Establish Mmp9 Gene Knockout Zebrafish Model And Explore The Role Of Mmp9 In Aluminum-induced Dementia Among The Model

Alzheimer disease(AD)is an age-related neurodegenerative disease characterized by learning and memory disorders.With the problem of aging seriously,AD becomes a very common disease among the world.In order to provide a new target for the treatment of Alzheimer's disease,we used the APPsw zebrafish model combined with aluminum poisoning to explore the role of mmp9 durring the development of AD,as well.as,established mmp9 knockout zebrafish model using Crispr/Cas 9.The research contents and results are as follows:1.Verification of mmp9 gene up-regulation in aluminum poisoning zebrafish modelWild-type zebrafish were treated with 100?g/L of AICl3 at pH==5.8 for 40 days.RT-qPCR showed up-regulation of mmp9 mRNA transcription level in the zebrafish brain(p<0.05).2.Establishment of mmp9 knockout zebrafish modelmmp9 knockout target sites were screened using the CHOPCHOP website.The pT7-mmp9-gRNA in vitro transcription template was constructed and transcribed in vitro.Mixed gRNA(final concentration 50 ng/?L)and Cas9 mRNA(final concentration 300 ng/?L)to inject into wild type AB strain and APPsw transgenic line zebrafish fertilized eggs.Useing T7E I enzyme digestion and sanger sequencing to verify the knockout effect.Using genetic methods,the F2 generation mmp9 gene knockout was successfully obtained.The hatching rate,phenotype and the movement of the knockout strain zebrafish was no significant difference among the three strains.3.The combined effect of aluminum poisoning and APPsw to explore the mmp9 gene's founcationSix months old wild type AB zebrafish,APPsw transgenic zebrafish and F1 generation APPsw-mmp9-KO zebrafish,each line was divided into two groups:circulating culture water treatment group(control group),AICl3 treatment group.AICl3 treatment:100?g/L AlCl3 solution with pH==5.8 was dealt for 20 days.The behavioral changes of zebrafish were detected by Noldus.HE staining,Nissl staining,immunohistochemistry and transmission electron microscopy were used to observe the pathological changes of zebrafish.RT-qPCR was used to detect the changes of gene transcription level to explore the joint effect of aluminum poisoning and APPsw mutant genes,as well as the role of mmp9 in the pathogenesis of the disease.The results showed that compared with the normal group and the APPsw group,the behavior of the APPsw zebrafish was abnormal after the treatment with AICl3,and the latency of finding the nutrient-rich region was significantly prolonged(p<0.01),and the granulosa cells of the terminal brain and cortex were decreased.There were voids,the number of neurons was significantly reduced(p<0.01),the boundary of the nuclear membrane disappeared,the number of organelles decreased,and the deposition of A?1-42 increased,mainly involving the membrane and blood vessels.In terms of gene transcription level,claudin 2,claudin 5b,IL-1?,ERK1,mmp2 and mmp14 were up-regulated and claudin h was down-regulate.Compared with the APPsw-mmp9-KO with AICl3 treatment group,the APPsw-mmp9-KO with AICl3 treatment group improved the delay time(p<0.05),but the granule cells decreased and the cavity decreased,the number of neurons decreased,and the nuclear membrane disappeared.Immunohistochemistry showed that compared to APPsw with AICl3 treatment group,the deposition of A?1-42 APPsw-mmp9-KO with AICl3 treatment group was reduced,mainly involving the membrane.The gene transcription level,claudin 2,claudin 5b,IL-1? ERK1,mmp2 and mmp14 were down-regulated significantly in the APPsw-mmp9-KO with AlC13 treatment group(p<0.05).The results indicate that the synergistic effect between acidic AlC13 and APPsw can accelerate the development of AD.After mmp9 gene knockout,the body's inflammation level is reduced,as well as the synergy between acidic AICI3 and APPsw,and slow down the disease process.