内源性危险信号低分子量透明质酸作为新型疫苗佐剂

发布时间：2018-05-16 13:44

本文选题：内源性危险信号 + 佐剂　；参考：《中国协和医科大学》2008年硕士论文

【摘要】： 感染性疾病如HBV,HCV,HIV以及肿瘤是全球波及范围最广,受染人群最多,严重危害人类健康的重要疾病。预防和治疗感染性疾病及肿瘤最有效且最经济的手段是接种疫苗。疫苗包括在预防性疫苗和治疗性疫苗。目前研究的基因工程疫苗,亚单位疫苗,合成肽抗原疫苗其主要问题为免疫原性差,且疫苗提呈的靶向性差。这就需要给疫苗特别是治疗性疫苗添加佐剂以增强疫苗抗原的特异性体液免疫应答和CTL(cytotoxic T lymphocyte)细胞免疫应答,从而起到既预防又治疗病毒感染、肿瘤性疾病的作用。疫苗佐剂能够选择性的刺激免疫应答而提高免疫效果。但是目前大多数疫苗佐剂只能诱导体液免疫应答,而产生的细胞免疫效应较弱甚至没有。因此,迫切需要开发新型人用疫苗佐剂,使其能够有效提高抗原免疫原性,且通过交叉递呈途径将外源性抗原加工处理增强细胞免疫应答。为了更好地解释免疫识别机制,Polly Matzinger于1994年提出危险信号模式理论,在该模式中,免疫系统的功能是参与识别危险信号分子与非危险信号分子。细胞损伤释放的危险信号分子可以激活抗原呈递细胞(Antigen Presenting Cells,APCs)进而有效的激活T/B细胞产生相应的免疫应答。这也就表明受损细胞释放的危险信号分子具有潜在的免疫佐剂效应。内源性危险信号具有交叉递呈抗原,诱导细胞免疫的特性,提示内源性危险信号可作为治疗性疫苗佐剂诱导特异性的细胞免疫应答。本研究首次选用内源性危险信号—细胞外基质降解产物低分子量透明质酸作为新型疫苗佐剂,低分子量透明质酸是指分子量为＜10~6;分别与HAV抗原和HBV抗原混合后免疫ICR小鼠;并设HAV,HBV单独抗原免疫组和Al(OH)_3佐剂组为对照组。经ELISA法和LDH法分别检测抗HAV-IgG,抗HBV-IgG抗体滴度和CTL细胞免疫效应,得到不同时间的抗HAV-IgG,抗HBV-IgG的抗体滴度和CTL细胞杀伤活性。此外,我们还选用了2.0mg低分子量透明质酸+HBV抗原免疫组并设铝佐剂为对照组,在免疫后第七天采血检测抗-HBV抗体滴度,其特异性抗HBV IgG水平分别为1:30.84±1.43,而常规铝佐剂组为1:9.46±3.99两者相比具有统计学意义(P＜0.05)。本研究初步获得内源性危险信号分子低分子量透明质酸能显著增强HAV抗原和HBV抗原的抗体水平及HBV抗原的CTL细胞免疫效应的结果。并证实内源性危险信号分子低分子量透明质酸产生的抗HAV IgG和抗HBV IgG的抗体水平显著高于铝佐剂组(P＜0.05)。此外低分子量透明质酸产生的抗体水平比铝佐剂要早。此外,我们用低分子量透明质酸制备成纳米微球,并包被HBV抗原免疫ICR小鼠。初步探索了其免疫增强效应,结果发现与非纳米微球实验组及常规铝佐剂组相比,低分子量透明质酸纳米微球显著增强抗HBV-IgG的水平(P＜0.05)。我们在佐剂的安全试验中还发现,常规铝佐剂组的小鼠出现毛发脱落,病理结果显示,脑实质有一小胶质细胞小结,提示铝佐剂有可能对于神经系统有损伤,肝肾未见异常;而低分子量透明质酸实验组和包被有HBsAg的低分子量透明质酸纳米微球实验组的毒性实验和过敏反应实验中均未观察到异常反应。肝、脑、肾病理结果均未见异常。这就提示:低分子量透明质酸安全无毒,可适合应有于人。总之,本实验中,我们发现低分子量透明质酸作为免疫佐剂,既能有效增强特异性体液免疫应答,又能显著诱导CTL细胞免疫效应;另外,用其制备的纳米微球也能显著诱导产生强的免疫效应。
[Abstract]:Infectious diseases such as HBV, HCV, HIV and tumor are the world's most widespread, most infected people, important diseases that seriously harm human health. Vaccination is the most effective and economical means to prevent and treat infectious diseases and tumors. Vaccines include preventive vaccines and therapeutic vaccines. The main problem of the unit vaccine, the main problem of the synthetic peptide antigen vaccine is the poor immunogenicity and the poor targeting of the vaccine. It is necessary to add adjuvant to the vaccine, especially the therapeutic vaccine, to enhance the specific humoral immune response of the vaccine antigen and the immune response of the CTL (cytotoxic T lymphocyte) cells, thus preventing and treating the virus infection, The vaccine adjuvant can selectively stimulate the immune response and improve the immune response. But most of the vaccine adjuvant can only induce the humoral immune response, and the cell immune effect is weak or not. Therefore, it is urgent to develop a new human vaccine adjuvant, so that it can effectively improve the antigen immunogen. In addition, the processing of exogenous antigens enhanced the cellular immune response through cross presentation.
In order to better explain the immune recognition mechanism, Polly Matzinger proposed the dangerous signal model theory in 1994, in which the function of the immune system is to identify the dangerous signal molecules and non dangerous signal molecules. The dangerous signal molecules of the release of cell damage can activate the Antigen Presenting Cells (APCs). Effective activation of T/B cells to produce a corresponding immune response. This also indicates that the dangerous signal molecules released by the damaged cells have a potential immune adjuvant effect. Endogenous risk signals have cross presenting antigen, inducing the characteristics of cellular immunity, suggesting that endogenous risk signals can be used as a therapeutic vaccine adjuvant to induce specific cellular immunity. A pestilence response.
This study first selected endogenous risk signals - low molecular weight hyaluronic acid from extracellular matrix degradation products as a new vaccine adjuvant. Low molecular weight hyaluronic acid was the molecular weight of < 10~6; ICR mice were immunized with HAV antigen and HBV antigen respectively, and HAV, HBV independent antigen immunization group and Al (OH) _3 adjuvant group as control group. SA and LDH methods were used to detect anti HAV-IgG, anti HBV-IgG antibody titer and CTL cell immune effect, and to obtain anti HAV-IgG, anti HBV-IgG antibody titer and CTL cell killing activity at different time. Besides, we also selected 2.0mg low molecular weight hyaluronic acid +HBV antigen immunization group and set aluminum adjuvant as control group. Blood samples were collected seventh days after immunization. The level of anti -HBV antibody titer, its specific anti HBV IgG level was 1:30.84 + 1.43, while the conventional aluminum adjuvant group was 1:9.46 + 3.99 (P < 0.05). This study preliminarily obtained the low molecular weight hyaluronic acid of endogenous risk molecules can significantly enhance the antibody level of HAV antigenic and HBV antigen and the CTL fine of HBV antigen. The results of cellular immune effect showed that the level of anti HAV IgG and anti HBV IgG produced by the low molecular weight hyaluronic acid of endogenous risk molecules was significantly higher than that of the aluminum adjuvant group (P < 0.05).
In addition, we prepared nanospheres with low molecular weight hyaluronic acid and immunized ICR mice with HBV antigen. The immune enhancement effect was preliminarily explored. The results showed that the low molecular weight hyaluronic acid nanospheres significantly increased the level of anti HBV-IgG (P < 0.05) compared with the non nanospheres experimental group and the conventional aluminum adjuvant group.
In the safety test of adjuvant, we also found that the mice in the routine aluminum adjuvant group appeared hair loss. The pathological results showed that there was a microglial nodule in the brain parenchyma, suggesting that the aluminum adjuvant might have damage to the nervous system and the liver and kidney were not abnormal, while the low molecular weight hyaluronic acid experimental group and the package were HBsAg with low molecular weight hyaluronic acid. The abnormal reaction was not observed in both the toxicity test and the allergic reaction test in the experimental group. The pathological results of liver, brain and kidney were not abnormal. This suggests that low molecular weight hyaluronic acid is safe and nontoxic, and it can be suitable for people.
In this experiment, we found that the low molecular weight hyaluronic acid, as an immune adjuvant, can not only enhance the specific humoral immune response, but also significantly induce the immune effect of CTL cells. In addition, the nanospheres prepared by it can also significantly induce strong immune effects.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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