【摘要】： 研究目的： 本課題組構(gòu)建的靶向免疫防齲DNA疫苗經(jīng)過多年的發(fā)展,在實驗室研究中顯示出較強的粘膜免疫效果和防齲保護(hù)作用。但是關(guān)于靶向免疫防齲DNA疫苗如何增強免疫反應(yīng)的機制,尚有待進(jìn)一步研究。本實驗的研究目的是探討靶向防齲DNA疫苗加強粘膜免疫的效果的生物學(xué)機制,為今后更好的促進(jìn)靶向防齲DNA疫苗研究和應(yīng)用提供的深入的分子機制研究基礎(chǔ)。 研究方法： 本實驗研究首先構(gòu)建編碼小鼠細(xì)胞毒性T淋巴細(xì)胞抗原4(cytotoxic T lymphocyte antigen 4, CTLA4)胞外區(qū)、人IgG1鉸鏈區(qū)與Fc段、變形鏈球菌(Streptococcus mutans) PAc區(qū)和GLU區(qū)的靶向防齲質(zhì)粒(pmGJA-P),構(gòu)建編碼人CD5分子引導(dǎo)序列、人IgGl鉸鏈區(qū)與Fc段、PAc區(qū)和GLU區(qū)的非靶向防齲質(zhì)粒做為對照(pCDA-P).然后,將BALB/c小鼠分成三組,每組20只,分別經(jīng)過鼻腔滴注途徑給予靶向防齲疫苗(給予pmGJA-P鼻腔滴注),非靶向防齲疫苗(給予pCDA-P滴注)和空質(zhì)粒組(給予PCI滴注,陰性對照組)。比較各種質(zhì)粒在各試驗組誘導(dǎo)的抗體反應(yīng),抗原提呈細(xì)胞的形成,淋巴細(xì)胞增殖反應(yīng)。此外,比較不同組小鼠頭頸部淋巴結(jié)里樹突狀細(xì)胞(Dendritic cells, DCs)的表型變化,并應(yīng)用抗原提呈細(xì)胞功能芯片分析不同實驗組的DCs細(xì)胞在給予疫苗免疫前后基因的變化,實時定量PCR驗證芯片分析的結(jié)果。 實驗結(jié)果： 1特異性抗體反應(yīng)： 本研究檢測了14天和28天時各組的唾液和血清中抗PAc和抗GLU抗體水平。結(jié)果顯示靶向疫苗組唾液中兩種抗體水平均明顯高于其他組。另外,靶向疫苗組血清中的抗PAc和抗GLU抗體水平也明顯高于其他組。非靶向組的唾液和血清中抗PAc和抗GLU抗體水平則顯著高于空質(zhì)粒組。 2抗原提呈細(xì)胞： 通過運用抗原特異性的ELISPOT assay,本實驗分析了從頭頸部淋巴結(jié)和脾臟分離的單核細(xì)胞產(chǎn)生抗原提呈細(xì)胞的能力。從靶向疫苗組和非靶向疫苗組的頭頸部淋巴結(jié)和脾臟分離的單核細(xì)胞產(chǎn)生特異性抗原提呈細(xì)胞的數(shù)目均顯著的多于空質(zhì)粒組。靶向疫苗組的頭頸部淋巴結(jié)和脾臟分離的單核細(xì)胞形成抗原提呈細(xì)胞的數(shù)目則高于非靶向疫苗組。 3淋巴細(xì)胞增值實驗 從靶向疫苗組和非靶向疫苗組的頭頸部淋巴結(jié)分離的淋巴結(jié)細(xì)胞懸液的淋巴細(xì)胞增殖能力要明顯高于空質(zhì)粒組,兩個使用疫苗的組的脾細(xì)胞淋巴細(xì)胞增殖能力也要明顯高于空質(zhì)粒組。 4細(xì)胞因子： 給予靶向和非靶向疫苗的兩組,其頭頸部淋巴結(jié)和脾臟分離的細(xì)胞懸液經(jīng)抗原刺激之后,產(chǎn)生的分泌的IFN-γ和IL-4細(xì)胞要明顯多于空質(zhì)粒組。靶向和非靶向疫苗的兩組比較,分泌IFN-γ淋巴細(xì)胞的數(shù)目無顯著性差異。但是,無論頭頸部淋巴結(jié)還是脾臟分離的細(xì)胞懸液,靶向疫苗組分泌IL-4的細(xì)胞的數(shù)目要明顯多于非靶向疫苗組。 從兩個疫苗組的小鼠頭頸部和脾臟分離的細(xì)胞懸液經(jīng)過刺激后,其IFN-γ,IL-4和IL-5的mRNA表達(dá)水平明顯高于空質(zhì)粒組。IFN-γ在兩個疫苗組的表達(dá)水平無明顯差異。但是IL-4和IL-5 mRNA的表達(dá)水平,則是靶向疫苗組明顯高于非靶向疫苗組。這些結(jié)果表明靶向和非靶向組都產(chǎn)生了Th 1/Th2混合型的免疫反應(yīng)。靶向疫苗組產(chǎn)生更高的Th2型的細(xì)胞因子表達(dá),相比非靶向組加強了Th2型的免疫反應(yīng)。 5頭頸部淋巴結(jié)和脾臟分離的樹突狀細(xì)胞的表型分析： 從頭頸部淋巴結(jié)和脾臟中分離的單核細(xì)胞的中CD11c陽性的DCs在靶向疫苗組要明顯多于非靶向組。此外,在靶向組CD11c陽性的細(xì)胞的MHCⅡ、CD80和CD86表達(dá)更高。而非靶向疫苗組與空質(zhì)粒組比較,其CD11c陽性細(xì)胞的數(shù)目,MHCⅡ、CD80和CD86的表達(dá)均無明顯差異。 6芯片分析 有8個基因達(dá)到我們選定候選基因的標(biāo)準(zhǔn),所有候選基因又通過Real-timePCR進(jìn)行驗證,其中有7個候選基因的表達(dá)得到Real-time PCR的證實。這些差異基因涉及DCs的成熟,抗原攝取、提呈,生存。 實驗結(jié)論： 我們結(jié)果顯示,靶向防齲疫苗可能通過提高抗原呈遞的效果,促進(jìn)DC細(xì)胞的成熟,并由此提高DCs對抗原的攝取和提呈能力,促進(jìn)DCs的生存,進(jìn)而加強DCs對T、B細(xì)胞的激活,加強粘膜免疫。
[Abstract]:The purpose of the study is:
The target immune anti caries DNA vaccine developed by our group has shown strong mucosal immune effect and protection against caries in laboratory studies. However, further research on how to enhance the immune response of target immunization DNA vaccine is still to be further studied. The purpose of this experiment is to explore the DNA epidemic of targeting anti caries. The biological mechanism of the vaccine enhancement of mucosal immunity is the basis for further research and application of molecular mechanism for promoting the research and application of DNA vaccine against caries prevention.
Research methods:
In this experimental study, we first constructed the cytotoxic T lymphocyte antigen 4 (cytotoxic T lymphocyte antigen 4, CTLA4), human IgG1 hinges and Fc segments, the PAc region of the Streptococcus mutans (Streptococcus mutans) and the targeted anti caries plasmid of the GLU region. The non targeted caries resistant plasmids in the GLU area were compared (pCDA-P). Then, the BALB/c mice were divided into three groups, 20 in each group. The targeted caries vaccine (given pmGJA-P nasal drip), non targeted caries vaccine (pCDA-P drip) and air particle group (PCI drip, negative control group) were given by nasal drip pathway respectively. The various plasmids were compared. The antibody reaction induced by the experimental group, the formation of antigen presenting cells and the lymphocyte proliferation reaction. In addition, the phenotypic changes of Dendritic cells (DCs) in the head and neck lymph nodes of different groups were compared, and the antigen presenting cell function chip was used to analyze the changes of the DCs cells in different test groups before and after immunization. The results of the real-time quantitative PCR verification chip analysis.
Experimental results:
1 specific antibody response:
The study detected the anti PAc and anti GLU antibody levels in saliva and serum of each group at 14 days and 28 days. The results showed that the level of two antibodies in the saliva of the target vaccine group was significantly higher than that of the other groups. In addition, the anti PAc and anti GLU antibody levels in the serum of the target vaccine group were significantly higher than those of the other groups. The anti PAc and anti PAc in the saliva and serum of the non targeted group were PAc and anti. The level of GLU antibody was significantly higher than that in the empty plasmid group.
2 antigen presenting cells:
By using the antigen specific ELISPOT assay, the ability to produce antigen presenting cells from the mononuclear cells separated from the head and neck lymph nodes and the spleen was analyzed. The number of specific antigen presenting cells produced from the target vaccine group and the mononuclear cells separated from the head and neck lymph nodes and the spleen of the non target vaccine group were significantly more than that of the mononuclear cells. The number of mononuclear cells isolated from the head and neck lymph nodes and spleen of the targeted vaccine group was higher than that of the non targeted vaccine group.
3 lymphocyte increment experiment
Lymphoid cell suspension from the head and neck lymph nodes isolated from the target vaccine group and the non target vaccine group was significantly higher than that in the empty plasmid group, and the proliferation ability of the splenocytes in the two vaccine groups was significantly higher than that in the empty plasmid group.
4 cytokine:
In two groups of targeted and non targeted vaccines, the secreted IFN- gamma and IL-4 cells secreted in the head and neck lymph nodes and the splenic cell suspension were significantly more than those in the empty plasmid group. There were no significant differences in the number of IFN- gamma lymphocytes secreted by the target and non targeted vaccines, however, no matter the number of IFN- gamma lymphocytes was secreted. The number of cells secreting IL-4 in the targeted vaccine group was significantly higher than that in the non targeted vaccine group.
The expression level of IFN- gamma, IL-4 and IL-5 was significantly higher than that of the empty plasmid group.IFN- gamma in the two vaccine groups, but the expression level of IL-4 and IL-5 mRNA was significantly higher than that of the non targeted vaccine group, but the expression level of IL-4 and IL-5 mRNA was significantly higher than that of the IL-4 and IL-5 mRNA. The results showed that both the target and non target groups produced a mixed Th 1/Th2 immune response. The target vaccine group produced a higher expression of Th2 type cytokine, and enhanced the immune response of the Th2 type compared to the non targeted group.
Phenotypic analysis of dendritic cells isolated from 5 cervical lymph nodes and spleen:
The median CD11c positive DCs of the mononuclear cells isolated from the neck lymph node and the spleen was significantly more than the non targeting group. In addition, the expression of MHC II, CD80 and CD86 in the target group CD11c positive cells was higher. The number of CD11c positive cells, the expression of MHC II, CD80 and CD86 were both in the non targeted vaccine group and the empty plasmid group. There is no obvious difference.
6 chip analysis
8 genes reached the standard of our selected candidate genes, and all the candidate genes were verified by Real-timePCR, of which 7 of the candidate genes were expressed by Real-time PCR, which involved the maturation of DCs, the antigen uptake, the presentation, and survival.
Experimental conclusions:
The results show that the targeted anti caries vaccine may promote the maturation of DC cells by improving the effect of antigen presentation, and thus improve the ability of DCs to absorb and present the antigen, promote the survival of DCs, and then strengthen the activation of T, B cells and strengthen the mucosal immunity.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

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相關(guān)期刊論文 前2條

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本文編號：2124183