| Alzheimer's disease (AD) is a progressive neurodegeneration. Although AD pathogenesis is complex and remains unclear, aggregation ofβ-amyloid (Aβ) is considered to be the primary event in a complex cascade eventually leading to neurodegeneration.In the previous research, serum antibodies were obtained from immunized mice. The strongest antibody response against Aβ42 (titer 1:3200) was achieved by GST-I-Aβ28 or GST-Aβ42 immunization. GST-Aβ42 or GST-I-Aβ28-induced serum antibodies also showed the most protective and restorative effects on target cells in vitro by inhibiting or neutralizing Aβ42-induced cytotoxicity. It also been found that they could inhibit or reverse Aβ42 fibration by transmission electron microscopy. However, the most troubling problem in AD immunotherapy was the safety of the vaccines or antibody. In this thesis we found that the IgGl and IgG2b were the predominant serum antibody isotype responses by GST-I-Aβ28 immunization, whereas did IgG2a by GST-Aβ42 immunization. Thus, it indicated that GST-I-Aβ28 immunization in a mouse mainly evoked a stronger Th-2-type response, whereas GST-Aβ42 immunization did a stronger Th-1-type response. All of the above results indicated that GST-I-Aβ28 could be used as a safe and reliable antigen in AD immunotherapy. Moreover, GST-I-Aβ28 and GST-Aβ42 induced serum antibodies had higher specificity to Aβ42 monomers and Aβ42 oligomers than to Aβ42 protofibrils and Aβ42 mature fibrils, and exhibited the highest efficacy to block Aβ42 aggregation or fibrillogenesis and to disassemble Aβ42 aggregates in vitro. Meanwhile, these two serum antibodies cloud inhibit the adhesion of COS7 cell transfected with APP on Aβ42 (oligomer) matrix. It suggested that antibody blocking the interaction between Aβ42 and APP by binging with Aβ42 region of APP/C99,thus APP depended Aβ42 pathway.In recent years, the effects of Traditional Chinese Medicine (TCM) in AD therapy received a increasing attention. In this thesis, extracts from 20 kinds of natural herbal plants were obtained by hot water extraction,4 of them showed the effects on inhibiting or neutralizing Aβ42-induced cytotoxicity by MTT assay. Further research confirmed that Ginkgo flavonoids, Matricaria lactone (MN), Matricaria flavonoids (MH), Polygalaceae saponins, Wolfberry Polysaccharide, were the active components with these functions from these extracts, and MN, MH showed the strongest effects. Observed by transmission electron microscopy, MH, MH had the abilities on inhibiting and reversing A(342 fibril formation. Meanwhile, MN, MH cloud inhibit the adhesion of COS7 cell transfected with APP on Aβ42 (oligomer) matrix.MH, MN showed similar properties with anti-Aβ42 antibodies, while Aβ42 is a fragment of C99 which is a substrate of y-secretase. Consequently, we analyzed the effects of MH and MN on the interactions between PS1 and C99. According to this idea, we conducted the following experiment:(1) cell adhesion experiments:analysis of the impacts of MN, MH on the adhesion between COS-7 cells transfected with PS1-F, PS1-N, PS1-C and C99 matrix; (2) Co-immunoprecipitation:analysis of the impacts of MN, MH on the interaction between PS1 and C99. Meanwhile, NotchΔE, another important substrate of y-secretase, was used to repeat the experiments above. The results showed that:MN, MH not only inhibited the adhesion of PS 1-transfected COS-7 cells on C99 matrix, but also inhibited the interaction between PS1 and C99, however,the similar cases were not observed in NotchΔE experiments.In summary, in this thesis safety and recognition specificity of anti-Aβ42 antibodies obtained in preliminary studies were confirmed. Five lead compounds [Ginkgo flavonoids, Matricaria lactone (MN), Matricaria flavonoids (MH), Polygalaceae saponins, Wolfberry Polysaccharide] were screened from 20 kinds of TCM which had the inhibitory effects on the neurotoxic of Aβ42. Furthermore, we confirmed the possibility of MN,MH as y-secretase inhibitors。These results suggested that the MN and MN had significant effects on inhibiting Aβ42 oligomers'neurotoxicity and blocking the interaction between PS 1 and the C99.The results of this thesis provided a theoretical guidance to drug design and application of antibodies and natural medicines in AD therapy. |
