Aβ多肽的T细胞免疫刺激抑制研究及人源抗体基因真核表达体系的构建

发布时间：2018-07-10 18:36

本文选题：阿尔茨海默病 + β-淀粉样多肽　；参考：《中国协和医科大学》2009年硕士论文

【摘要】： 阿尔茨海默病(Alzheimer's disease,AD)是以记忆力减退、认知功能障碍为特征的中枢神经系统变性疾病,是老年人中常见的一种痴呆症。主要的病理学特征包括脑内的细胞外β-淀粉样多肽(β-amyloid peptide,Aβ)的沉积导致的老年斑、细胞内过度磷酸化tau蛋白导致的神经纤维缠结和神经元细胞的缺失营养坏死。近年来的研究充分表明,无论是采用Aβ作为抗原的主动免疫刺激或者直接注射抗Aβ抗体,都不但可以减少和阻抑AD转基因鼠中Aβ的沉积,还可以明显改善实验鼠的行为及认知障碍。目前免疫治疗已经成为治疗AD的重要途径,并取得了很大进展。 AD主动免疫治疗临床Ⅱ期研究中发现Aβ免疫后6%的患者大脑出现脑炎反应的毒副作用。目前导致脑膜脑炎的具体原因尚不清楚,但相关研究提示脑炎的发生可能是T淋巴细胞介导的自身免疫反应的结果,与免疫引起的T淋巴细胞亚型(Thelper cell 1/T helper cell 2,Th1/Th2)的平衡有关,如能降低或抑制Th1反应、增强Th2的反应,将增加AD免疫治疗的安全性。目前被动免疫研究结果大都是用单克隆抗体在动物试验中得到的,杂交瘤技术制备的单抗多为鼠源性,对于人体来说属于异种蛋白,且其不能有效地活化补体和Fc受体相关的效应系统,进一步的研究需要将鼠源抗体转换成人源化的抗体才能应用于人体,但这种方法技术复杂难度高,即使改造后也难以得到百分之百的人源化,且有可能在改造过程中使原来抗体的亲和力和特异性降低。针对AD免疫学治疗中存在的问题,本课题在上述两个方面进行了初步探讨。本课题第一部分利用不同的Aβ多肽序列与佐剂的组合,观察体液免疫和细胞免疫的水平高低,通过细胞因子的测定分析佐剂、抗原的序列长短及T细胞刺激表位这三种因素引起的T淋巴细胞亚型的不同刺激和影响,探讨导致免疫毒性反应的具体因素和抑制途径。将BALB/c小鼠随机分组:铝(Al)佐剂对照组、Aβ42+福氏佐剂(CFA)组、Aβ42+Al组、Aβ40+Al组、Aβ40(E22A)+Al组,每组8只。经初次免疫和3次加强免疫后,检测抗体效价,培养脾淋巴细胞,分别用各自抗原刺激,48h后用双抗夹心ELISA试剂盒检测培养液中IFN-γ、IL-2、TNF-α和IL-4的含量。72h后用CCK-8法检测脾淋巴细胞特异性增殖反应。结果表明,经Aβ免疫的4个试验组血清中均有特异性抗Aβ的抗体产生。脾淋巴细胞经各自抗原刺激后,均出现脾淋巴细胞特异性增殖反应。细胞培养上清中细胞因子检测结果显示,Aβ42+CFA组分泌的代表Th1型的细胞因子含量最高,Aβ40(E22A)+Al组则有显著降低(P＜0.01)。其中Aβ40及突变型+Al组与Aβ42+Al组相比,分泌的Th1型细胞因子也有明显降低(P＜0.05)。因此Aβ40(E22A)+Al组对T细胞的毒性作用最低,在机体的免疫反应中可能具有更好的安全性。本课题第二部分研究内容为抗Aβ人源抗体基因真核表达体系的构建。本室利用噬菌体展示技术筛选得到了抗Aβ的抗体,通过筛选直接得到人源抗体,避免了对通过杂交瘤技术产生的抗体的人源化改造过程。首先将scFv抗体基因与合适的信号肽基因及人IgG抗体恒定区基因重组,构建成全抗体基因表达体系,以增加抗体稳定性、延长半衰期、提高抗体性能。为了提高抗体的安全性,还将抗体基因的Fc段的3个重要位点进行突变,以期减少T细胞反应和少脑部的微血管出血反应。最后选用真核表达载体pcDNA3.1,将目的基因连入真核表达载体中,轻重链做共转染COS-7、CHO真核细胞表达,但是目前没有检测到抗体基因在COS-7及CHO中的表达,没有表达的原因可能与抗体基因的可变区序列及亲和力有关。 Aβ疫苗及抗体的安全性问题是当今AD免疫治疗的关键问题之一。本课题一方面从探讨降低免疫反应的T细胞毒性作用的途径和增加抗原抗体反应的人体安全性适应性入手,另一方面构建了抗Aβ人源抗体基因的真核表达体系,在AD的主动免疫和被动免疫两方面做了探索研究。今后,如果能筛选出适宜的抗原及佐剂的组合,使Th细胞发生向Th2的极化,那么极有可能避免临床试验中出现的脑膜脑炎不良反应的问题,为Aβ疫苗的临床应用打下良好基础。如果人源抗体通过改造在真核细胞内表达成功,将为AD的免疫治疗提供另一条有效途径。
[Abstract]:Alzheimer's disease (AD) is a degenerative disease of the central nervous system characterized by impairment of memory and cognitive dysfunction. It is a common type of dementia among the elderly. The main pathological features include the senile plaques caused by the deposition of extracellular beta amyloid peptide (beta -amyloid peptide, A beta) in the brain, and the over cell Phosphorylation of tau protein results in neurofibrillary tangles and loss of nutritional necrosis in neuronal cells.
Recent studies have fully demonstrated that the use of A beta as an active immunization stimulus or direct injection of anti A beta antibody can not only reduce and inhibit the deposition of A beta in AD transgenic mice, but also obviously improve the behavior and cognitive impairment of experimental mice. Great progress.
In the clinical stage II study of AD active immunotherapy, 6% of the patients with A beta immunization have been found to have the toxic and side effects of brain inflammation. The specific causes of meningitis are still unclear, but the related research suggests that the occurrence of encephalitis may be the result of T lymphocyte mediated autoimmune reaction, and the T lymphocyte subtype induced by immunization (Thel The balance of per cell 1/T helper cell 2 (Th1/Th2) is related to reducing or inhibiting Th1 reaction and enhancing Th2 reaction, which will increase the safety of AD immunotherapy.
At present, the results of passive immunization are mostly obtained by monoclonal antibodies in animal experiments. The monoclonal antibodies prepared by hybridoma technology are mostly rat origin, and they are heterologous proteins for human body, and they can not effectively activate the effect system related to complement and Fc receptor. Body can be applied to the human body, but this method is complicated and difficult. Even after the transformation, it is difficult to get one hundred percent of the human source, and it is possible to reduce the affinity and specificity of the original antibody in the process of transformation.
In view of the problems existing in the immunotherapy of AD, this topic has carried on the preliminary discussion in the above two aspects.
The first part of this project, using the combination of different A beta polypeptide sequence and adjuvant, observed the level of humoral immunity and cell immunity, and analyzed the different stimuli and effects of the T lymphocyte subtypes caused by the three factors, such as the length of the antigen and the T cell stimulation epitopes, and the effect of the cytotoxicity. BALB/c mice were randomly divided into two groups: Aluminum (Al) adjuvant control group, A beta 42+ Freund adjuvant (CFA) group, A beta 42+Al group, A beta 40+Al group, A beta 40 (E22A) +Al group, 8 rats in each group. After the first immunization and 3 immunization, the antibody titer was detected and the spleen cells were cultured with their respective antigens. The specific proliferation reaction of splenic lymphocyte was detected by CCK-8 method after IFN- gamma, IL-2, TNF- alpha and IL-4 in the culture solution. The results showed that there were specific anti A beta antibodies in the serum of 4 experimental groups with A beta immunization. Spleen lymphocyte specific proliferation reaction occurred after the spleen lymphocyte was stimulated by each antigen. Cytokine detection in cell culture supernatant showed that the content of Th1 type cytokines secreted in A beta 42+CFA group was the highest, while A beta 40 (E22A) +Al group decreased significantly (P < 0.01). The Th1 type cytokines secreted by A beta 40 and mutant +Al groups were also significantly lower than A beta 42+Al group. The toxicity is the lowest and may have better safety in the body's immune response.
The second part of this study is the construction of the eukaryotic expression system of anti A beta human antibody gene. In this room, the antibody against A beta is screened by using phage display technology, and the human antibody is obtained by screening directly, and the human transformation process of antibody produced by hybridoma technology is avoided. First, the scFv antibody gene is suitable for the antibody gene. In order to increase the stability of the antibody, prolong the half-life and improve the performance of the antibody, 3 important sites in the Fc segment of the antibody gene are mutated to reduce the T cell response and the microvascular hemorrhage response in the less brain in order to improve the antibody safety. Finally, the eukaryotic expression vector pcDNA3.1 was selected to connect the target gene into the eukaryotic expression vector, and the heavy chain was co transfected with COS-7 and CHO eukaryotic cells, but the expression of the antibody gene in COS-7 and CHO was not detected at present, and the reason that the expression was not expressed may be related to the variable region sequence and affinity of the antibody gene.
The safety of A beta vaccine and antibody is one of the key problems in AD immunotherapy today. On the one hand, we start with the approach to reduce the toxicity of T cells in the immune response and the adaptability of the human body to increase the antigen antibody response. On the other hand, the eukaryotic expression system of the anti A beta human antibody gene is constructed, and the initiative of the AD is in the initiative. Two aspects of immune and passive immunity have been explored. In the future, if a suitable combination of antigen and adjuvant can be screened to polarizing the Th cells to Th2, it is very possible to avoid the problems of meningitis in clinical trials and provide a good basis for the clinical use of the A beta vaccine. Successful expression in eukaryotic cells will provide another effective way for AD immunotherapy.
【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】：2009
【分类号】：R392

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