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。
Alzheimer's disease(AD) is a degenerative disease of the central nervous system, progressive dysmnesia and intelligent recession as main clinical feature.AD is the most prevalent form of dementia in old people.It is neuropathologically characterized by the deposition of extracellularβ-amyloid(Aβ)-containing plaques,intracellular neurofibrillary tangles,reduced synaptic density and neuronal loss in selected brain areas.
Research in recent years fully shows that both the use of Aβimmunization as an antigen to stimulate the immune response or direct injection of anti-Aβantibodies can not only reduce and inhibit Aβdeposition in AD transgenic mice,but also improve the mice's behavior and cognitive obstacles.At present,immunotherapy has become an important channel for the treatment of AD,and has been made great progress.
It is found that 6 percent of subjects suffered meningoencephalomyelitis after Aβimmunization in PhaseⅡclinical study.At present,the specific reasons for meningoencephalomyelitis is not clear,but some study suggests the occurrence of meningoencephalomyelitis may be triggered by the T lymphocyte-mediated autoimmune reaction and concern to T lymphocyte subtype(T helper cell 1/T helper cell 2,Th1/Th2) balance,If we can reduce or suppress the Th1 responseand enhance Th2 response,the safety of immunotherapy with AD will be increased.
At present,the results of the study of passive immunization are got by using monoclonal antibodies in the animal experiment.The antibodies was largely murine produced by the hybridoma technique.They are dissimilar proteins for the human body, and can not effectively activate complement and Fc receptor-related effects.Further research needs to convert murine antibodies to humanized antibodies,but this method is technically complex and difficult,even after the transformation the 100 percent of humanized antibodies can not got.The original antibody's affinity and specificity may be reduced after the process of transformation.
The first part of my study is to observe stimulation and regulation of T lymphocyte subtypes when using wild-type and mutant Aβ40 and Aβ42 with different adjuvants and to investigate possible ways to lower the toxicity of Aβimmune response.Forty BALB/c mice were randomized into five groups:Al adjuvant group,Aβ42+CFA group,Aβ42+Al group,Aβ40+Al group,Aβ40(E22A) +Al group.After the initial immunization and booster immunization 3 times,antibody titer were detected and the mice were killed. Their splenocytes were stimulated by respective antigen.After 48h quantity of IFN-γ, IL-2,TNF-αand IL-4 were tested.After 72h of culture,the proliferative response of splenocytes were detected by CCK-8 assay.The results showed that experimental groups generated specific anti-Aβantibodies and had proliferative response of spleen lymphocytes.Cell culture supernatant of cytokine detection results showed that Aβ42+CFA group had the highest secretion of Th1-type cytokines IFN-γ,IL-2,TNF-αwhen Aβ40(E22A) +Al group had the lowest.(P＜0.01).When Aβ40 and the mutant Aβ40+Al groups compared with Aβ42+Al group,secretion of IFN-γ,IL-2,TNF-αhave also decreased significantly(P＜0.05).The secretion of IL-4 was no significant difference.It is concluded that Aβ40(E22A) +Al group stimulate the lowest toxicity of T cells and may have better security in the immune response.
The second part of the study is to construct for human anti-Aβantibody eukaryotic expression system.Our lab used phage display technology to select anti-Aβantibodies.The antibody came from human phage library and avoided the adoption of technologies used to convert hybridoma antibody.First we overlap scFv antibody gene with a suitable signal peptide gene and constant gene to form a complete antibody gene, in order to increase the stability of antibodies,to extend the half-life and improve antibody performance.In order to reduce the T cell response and the brain microvascular bleeding,we mutated the three important sites of Fc.The target gene was cloned to the th eukaryotic expression vector pcDNA3.1.The light and heavy chain were cotransfected to COS-7 and CHO cells,but there is no antibody detected in cell supernatant.The reason for this may be related to the variable region of the antibody and its affinity.
The assurance of the safety of Aβvaccine or antibody is one of the key issues in today's AD immunotherapy.Our study concentrate on reducing the toxic effects of T cells and increasing the safety of antigen-antibody reaction on the one hand,and constructing an anti-Aβantibody eukaryotic expression system on the other hand.If we can find out the appropriate combination of antigen and adjuvant to make Th cells to polarize toTh2, it is very likely to avoid appearing of meningoencephalomyelitis in clinical trials.If the human antibody express successfully in eukaryotic cells,it may become an effective way for the immune therapy for AD.
引文
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