茯苓多糖PCP-I对炭疽保护性抗原的佐剂效应研究
发布时间：2018-08-05 13:37
【摘要】：疫苗是经济有效的预防传染病的手段,开发安全高效的新型疫苗是疫苗学研究的目标。亚单位疫苗和重组蛋白类疫苗的抗原是高纯度的蛋白质,疫苗的安全性得到了提高,但是存在免疫原性弱,激发的抗体反应不强,难以激发T细胞反应等不足。为了提高蛋白类疫苗的效力,研究者们一直在寻找高效的佐剂来增强抗原的免疫原性,诱导更强的免疫反应或改变免疫反应的类型。铝佐剂是使用最广泛、时间最长的人用疫苗佐剂。近年来,MF59、AS03、AS04和Virosomes等几种新佐剂逐渐获批人用。而植物多糖,因其较强的免疫调节功能,以及具有生物可降解和低毒性等优点,成为新型佐剂研究的一个热点。茯苓多糖PCP-I是从中药茯苓中提取纯化得到的一种均一多糖,由岩藻糖、甘露糖、葡萄糖和半乳糖组成。本研究以炭疽芽孢杆菌保护性抗原(PA)为模式抗原,从体液免疫反应和细胞免疫反应两方面对PCP-I的佐剂效应进行了系统评价;观察了PCP-I与寡聚脱氧核苷酸CpG构成复合佐剂的佐剂效应;分别使用炭疽致死毒素(LT)攻毒模型和芽孢攻毒模型,对PCP-I增强PA保护效力的能力进行了考察;从树突状细胞(DC)成熟、生发中心(GC)反应和外周血单核细胞(PBMC)基因表达差异三个角度,对PCP-I佐剂效应的机制进行了初步探讨。首先对小鼠免疫实验中PCP-I的量效关系进行了考察。将0.5μg PA分别与50、200、500μg PCP-I混合,免疫BALB/c小鼠,免疫二次,间隔两周,在二免后两周取血检测anti-PA抗体和抗炭疽毒素中和抗体。结果表明,PCP-I为200μg时,小鼠血清中的antiPA抗体(5.38×103)和中和抗体(8.7×101)显著高于50μg组(1.54×102,2.65×101),略高于500μg组(4.15×103,6.13×101)。据此,选择200μg PCP-I进行后续的实验。为评价PCP-I对体液免疫的影响,以0.5μg PA分别混合PCP-I和铝佐剂(Al),免疫BALB/c小鼠,并设PA无佐剂组和PBS组分别作为阴性和空白对照,免疫三次,间隔两周,在三免后两周取血进行相关检测。PA+PCP-I组的anti-PA抗体滴度(1.81×104)和抗体亲和力(0.81)显著高于PA组(1.60×103,0.52),而与PA+Al组(2.35×104,0.83)相近。对anti-PA抗体亚类进行分析,发现PA+PCP-I组的IgG1显著高于IgG2a(5.12×104,2.93×1 03),提示PCP-I是Th2偏向型佐剂。毒素中和实验结果表明,PA+PCP-I组的中和抗体(2.55×103)显著高于PA组(1.32×102),与PA+Al组(1.76×103)无显著性差异。为评价PCP-I对细胞免疫的影响,以5μg PA分别混合PCP-I和铝佐剂免疫小鼠,PA无佐剂组和PBS组作对照,在三免后两周取脾细胞进行细胞免疫相关检测。PA+PCP-I组小鼠脾脏中PA特异性记忆B细胞的频率(4.26%)显著高于PA组(0.87%),与PA+Al组(5.39%)无显著性差异,表明PCP-I能够增强B细胞记忆。使用CCK8检测脾细胞在体外接受PA刺激后的增殖情况,PA+PCP-I组小鼠脾细胞增殖指数(1.37)显著高于PA+Al组(1.05)和PA组(1.08),表明PCP-I能提高小鼠脾细胞再次接触抗原后的增殖能力。使用ELISPOT检测脾细胞中IL-4、IFN-γ分泌细胞的频率,PA+PCP-I组IL-4分泌细胞频率(92/106)高于PA+Al组(34/106),而IFN-γ分泌细胞的频率无明显差异。用细胞因子芯片对PA刺激培养的脾细胞上清中各细胞因子的含量进行检测,PA+PCP-I组IL-2、IL-4、IL-5的含量(63.5、23.0、1020.8pg/ml)显著高于PA+Al组(17.0、2.6、5.1pg/ml),表明PCP-I增强了记忆T细胞再次接触抗原后分泌相应细胞因子的能力。使用炭疽致死毒素(LT)对免疫后小鼠进行攻毒,观察存活率。PA组和PBS组小鼠全部死亡,PA+PCPI组小鼠存活率为75%。为评价PCP-I与CpG构成的复合佐剂的佐剂效应,使用0.5μg PA混合不同的佐剂免疫BALB/c小鼠,在三免后两周取血进行检测。PA+PCP-I+Cp G组的anti-PA抗体(1.02×105)显著高于PA+PCP-I组(1.81×104)和PA+CpG组(3.49×103);抗体亲和力(0.73)显著高于PA+CpG组(0.52),略低于PA+PCP-I组(0.81)。抗体亚类分析表明,PA+PCP-I+CpG组的IgG1与PA+PCP-I组相当(7.90×104,5.12×104),而IgG2a显著高于后者(5.12×104,2.93×103),说明联合佐剂使得PA特异性体液免疫反应呈现Th1/Th2均衡。PA+PCP-I+CpG组的中和抗体(1.38×104)显著高于PA+PCP-I组(2.55×103)和PA+CpG组(1.62×103)。上述结果说明复合佐剂能够以Th1/Th2均衡的方式提高anti-PA抗体,血清中和毒素的能力也显著提高。使用LT对小鼠进行攻毒,观察存活率。PA+PCP-I+CpG组的存活率与PA+PCP-I组相同(75%),略高于PA+CpG组(50%)。为更直接地对佐剂增强PA免疫保护的能力进行评价,用0.5μg PA混合不同的佐剂免疫对炭疽芽孢攻击敏感的A/J小鼠,三免后两周取血检测,三免后四周使用炭疽杆菌A16R株芽孢进行攻毒。检测结果显示,PA+PCP-I组的anti-PA抗体和中和抗体(7.18×103,2.42×102)低于PA+Al组(3.23×104,3.49×103),而PA+PCP-I+CpG组的抗体和中和抗体(3.23×104,2.29×103)与PA+Al组相当。芽孢攻毒后,PA+PCP-I+CpG组和PA+Al组小鼠全部存活,PA+PCP-I组小鼠存活率为83%。PCP-I单独使用的佐剂效应在A/J小鼠实验中不如BALB/c小鼠,由于A/J小鼠属于补体成分5(C5)缺陷型小鼠,推测PCP-I的佐剂效应可能与激活补体途径相关,而CpG的加入能够在一定程度上弥补C5缺失对PCP-I佐剂效应的不利影响。为探讨PCP-I的作用机制,从DC成熟、GC反应和PBMC基因表达差异三个角度进行了初步研究。分别使用PA+PCP-I、PA+Al、PA刺激培养骨髓来源的DC,之后用流式细胞仪检测DC表面CD80和MHC-II的表达。PA+PCP-I刺激组DC中CD80阳性细胞的比例(82.2%)显著高于PA组(51.7%),略高于PA+Al组(65.1%);PA+PCP-I组DC中MHC-II阳性细胞的比例(74.9%)显著高于PA组(46.8%)和PA+Al组(56.1%)。说明PCP-I能够诱导DC上调CD80和MHC-II表达,促进DC成熟。使用5μg PA分别混合PCP-I和铝佐剂免疫BALB/c小鼠,免疫两次,间隔两周,在二免后7天取脾细胞利用流式细胞术检测GC B细胞和滤泡状辅助T细胞(Tfh)的频率。PA+PCP-I组小鼠脾脏中GC B细胞(7.73%)频率显著高于PA组(6.30%),Tfh细胞的频率略高于后者(4.97%vs 4.20%),提示PCP-I能促进生发中心反应。使用转录组测序技术,观察PA+PCP-I、PA+Al和PA免疫3天后PBMC中基因表达的差异。通过数据分析,PA+PCP-I组和PA组之间比较筛选到66个差异基因,其中53个基因与PA+Al组和PA组间差异基因重叠,13个基因是PCP-I特异的差异基因。对这13个基因进行功能注释,发现Il1r2、Clec4e、Stab1和C5ar1与免疫反应相关,其中C5ar1编码C5aR,参与补体C5a对特异性免疫的调节,与PCP-I的佐剂效应与C5相关的假设相符。综上所述,PCP-I作为一种植物多糖类疫苗佐剂,其增强体液免疫反应能力与铝佐剂相当,增强细胞免疫反应的能力强于铝佐剂;当PCP-I与CpG构成复合佐剂后,以Th1/Th2均衡的方式进一步增强了体液免疫反应;初步的机制分析发现,PCP-I可通过促进DC成熟和增强生发中心反应,来增强抗原特异性免疫反应,其作用机制还可能与补体C5和C5aR相关。
[Abstract]:Vaccine is an economical and effective means to prevent infectious diseases. Developing a safe and efficient new vaccine is the target of the study of vaccine. The antigen of subunit vaccine and recombinant protein vaccine is high purity protein. The safety of the vaccine has been improved, but the immunogenicity is weak, the activated antibody reaction is not strong and it is difficult to stimulate the T cell response. In order to improve the effectiveness of the protein vaccine, researchers have been looking for an efficient adjuvant to enhance the immunogenicity of the antigen, to induce a stronger immune response or to change the type of immune response. Aluminum adjuvant is the most widely used and longest human vaccine adjuvant. In recent years, several new adjuvant, such as MF59, AS03, AS04 and Virosomes, have been used. Plant polysaccharide has become a hot spot in the research of new adjuvant because of its strong immune regulation function and its biodegradability and low toxicity. Tuckahoe polysaccharide PCP-I is a kind of homogeneous polysaccharide extracted from Poria cocos, which consists of fucose, mannose, glucose and galactose. This study is based on carbon. Bacillus subtilis protective antigen (PA) is a model antigen, and the adjuvant effect of PCP-I is systematically evaluated from two aspects of humoral immune response and cell immune response. The adjuvant effect of PCP-I and oligo deoxydeoxynucleotide CpG compound adjuvant is observed. The attack model of anthrax lethal toxin (LT) and the spore attack model are used respectively, and PCP-I The ability to enhance the effectiveness of PA protection was investigated. The mechanism of the effect of PCP-I adjuvant effect was preliminarily discussed from three angles: dendritic cells (DC) maturation, germinal center (GC) reaction and peripheral blood mononuclear cell (PBMC) gene expression difference. First, the quantitative effect relationship of PCP-I was investigated in the mice immunization experiment. The 0.5 g PA and 50,2 were respectively compared with 50,2. 00500 micron g PCP-I, immune BALB/c mice, immunized two times, interval two weeks, and two weeks after two immunization to detect anti-PA antibody and anti anthrax neutralization antibody. The results showed that the antiPA antibody (5.38 * 103) and neutralizing antibody (8.7 * 101) in the serum of the mice were significantly higher than that of 50 u g group (1.54 * 102,2.65 x 101), slightly higher than that of the 500 mu g group. 4.15 * 103,6.13 x 101). According to this, 200 mu g PCP-I was selected for subsequent experiments. In order to evaluate the effect of PCP-I on humoral immunity, 0.5 mu g PA was mixed with PCP-I and aluminum adjuvant (Al), and BALB/c mice were immunized with PA without adjuvant and PBS group as negative and blank control, three times of immunization, two weeks interval, and two weeks after three immunity. The titer of anti-PA antibody (1.81 * 104) and antibody affinity (0.81) in.PA+PCP-I group were significantly higher than that in group PA (1.60 x 103,0.52), and similar to that of PA+Al group (2.35 x 104,0.83). The analysis of anti-PA antibody subclass showed that IgG1 in PA+PCP-I group was significantly higher than IgG2a (5.12 x 104,2.93 x 103), suggesting that PCP-I is a biased adjuvant. The results showed that the neutralization antibody (2.55 x 103) in the PA+PCP-I group was significantly higher than that in the PA group (1.32 x 102), and there was no significant difference with the group PA+Al (1.76 x 103). In order to evaluate the effect of PCP-I on cell immunity, the mice were immunized with 5 mu g PA and PCP-I and aluminum adjuvant respectively, and the PA free adjuvant group was compared with the PBS group, and the spleen cells were taken for cellular immune correlation in the two week after three immunity. The frequency of PA specific memory B cells in the spleen of.PA+PCP-I group (4.26%) was significantly higher than that in group PA (0.87%), and there was no significant difference from PA+Al group (5.39%), indicating that PCP-I could enhance the memory of B cells. The proliferation of splenocytes after PA stimulation in vitro was detected with CCK8, and the splenocytes proliferation index (1.37) in PA+PCP-I group was significantly higher than that of PA+Al. Group (1.05) and group PA (1.08) showed that PCP-I could increase the proliferation ability of mouse splenocytes after re contact with antigen. The frequency of IL-4, IFN- gamma secreting cells in splenocytes and the frequency of IL-4 secreting cells in group PA+PCP-I (92/106) were higher than that of group PA+Al (34/106) with ELISPOT, and the frequency of IFN- gamma secretory cells was not significantly different. The content of cytokines in the supernatant of splenocytes in stimulated culture was detected. The content of IL-2, IL-4, and IL-5 in group PA+PCP-I (63.5,23.01020.8pg/ml) was significantly higher than that in group PA+Al (17.0,2.6,5.1pg/ml), indicating that PCP-I enhanced the ability to secrete the corresponding Cytokine after the recontact of the antigen of the memory T cells. The use of anthrax lethal toxin (LT) to immune mice. The survival rate of group.PA and PBS mice was all dead. The survival rate of group PA+PCPI was 75%. as the adjuvant effect of the compound adjuvant of PCP-I and CpG, and the BALB/c mice were immunized with 0.5 mu g PA mixed with different adjuvant. The antibody (1.02 * 105) of.PA+ PCP-I+Cp G group was significantly higher than that of the.PA+ PCP-I+Cp G group (1.02 x 105). Group A+PCP-I (1.81 x 104) and group PA+CpG (3.49 x 103), antibody affinity (0.73) was significantly higher than group PA+CpG (0.52), slightly lower than group PA+PCP-I (0.81). Antibody subclass analysis showed that IgG1 in PA+PCP-I+CpG group was equivalent to group PA+PCP-I (7.90 x 104,5.12 x 104), and IgG2a was significantly higher than that of the latter (5.12 * 104,2.93 x 103), indicating that the combined adjuvant made PA specific body fluids. The immunoreaction showed that the neutralizing antibody (1.38 x 104) of the Th1/Th2 balanced.PA+PCP-I+CpG group was significantly higher than that of the PA+PCP-I group (2.55 x 103) and the PA+CpG group (1.62 x 103). The results indicated that the compound adjuvant could improve the anti-PA antibody in the Th1/Th2 balanced manner and the ability to neutralize the toxin in the serum significantly. The mice were attacked by LT and observed survival. The rate of survival in group.PA+PCP-I+CpG was the same as that in group PA+PCP-I (75%), slightly higher than that in group PA+CpG (50%). It was more direct to evaluate the ability of the adjuvant to enhance the immune protection of PA, with 0.5 mu g PA mixed with different adjuvants to attack the sensitive A/J mice with anthrax spore, three after two weeks of immunization, and three weeks after the use of Bacillus anthracis A16R plant buds. The results showed that the anti-PA and neutralizing antibodies (7.18 x 103,2.42 * 102) of the PA+PCP-I group were lower than that of the PA+Al group (3.23 x 104,3.49 * 103), while the antibody and neutralizing antibody (3.23 * 104,2.29 x 103) in the PA+PCP-I+CpG group were equivalent to that of the PA+Al group. The adjuvant effect of 83%.PCP-I alone is not as good as BALB/c mice in A/J mice. Because A/J mice belong to complement component 5 (C5) deficient mice, it is speculated that the adjuvant effect of PCP-I may be related to the activation of complement pathway, and CpG can make up for the adverse effect of C5 deletion on the PCP-I adjuvant effect to a certain extent. This is to discuss PCP-I. The mechanism of action was preliminarily studied from three angles: DC maturation, GC reaction and PBMC gene expression difference. PA+PCP-I, PA+Al, PA were used to stimulate the culture of DC in bone marrow, and then the expression of CD80 and MHC-II in DC surface was detected by flow cytometry (82.2%) in.PA+PCP-I stimulus group (51.7%), slightly higher than that in the group (51.7%). Higher than group PA+Al (65.1%), the proportion of MHC-II positive cells in group DC (74.9%) in group DC was significantly higher than that in group PA (46.8%) and PA+Al group (56.1%). It indicated that PCP-I could induce DC to increase CD80 and MHC-II expression and promote DC maturation. 5 micron g and aluminum adjuvant immunized respectively, two times, interval two weeks, and splenocytes for 7 days after two immunization. The frequency of flow cytometry was used to detect the frequency of GC B cells and follicular assisted T cells (Tfh) in.PA+PCP-I group. The frequency of GC B cells (7.73%) in the spleen of the mice was significantly higher than that of the PA group (6.30%). The frequency of Tfh cells was slightly higher than that of the latter (4.97%vs 4.20%), suggesting that PCP-I could promote the reaction of the germinal center. The difference of gene expression in post PBMC was analyzed. Through data analysis, 66 different genes were screened from group PA+PCP-I and PA, of which 53 genes overlapped with PA+Al and PA groups, and 13 genes were PCP-I specific differentially genes. The 13 genes were annotated by function, and Il1r2, Clec4e, Stab1 and C5ar1 were associated with immune response, C5ar1, which encodes C5aR, participates in the regulation of complement C5a to specific immunity, which is consistent with the C5 related hypothesis of the adjuvant effect of PCP-I. As a plant polysaccharide vaccine adjuvant, PCP-I has the same ability to enhance the humoral immune response as aluminum adjuvant, which is stronger than the aluminum adjuvant; when PCP-I and CpG constitute a compound. After the adjuvant, the humoral immune response was further enhanced by Th1/Th2 equilibrium. Preliminary mechanism analysis showed that PCP-I could enhance the antigen specific immune response by promoting the maturation of DC and enhancing the reaction of the germinal center, and its mechanism may also be related to the complement of C5 and C5aR.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
本文编号：2165960
[Abstract]:Vaccine is an economical and effective means to prevent infectious diseases. Developing a safe and efficient new vaccine is the target of the study of vaccine. The antigen of subunit vaccine and recombinant protein vaccine is high purity protein. The safety of the vaccine has been improved, but the immunogenicity is weak, the activated antibody reaction is not strong and it is difficult to stimulate the T cell response. In order to improve the effectiveness of the protein vaccine, researchers have been looking for an efficient adjuvant to enhance the immunogenicity of the antigen, to induce a stronger immune response or to change the type of immune response. Aluminum adjuvant is the most widely used and longest human vaccine adjuvant. In recent years, several new adjuvant, such as MF59, AS03, AS04 and Virosomes, have been used. Plant polysaccharide has become a hot spot in the research of new adjuvant because of its strong immune regulation function and its biodegradability and low toxicity. Tuckahoe polysaccharide PCP-I is a kind of homogeneous polysaccharide extracted from Poria cocos, which consists of fucose, mannose, glucose and galactose. This study is based on carbon. Bacillus subtilis protective antigen (PA) is a model antigen, and the adjuvant effect of PCP-I is systematically evaluated from two aspects of humoral immune response and cell immune response. The adjuvant effect of PCP-I and oligo deoxydeoxynucleotide CpG compound adjuvant is observed. The attack model of anthrax lethal toxin (LT) and the spore attack model are used respectively, and PCP-I The ability to enhance the effectiveness of PA protection was investigated. The mechanism of the effect of PCP-I adjuvant effect was preliminarily discussed from three angles: dendritic cells (DC) maturation, germinal center (GC) reaction and peripheral blood mononuclear cell (PBMC) gene expression difference. First, the quantitative effect relationship of PCP-I was investigated in the mice immunization experiment. The 0.5 g PA and 50,2 were respectively compared with 50,2. 00500 micron g PCP-I, immune BALB/c mice, immunized two times, interval two weeks, and two weeks after two immunization to detect anti-PA antibody and anti anthrax neutralization antibody. The results showed that the antiPA antibody (5.38 * 103) and neutralizing antibody (8.7 * 101) in the serum of the mice were significantly higher than that of 50 u g group (1.54 * 102,2.65 x 101), slightly higher than that of the 500 mu g group. 4.15 * 103,6.13 x 101). According to this, 200 mu g PCP-I was selected for subsequent experiments. In order to evaluate the effect of PCP-I on humoral immunity, 0.5 mu g PA was mixed with PCP-I and aluminum adjuvant (Al), and BALB/c mice were immunized with PA without adjuvant and PBS group as negative and blank control, three times of immunization, two weeks interval, and two weeks after three immunity. The titer of anti-PA antibody (1.81 * 104) and antibody affinity (0.81) in.PA+PCP-I group were significantly higher than that in group PA (1.60 x 103,0.52), and similar to that of PA+Al group (2.35 x 104,0.83). The analysis of anti-PA antibody subclass showed that IgG1 in PA+PCP-I group was significantly higher than IgG2a (5.12 x 104,2.93 x 103), suggesting that PCP-I is a biased adjuvant. The results showed that the neutralization antibody (2.55 x 103) in the PA+PCP-I group was significantly higher than that in the PA group (1.32 x 102), and there was no significant difference with the group PA+Al (1.76 x 103). In order to evaluate the effect of PCP-I on cell immunity, the mice were immunized with 5 mu g PA and PCP-I and aluminum adjuvant respectively, and the PA free adjuvant group was compared with the PBS group, and the spleen cells were taken for cellular immune correlation in the two week after three immunity. The frequency of PA specific memory B cells in the spleen of.PA+PCP-I group (4.26%) was significantly higher than that in group PA (0.87%), and there was no significant difference from PA+Al group (5.39%), indicating that PCP-I could enhance the memory of B cells. The proliferation of splenocytes after PA stimulation in vitro was detected with CCK8, and the splenocytes proliferation index (1.37) in PA+PCP-I group was significantly higher than that of PA+Al. Group (1.05) and group PA (1.08) showed that PCP-I could increase the proliferation ability of mouse splenocytes after re contact with antigen. The frequency of IL-4, IFN- gamma secreting cells in splenocytes and the frequency of IL-4 secreting cells in group PA+PCP-I (92/106) were higher than that of group PA+Al (34/106) with ELISPOT, and the frequency of IFN- gamma secretory cells was not significantly different. The content of cytokines in the supernatant of splenocytes in stimulated culture was detected. The content of IL-2, IL-4, and IL-5 in group PA+PCP-I (63.5,23.01020.8pg/ml) was significantly higher than that in group PA+Al (17.0,2.6,5.1pg/ml), indicating that PCP-I enhanced the ability to secrete the corresponding Cytokine after the recontact of the antigen of the memory T cells. The use of anthrax lethal toxin (LT) to immune mice. The survival rate of group.PA and PBS mice was all dead. The survival rate of group PA+PCPI was 75%. as the adjuvant effect of the compound adjuvant of PCP-I and CpG, and the BALB/c mice were immunized with 0.5 mu g PA mixed with different adjuvant. The antibody (1.02 * 105) of.PA+ PCP-I+Cp G group was significantly higher than that of the.PA+ PCP-I+Cp G group (1.02 x 105). Group A+PCP-I (1.81 x 104) and group PA+CpG (3.49 x 103), antibody affinity (0.73) was significantly higher than group PA+CpG (0.52), slightly lower than group PA+PCP-I (0.81). Antibody subclass analysis showed that IgG1 in PA+PCP-I+CpG group was equivalent to group PA+PCP-I (7.90 x 104,5.12 x 104), and IgG2a was significantly higher than that of the latter (5.12 * 104,2.93 x 103), indicating that the combined adjuvant made PA specific body fluids. The immunoreaction showed that the neutralizing antibody (1.38 x 104) of the Th1/Th2 balanced.PA+PCP-I+CpG group was significantly higher than that of the PA+PCP-I group (2.55 x 103) and the PA+CpG group (1.62 x 103). The results indicated that the compound adjuvant could improve the anti-PA antibody in the Th1/Th2 balanced manner and the ability to neutralize the toxin in the serum significantly. The mice were attacked by LT and observed survival. The rate of survival in group.PA+PCP-I+CpG was the same as that in group PA+PCP-I (75%), slightly higher than that in group PA+CpG (50%). It was more direct to evaluate the ability of the adjuvant to enhance the immune protection of PA, with 0.5 mu g PA mixed with different adjuvants to attack the sensitive A/J mice with anthrax spore, three after two weeks of immunization, and three weeks after the use of Bacillus anthracis A16R plant buds. The results showed that the anti-PA and neutralizing antibodies (7.18 x 103,2.42 * 102) of the PA+PCP-I group were lower than that of the PA+Al group (3.23 x 104,3.49 * 103), while the antibody and neutralizing antibody (3.23 * 104,2.29 x 103) in the PA+PCP-I+CpG group were equivalent to that of the PA+Al group. The adjuvant effect of 83%.PCP-I alone is not as good as BALB/c mice in A/J mice. Because A/J mice belong to complement component 5 (C5) deficient mice, it is speculated that the adjuvant effect of PCP-I may be related to the activation of complement pathway, and CpG can make up for the adverse effect of C5 deletion on the PCP-I adjuvant effect to a certain extent. This is to discuss PCP-I. The mechanism of action was preliminarily studied from three angles: DC maturation, GC reaction and PBMC gene expression difference. PA+PCP-I, PA+Al, PA were used to stimulate the culture of DC in bone marrow, and then the expression of CD80 and MHC-II in DC surface was detected by flow cytometry (82.2%) in.PA+PCP-I stimulus group (51.7%), slightly higher than that in the group (51.7%). Higher than group PA+Al (65.1%), the proportion of MHC-II positive cells in group DC (74.9%) in group DC was significantly higher than that in group PA (46.8%) and PA+Al group (56.1%). It indicated that PCP-I could induce DC to increase CD80 and MHC-II expression and promote DC maturation. 5 micron g and aluminum adjuvant immunized respectively, two times, interval two weeks, and splenocytes for 7 days after two immunization. The frequency of flow cytometry was used to detect the frequency of GC B cells and follicular assisted T cells (Tfh) in.PA+PCP-I group. The frequency of GC B cells (7.73%) in the spleen of the mice was significantly higher than that of the PA group (6.30%). The frequency of Tfh cells was slightly higher than that of the latter (4.97%vs 4.20%), suggesting that PCP-I could promote the reaction of the germinal center. The difference of gene expression in post PBMC was analyzed. Through data analysis, 66 different genes were screened from group PA+PCP-I and PA, of which 53 genes overlapped with PA+Al and PA groups, and 13 genes were PCP-I specific differentially genes. The 13 genes were annotated by function, and Il1r2, Clec4e, Stab1 and C5ar1 were associated with immune response, C5ar1, which encodes C5aR, participates in the regulation of complement C5a to specific immunity, which is consistent with the C5 related hypothesis of the adjuvant effect of PCP-I. As a plant polysaccharide vaccine adjuvant, PCP-I has the same ability to enhance the humoral immune response as aluminum adjuvant, which is stronger than the aluminum adjuvant; when PCP-I and CpG constitute a compound. After the adjuvant, the humoral immune response was further enhanced by Th1/Th2 equilibrium. Preliminary mechanism analysis showed that PCP-I could enhance the antigen specific immune response by promoting the maturation of DC and enhancing the reaction of the germinal center, and its mechanism may also be related to the complement of C5 and C5aR.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 巫亚俊;李帅;李海霞;麻浩;赵修南;刘坤路;武军华;刘园园;单俊杰;王玉霞;;茯苓多糖PCP-Ⅰ对乙肝疫苗抗原的佐剂活性[J];国际药学研究杂志;2016年02期
2 李帅;王玉霞;李海霞;贾培媛;刘坤璐;贾启燕;单俊杰;马百平;;茯苓总多糖对H1N1流感疫苗和乙肝疫苗抗原的佐剂作用[J];中国药理学与毒理学杂志;2015年01期
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