本文選題：阿爾茨海默病 + β-淀粉樣多肽�。� 參考：《中國協(xié)和醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 阿爾茨海默病(Alzheimer's disease,AD)是以記憶力減退、認(rèn)知功能障礙為特征的中樞神經(jīng)系統(tǒng)變性疾病,是老年人中常見的一種癡呆癥。主要的病理學(xué)特征包括腦內(nèi)的細(xì)胞外β-淀粉樣多肽(β-amyloid peptide,Aβ)的沉積導(dǎo)致的老年斑、細(xì)胞內(nèi)過度磷酸化tau蛋白導(dǎo)致的神經(jīng)纖維纏結(jié)和神經(jīng)元細(xì)胞的缺失營養(yǎng)壞死。 近年來的研究充分表明,無論是采用Aβ作為抗原的主動(dòng)免疫刺激或者直接注射抗Aβ抗體,都不但可以減少和阻抑AD轉(zhuǎn)基因鼠中Aβ的沉積,還可以明顯改善實(shí)驗(yàn)鼠的行為及認(rèn)知障礙。目前免疫治療已經(jīng)成為治療AD的重要途徑,并取得了很大進(jìn)展。 AD主動(dòng)免疫治療臨床Ⅱ期研究中發(fā)現(xiàn)Aβ免疫后6%的患者大腦出現(xiàn)腦炎反應(yīng)的毒副作用。目前導(dǎo)致腦膜腦炎的具體原因尚不清楚,但相關(guān)研究提示腦炎的發(fā)生可能是T淋巴細(xì)胞介導(dǎo)的自身免疫反應(yīng)的結(jié)果,與免疫引起的T淋巴細(xì)胞亞型(Thelper cell 1/T helper cell 2,Th1/Th2)的平衡有關(guān),如能降低或抑制Th1反應(yīng)、增強(qiáng)Th2的反應(yīng),將增加AD免疫治療的安全性。 目前被動(dòng)免疫研究結(jié)果大都是用單克隆抗體在動(dòng)物試驗(yàn)中得到的,雜交瘤技術(shù)制備的單抗多為鼠源性,對于人體來說屬于異種蛋白,且其不能有效地活化補(bǔ)體和Fc受體相關(guān)的效應(yīng)系統(tǒng),進(jìn)一步的研究需要將鼠源抗體轉(zhuǎn)換成人源化的抗體才能應(yīng)用于人體,但這種方法技術(shù)復(fù)雜難度高,即使改造后也難以得到百分之百的人源化,且有可能在改造過程中使原來抗體的親和力和特異性降低。 針對AD免疫學(xué)治療中存在的問題,本課題在上述兩個(gè)方面進(jìn)行了初步探討。 本課題第一部分利用不同的Aβ多肽序列與佐劑的組合,觀察體液免疫和細(xì)胞免疫的水平高低,通過細(xì)胞因子的測定分析佐劑、抗原的序列長短及T細(xì)胞刺激表位這三種因素引起的T淋巴細(xì)胞亞型的不同刺激和影響,探討導(dǎo)致免疫毒性反應(yīng)的具體因素和抑制途徑。將BALB/c小鼠隨機(jī)分組:鋁(Al)佐劑對照組、Aβ42+福氏佐劑(CFA)組、Aβ42+Al組、Aβ40+Al組、Aβ40(E22A)+Al組,每組8只。經(jīng)初次免疫和3次加強(qiáng)免疫后,檢測抗體效價(jià),培養(yǎng)脾淋巴細(xì)胞,分別用各自抗原刺激,48h后用雙抗夾心ELISA試劑盒檢測培養(yǎng)液中IFN-γ、IL-2、TNF-α和IL-4的含量。72h后用CCK-8法檢測脾淋巴細(xì)胞特異性增殖反應(yīng)。結(jié)果表明,經(jīng)Aβ免疫的4個(gè)試驗(yàn)組血清中均有特異性抗Aβ的抗體產(chǎn)生。脾淋巴細(xì)胞經(jīng)各自抗原刺激后,均出現(xiàn)脾淋巴細(xì)胞特異性增殖反應(yīng)。細(xì)胞培養(yǎng)上清中細(xì)胞因子檢測結(jié)果顯示,Aβ42+CFA組分泌的代表Th1型的細(xì)胞因子含量最高,Aβ40(E22A)+Al組則有顯著降低(P＜0.01)。其中Aβ40及突變型+Al組與Aβ42+Al組相比,分泌的Th1型細(xì)胞因子也有明顯降低(P＜0.05)。因此Aβ40(E22A)+Al組對T細(xì)胞的毒性作用最低,在機(jī)體的免疫反應(yīng)中可能具有更好的安全性。 本課題第二部分研究內(nèi)容為抗Aβ人源抗體基因真核表達(dá)體系的構(gòu)建。本室利用噬菌體展示技術(shù)篩選得到了抗Aβ的抗體,通過篩選直接得到人源抗體,避免了對通過雜交瘤技術(shù)產(chǎn)生的抗體的人源化改造過程。首先將scFv抗體基因與合適的信號(hào)肽基因及人IgG抗體恒定區(qū)基因重組,構(gòu)建成全抗體基因表達(dá)體系,以增加抗體穩(wěn)定性、延長半衰期、提高抗體性能。為了提高抗體的安全性,還將抗體基因的Fc段的3個(gè)重要位點(diǎn)進(jìn)行突變,以期減少T細(xì)胞反應(yīng)和少腦部的微血管出血反應(yīng)。最后選用真核表達(dá)載體pcDNA3.1,將目的基因連入真核表達(dá)載體中,輕重鏈做共轉(zhuǎn)染COS-7、CHO真核細(xì)胞表達(dá),但是目前沒有檢測到抗體基因在COS-7及CHO中的表達(dá),沒有表達(dá)的原因可能與抗體基因的可變區(qū)序列及親和力有關(guān)。 Aβ疫苗及抗體的安全性問題是當(dāng)今AD免疫治療的關(guān)鍵問題之一。本課題一方面從探討降低免疫反應(yīng)的T細(xì)胞毒性作用的途徑和增加抗原抗體反應(yīng)的人體安全性適應(yīng)性入手,另一方面構(gòu)建了抗Aβ人源抗體基因的真核表達(dá)體系,在AD的主動(dòng)免疫和被動(dòng)免疫兩方面做了探索研究。今后,如果能篩選出適宜的抗原及佐劑的組合,使Th細(xì)胞發(fā)生向Th2的極化,那么極有可能避免臨床試驗(yàn)中出現(xiàn)的腦膜腦炎不良反應(yīng)的問題,為Aβ疫苗的臨床應(yīng)用打下良好基礎(chǔ)。如果人源抗體通過改造在真核細(xì)胞內(nèi)表達(dá)成功,將為AD的免疫治療提供另一條有效途徑。
[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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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