性CD4~+和CD8~+ T細(xì)胞的IFN-γ分泌,而且能夠誘生抗原特異性CD8~+ CTL反應(yīng)。與此不同,盡管A型CpG-ODN能夠在一定程度上促進(jìn)抗原特異性CD8~+ CTL反應(yīng),但并不增強(qiáng)抗原特異性CD4~+和CD8~+ T細(xì)胞的IFN-γ分泌。與此相一致,B和C型CpG-ODN在小鼠李斯特菌攻擊試驗(yàn)中也顯示了比A型CpG-ODN更強(qiáng)的抗原特異性免疫保護(hù)作用。 綜上所述,在三種類型CpG-ODN中,B或C型CpG-ODN與Al(OH)3組成的復(fù)合佐劑在小鼠體內(nèi)具有更強(qiáng)的促進(jìn)蛋白抗原通過(guò)交叉提呈和交叉致敏誘生CD8~+ CTL反應(yīng)的能力。
[Abstract]:CD8 ~ CTL reaction plays an important role in the treatment of diseases such as tumor and chronic infection. However, the use of DNA vaccine and vector live vaccine to induce cellular immunity through endogenous antigen processing is often of poor safety. The immune effect is not ideal and the anti-carrier effect is not good. Exogenous antigens such as protein and polypeptide can induce CD8 ~ CTL reaction by cross-presentation and cross-sensitization. But effective vaccine adjuvant is needed to enhance antigen presentation and costimulatory signal and to provide Th1 cytokine environment. In this paper, we preliminarily evaluate the effect of a new vaccine adjuvant, CpG-ODN and Al(OH)3 or Montanide ISA 720, in mice. The ability of protein entry antigen to induce CD8 ~ CTL reaction by cross presentation and cross sensitization, This provides a theoretical basis for the design of novel therapeutic vaccines based on protein and polypeptide antigens. In this study, chicken ovalbumin (ovalbumin) was first used as model antigen. CpG-ODN ISA 720CpG-ODN Al(OH)3 and CpG-ODN Montanide ISA 720 were used as vaccine adjuvants to immunize C57BL / 6 mice intramuscularly. The cellular immune effect was evaluated by intracellular cytokine staining and CTL killing in vivo. In vivo CTL killing and listeria attack methods were used to evaluate the cellular immune adjuvant effect of three kinds of complex adjuvants of CpG-ODN and Al(OH)3 with different chemical structure and biological activity on OVA antigen. The results of two-dose immunization showed that the CpG-ODN or Montanide ISA 720 alone could not effectively induce CpG-ODN or Montanide ISA 720 to enhance the IFN- 緯 secretion and CTL activity of antigen-specific CD8T cells compared with the control group without adjuvant. But the antigen-specific CD4T cell response was not enhanced. The two kinds of complex adjuvants had stronger cellular immune adjuvant effect than single adjuvant, and CpG-ODN Montanide ISA 720 could only enhance IFN- 緯 secretion of antigen-specific CD4- and CD8T cells. CpG-ODN Al(OH)3 could not only enhance IFN- 緯 reaction of antigen-specific CD4 ~ and CD8T cells, but also enhance CD8 ~ CTL reaction. It can not only promote IFN- 緯 secretion of antigen-specific CD4- and CD8T cells, but also induce antigen-specific CD8- CTL reaction, although type A CpG-ODN can promote antigen-specific CD8- CTL reaction to some extent. However, the IFN- 緯 secretion of antigen-specific CD4- and CD8T cells was not enhanced. In accordance with this, B and C CpG-ODN also showed stronger antigen-specific immune protection than type A CpG-ODN in Listeria mutans attack test. In conclusion, the complex adjuvant composed of B or C type CpG-ODN and Al(OH)3 in three types of CpG-ODN has a stronger ability to induce CD8 ~ CTL reaction by cross-presentation and cross-sensitization of protein antigen in mice.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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本文編號(hào)：1511045