【作者】 许道军；

【导师】 薛立群； 余兴龙；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2010， 博士

【摘要】 佐剂在免疫中的主要作用是增强机体抗原特异性免疫应答水平和持续时间,提高疫苗的接种效果。佐剂作为一种强烈的免疫刺激剂,能够在注射的局部或全身激发强烈而持久的免疫应答,诱导不同的细胞因子的分泌,进而对局部或全身的CD4+T细胞亚群的平衡状态产生一定的影响。佐剂对免疫平衡状态的改变可能对免疫系统产生一定的负面影响,造成一定程度的免疫性疾病或者抑制免疫系统对其它病原体的免疫应答。在当前我国畜牧业中,由于接种疫苗的种类以及接种次数均有所增加,造成佐剂的使用量相应增加。系统而全面的评估佐剂对于整体免疫系统的影响显得尤为重要。尽管已有较多研究从佐剂对Th1/Th2免疫平衡的影响这一角度进行了相关的研究,但是系统的研究单纯的佐剂对CD4+T细胞亚群的影响的研究还不多见,佐剂对Tregs的影响的研究也还匮乏。为了较全面的研究佐剂对CD4+T细胞亚群的影响,本研究采用单纯的弗氏完全佐剂及Al(OH)3佐剂这二种常见的佐剂作为研究对象,利用Balb/c小鼠作为实验动物,对该二种佐剂处理后的第14d后的脾脏内调节性T细胞及CD4+T细胞各亚群的相关基因进行了相关的研究。以期为系统的评估佐剂对整体免疫系统的影响提供理论基础。本研究的主要的研究内容及结果如下：实验选取30只Balb/c小鼠随机分为弗氏完全佐剂组、Al(OH)3佐剂组和对照组。三组小鼠分别经腹腔一次性注射生理盐水乳化的完全弗氏佐剂(1：1乳化)、1mg/mL的Al(OH)3悬混液、生理盐水各200μl。在佐剂处理后的14d麻醉小鼠后采集血液分离血清,同时无菌分离脾脏后进行Tregs流式细胞分析及CD4+T细胞相关基因的表达谱分析。采用流式细胞仪对弗氏佐剂和氢氧化铝佐剂处理小鼠的脾脏内CD4+T细胞、CD4+CD25-Foxp3+T细胞、CD4+Foxp3+T细胞以及CD4+CD25+Foxp3+T细胞进行分析。结果显示弗氏佐剂脾脏内CD4+CD25-Foxp3+T细胞、CD4+Foxp3+T细胞以及CD4+CD25+Foxp3+T细胞占总CD4+T细胞的百分比升高,而脾脏内CD4+T细胞占总脾细胞的百分比下降,与对照组比较差异均极显著(p<0.01)；Al(OH)3佐剂组的这些指标与对照组比较差异均不显著(p>0.05)；采用real-time RT-PCR及免疫组化的方法检测了三组小鼠脾脏内Foxp3基因的表达水平及Foxp3蛋白含量。结果显示三组小鼠脾脏内的Foxp3基因的表达水平及蛋白浓度差异不显著(p>0.05)。各组小鼠脾细胞的流式细胞散点图还表明弗氏佐剂组脾脏内一群CD4表达阴性,但是侧向角散射值较大的细胞类型显著增高,初步推测该类细胞为巨噬细胞。采用real-time RT-PCR基因芯片技术对弗氏佐剂和氢氧化铝佐剂处理小鼠的脾脏内Th1/Th2/Th3相关的共84个基因的表达水平进行了检测,结果显示弗氏佐剂组小鼠表达水平显著下降的基因有：BCL6、CCL5、CCR3、CCR4、CD28、CD4、CD80、CD86、CSF2、CTLA4、CXCR3、GATA3、ICOS、IFNG、IL-10、IL12RB2、IL-18、IL18BP、IL1R1、IL-2、IL27RA、IL2RA、I-L4、IL4RA、IRF1 JAK1、MAF、MAPK9、NFATC1、NFATC3、NFKB1、PTPRC、SOCS5、STAT1 STAT4；表达水平显著上升的基因有：IL-17 IL-23a、CEBPB、GFI1、IL-9、SPP1、YY1其余的基因表达水平差异不显著。氢氧化铝佐剂组与对照组比较仅IL-2基因表达水平显著上升,其余的差异均不显著。对弗氏佐剂和氢氧化铝佐剂处理小鼠的血清中的TGF-β1和IL-17的含量进行了ELISA检测,结果表明：弗氏佐剂组小鼠血清中TGF-β1和IL-17的含量显著提高,与对照组比较差异显著(p<0.05),而氢氧化铝佐剂组与对照组比较差异不显著(p>0.05)。对小鼠脾脏指数及显微切片观察结果表明：弗氏佐剂组脾脏指数显著提高,同时弗氏佐剂组脾脏内巨噬细胞显著增多。氢氧化铝佐剂组与对照组比较差异不显著。通过对以上实验结果的综合分析,表明单纯的弗氏佐剂腹腔处理小鼠,能显著增加小鼠脾脏内巨噬细胞数量,提高脾脏指数,同时还能够显著提高小鼠脾脏内Tregs的百分比,及Th17细胞相关的IL-17和IL-23的表达水平,并对脾脏内的Th1、Th2及总的CD4+T细胞相关的基因表达具有一定的抑制作用。而氢氧化铝佐剂对上述指标的差异均不显著。初步推测弗氏佐剂虽然能够有效的提高机体免疫系统针对抗原特异性的免疫应答水平,但是其对整体免疫系统的功能可能具有一定程度的抑制作用。更多还原

【Abstract】 Adjuvants are used to boost the potency and longevity of specific immune response to antigens. As a strong agent in improving the body’s specific immune response, adjuvants are able to stimulate a strong and lasting immune response where the local or systemic of body, which induces different cytokines which have an effect on the local or systemic CD4+T cell subsets balance. Changes in the immune balance caused by adjuvants may have some negative impacts on the immune system, which might cause a certain degree of immunity disease or suppress the immune response to other pathogens. With the type of vaccination and inoculation times increasing in the livestock industry in our country, which results in a corresponding increase in the use of adjuvant. The situation is even more prominent especially to evaluate the effects of adjuvants on systemic immune system.Although numerous reports indicated that adjuvants had a significant effect on the immune system of Thl/Th2 immune balance, there were less studies on a newly found CD4+CD25+regulatory T cells (Tregs) and Th17 cells which following the Thl and Th2. Little information is available on cytokine induction by adjuvant only. To comprehensively understand the effects of adjuvant on CD4+T cell subsets, Freund’s complete adjuvant and Al (OH)3 adjuvant were used to evaluate their effects on Tregs and gene expression profile in spleen of Balb/c mice. Thirty Balb/c mice were randomly assigned to three groups; CFA group, AHA group and control group. Three groups were administered intraperitoneally with 200μL CFA (emulsified with PBS in a ratio of 1:1), AHA(1 mg/mL) or PBS respectively. All mice were necropsied at Day 14. This studies might provide a theoretical basis for assessing the impact of adjuvants on immune system. The main research contents and results as follows:The frequencies of CD4+Foxp3+T cells, CD4+CD25- Foxp3+T cells and CD4+CD25+Foxp3+T cells in splenocytes were assessed by flow cytometry and Foxp3 mRNA levels were detected using reverse transcription-PCR (RT-PCR) and immunohistochemical analysis. The result showed that CFA decreased the proportion of CD4+T cell in splenocytes and promoted the percent of CD4+Foxp3+T cells, CD4+CD25-Foxp3+T cells and CD4+CD25+Foxp3+T cells in CD4+T cells in splenocytes on day 14 (p< 0.01). But the levels of Foxp3 mRNA were no difference among the three groups(p> 0.05). AHA has no effect on foregoing index(p> 0.05). Results from the figures of flow cytometry analysis, there were a group of cells with CD4 negative, and with large side-angle scattering and the cells number was significantly increased in Freund’s adjuvant group compared with the other groups. Based on the above characteristics, these cells might be macrophages.Eighty-four gene expression profiles which related to Thl/Th2/Th3 were detected by real-time RT-PCR gene chip technology. The results showed that the expressions of genes BCL6, CCL5, CCR3, CCR4, CD28, CD4, CD80, CD86, CSF2, CTLA4, CXCR3, GATA3, ICOS, IFNG, IL-10, IL-12RB2, IL-18, IL-18BP, IL-1R1, IL-2, IL-27RA, IL-2RA, IL-4, IL-4RA, IRF1, JAK1, MAF, MAPK9, NFATC1, NFATC3, NFKB1, PTPRC, SOCS5, STAT1 and STAT4 were strongly down-regulated in Freund’s adjuvant group; but IL-17, IL-23A, CEBPB, GFI1, IL-9, SPP1 and YY1 gene were significantly up-regulated. The gene expression levels of other genes was not significant. There was only IL-2 expression increased significantly in Aluminum hydroxide adjuvant group compared with the control group.The serum concentration of TGF-p and IL-17 of mice treated with Freund’s adjuvant and aluminum hydroxide adjuvant were detected by ELISA. The results showed that the serum levels of TGF-βand IL-17 in were increased significantly in Freund’s adjuvant group compared with the control group, and there was no significant difference between the aluminum hydroxide adjuvant group and the control group. Aluminum hydroxide adjuvant has no effect on the spleen index and the percentage of macrophages in spleen.The above results indicated that the Freund’s adjuvant could significantly increase the percentage of macrophages in the spleen, Tregs spleen index and the serum concentration of IL-17 IL-23. It showed some inhibition functions on the levels of gene expression involvement with Thl, Th2, and total CD4+T cells to a certain extent. The effect of aluminum hydroxide adjuvant on the index were not significant different. We suggested that CFA could improve the level of immune system for antigen-specific immune response, but the overall function of the immune system might be inhibited to a certain extent.更多还原