靶向防龋DNA疫苗增强粘膜免疫的分子机制研究
发布时间：2018-07-15 13:11
【摘要】： 研究目的： 本课题组构建的靶向免疫防龋DNA疫苗经过多年的发展,在实验室研究中显示出较强的粘膜免疫效果和防龋保护作用。但是关于靶向免疫防龋DNA疫苗如何增强免疫反应的机制,尚有待进一步研究。本实验的研究目的是探讨靶向防龋DNA疫苗加强粘膜免疫的效果的生物学机制,为今后更好的促进靶向防龋DNA疫苗研究和应用提供的深入的分子机制研究基础。 研究方法： 本实验研究首先构建编码小鼠细胞毒性T淋巴细胞抗原4(cytotoxic T lymphocyte antigen 4, CTLA4)胞外区、人IgG1铰链区与Fc段、变形链球菌(Streptococcus mutans) PAc区和GLU区的靶向防龋质粒(pmGJA-P),构建编码人CD5分子引导序列、人IgGl铰链区与Fc段、PAc区和GLU区的非靶向防龋质粒做为对照(pCDA-P).然后,将BALB/c小鼠分成三组,每组20只,分别经过鼻腔滴注途径给予靶向防龋疫苗(给予pmGJA-P鼻腔滴注),非靶向防龋疫苗(给予pCDA-P滴注)和空质粒组(给予PCI滴注,阴性对照组)。比较各种质粒在各试验组诱导的抗体反应,抗原提呈细胞的形成,淋巴细胞增殖反应。此外,比较不同组小鼠头颈部淋巴结里树突状细胞(Dendritic cells, DCs)的表型变化,并应用抗原提呈细胞功能芯片分析不同实验组的DCs细胞在给予疫苗免疫前后基因的变化,实时定量PCR验证芯片分析的结果。 实验结果： 1特异性抗体反应： 本研究检测了14天和28天时各组的唾液和血清中抗PAc和抗GLU抗体水平。结果显示靶向疫苗组唾液中两种抗体水平均明显高于其他组。另外,靶向疫苗组血清中的抗PAc和抗GLU抗体水平也明显高于其他组。非靶向组的唾液和血清中抗PAc和抗GLU抗体水平则显著高于空质粒组。 2抗原提呈细胞： 通过运用抗原特异性的ELISPOT assay,本实验分析了从头颈部淋巴结和脾脏分离的单核细胞产生抗原提呈细胞的能力。从靶向疫苗组和非靶向疫苗组的头颈部淋巴结和脾脏分离的单核细胞产生特异性抗原提呈细胞的数目均显著的多于空质粒组。靶向疫苗组的头颈部淋巴结和脾脏分离的单核细胞形成抗原提呈细胞的数目则高于非靶向疫苗组。 3淋巴细胞增值实验 从靶向疫苗组和非靶向疫苗组的头颈部淋巴结分离的淋巴结细胞悬液的淋巴细胞增殖能力要明显高于空质粒组,两个使用疫苗的组的脾细胞淋巴细胞增殖能力也要明显高于空质粒组。 4细胞因子： 给予靶向和非靶向疫苗的两组,其头颈部淋巴结和脾脏分离的细胞悬液经抗原刺激之后,产生的分泌的IFN-γ和IL-4细胞要明显多于空质粒组。靶向和非靶向疫苗的两组比较,分泌IFN-γ淋巴细胞的数目无显著性差异。但是,无论头颈部淋巴结还是脾脏分离的细胞悬液,靶向疫苗组分泌IL-4的细胞的数目要明显多于非靶向疫苗组。 从两个疫苗组的小鼠头颈部和脾脏分离的细胞悬液经过刺激后,其IFN-γ,IL-4和IL-5的mRNA表达水平明显高于空质粒组。IFN-γ在两个疫苗组的表达水平无明显差异。但是IL-4和IL-5 mRNA的表达水平,则是靶向疫苗组明显高于非靶向疫苗组。这些结果表明靶向和非靶向组都产生了Th 1/Th2混合型的免疫反应。靶向疫苗组产生更高的Th2型的细胞因子表达,相比非靶向组加强了Th2型的免疫反应。 5头颈部淋巴结和脾脏分离的树突状细胞的表型分析： 从头颈部淋巴结和脾脏中分离的单核细胞的中CD11c阳性的DCs在靶向疫苗组要明显多于非靶向组。此外,在靶向组CD11c阳性的细胞的MHCⅡ、CD80和CD86表达更高。而非靶向疫苗组与空质粒组比较,其CD11c阳性细胞的数目,MHCⅡ、CD80和CD86的表达均无明显差异。 6芯片分析 有8个基因达到我们选定候选基因的标准,所有候选基因又通过Real-timePCR进行验证,其中有7个候选基因的表达得到Real-time PCR的证实。这些差异基因涉及DCs的成熟,抗原摄取、提呈,生存。 实验结论： 我们结果显示,靶向防龋疫苗可能通过提高抗原呈递的效果,促进DC细胞的成熟,并由此提高DCs对抗原的摄取和提呈能力,促进DCs的生存,进而加强DCs对T、B细胞的激活,加强粘膜免疫。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
本文编号：2124183
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 ;Recent advances in DNA vaccine of hepatitis virus[J];Hepatobiliary & Pancreatic Diseases International;2002年02期
2 ;Enhanced efficacy of CTLA-4 fusion anti-caries DNA vaccines in gnotobiotic hamsters[J];Acta Pharmacologica Sinica;2007年08期
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