| Alzheimer's disease (AD), commonly known as senile dementia, is the fourth-ranking cause of death in the modern world.β-amyloid peptide (Aβ) and amyloid precursor protein (APP) are closely related to the occurrence and development of AD. In vivo, APP is proteolytically cleaved byβ-secretase andγ-secretase sequentially and releases Aβ, the latter can aggregate to form senile plaques, with neurotoxicity, is a major pathogenic factor in AD.In order to overcome the serious side effects caused by previous immunotherapy of AD, experimental immunotherapy study with different length of Aβwas designed and carried out in this thesis. Effects of serum antibodies induced by different length of Aβon Aβ42 aggregation and cytotoxicity were analyzed. Three recombinant expression vectors pET-41b(+)-Aβ28, pET-41b(+)-Aβ35, pET-41b(+)-Aβ42 were first constructed, and then three recombinant proteins Aβ28H8, Aβ35H8, Aβ42H8 were expressed and purified. Aβ28H8, Aβ35H8, Aβ42H8 and Aβ42 were used as immunogens to immune BALB/c mice respectively. The antisera were collected from the mice that were received a total of seven injections. The titers of serum antibodies induced by Aβ28H8, Aβ42H8 and Aβ42 against Aβ42 oligomers were 1:6400, whereas the one by Aβ35H8 was 1:3200. The levels of serum antibody concentration were consistent with the levels of the corresponding antibody titers. The analysis of isotypes of serum antibodies showed that Aβ28 mainly induced humoral immune response, while Aβ42 and Aβ35 mainly induced cellular immune response. It was indicated that immunotherapy for AD with Aβ28 as immunogen could reduce the inflammation caused by cellular immune response. Reactive specificities of serum antibodies with different aggregation forms of Aβ42 were determined by indirect ELISA. The results showed that all the serum antibodies could identify and react with various forms of Aβ42 including Aβ42 monomers, Aβ42 oligomers, Aβ42 protofibrils and Aβ42 mature fibers. This indicated that these antibodies had wide reactogenicities with all existing forms of Aβ42. The inhibitory or induction effects of serum antibodies on Aβ42 aggregation or on the disassembly of Aβ42 fibrils in vitro were observed by electron microscopy. The results showed that serum antibodies could not only prevent Aβ42 aggregation, but also disassemble the preformed Aβ42 oligomers, Aβ42 protofibrils or Aβ42 fibrils and ultimately induced Aβ42 aggregates into much smaller globular units in vitro. There were significant abilities of serum antibodies to inhibit and neutralize Aβ42 cytotoxicity in PC12 cells. These results indicated that Aβ28 was an effective, more importantly, a safer immunogen than Aβ42.In order to explore the pathogenic mechanism of AD and to reveal the effects of the interactions between APP on occurrence and development of AD, the key sites in APP for the interactions APP were investigated. COS-7 cells were first transfected with different tagged APP, and then we confirmed that APP can interact with each other by using immunofluorescence, co-IP, wound healing assay, transwell assay, and cell aggregation assay etc. These interactions between APPs could inhibit the migration of target cells, promote the adhesion of target cell, and mediate the adhesion between target cells or target cells and different Aβmatrix including Aβ4-8, Aβ11, Aβ28, Aβ35, Aβ42, C99. These effects mediated by interactions between APP could be inhibited by Aβ42 or the serum antibodies induced by Aβ28. These results suggested that the extracellular region of Aβ42, especially Aβ4-8, might play an important role in the interactions between APPs. It was supposed that the adhesion between neurons and extracellular Aβ42 aggregates was mediated by the interactions between molecules including Aβ4-8 region, leading to neurons dysfunction, and eventually AD.These conclusions obtained in this thesis are significant for providing the theoretical guidance for vaccine design involved in the immunization of prevention and treatment of AD, and for laying the theoretical foundation to reveal the pathogenic mechanism of AD.
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