Modeling Of Cerebrovascular Disease In Non-Human Primates

Non-human primates are indispensable resources and one of the effective experimental materials in exploring the origin of life,medical and health care,drug development,clinical trials etc.Using current science and technology to establish the non-human primates Wild Animal Germplasm resources system,not only strengthen the cooperation between the non-human primate resources and the related fields such as basic and clinical medicine,drug research and development,accelerate the transformation of the wild animal resources to the social value of scientific research,but also conducive to the protection and utilization of the non-human primate wild animal resources.Cerebrovascular disease is an important disease that leads to human death and disability.So far,there is a lack of animal models with highly similar genetic background and high repeatability to humans,and in particular,animal models for the effective mechanism evaluation and new target drug research of cerebrovascular disease.Using gene editing and other modern technological methods,we can establish the non-human primate cerebrovascular disease animal model,but the key to solve the above problems is choosing which gene or site.The inactivation and maladjustment of transcription factor RBPJ,can affects the function of notch and other signals pathways,that lead to the change of recognition markers of peripheral cells,trigger the fundamental changes of endothelial cell behavior,and cause brain ischemic damages.However,the relationship between RBPJ and cerebrovascular disease,and the feasibility of RBPJ as a target for non-human primates cerebrovascular disease model establishing are not clear.Therefore,in this project we want evaluates the relationship between RBPJ factor and cerebrovascular diseases,and the feasibility of RBPJ factor serves as the target for non-human primates' cerebrovascular diseases model establishing.1.Based on the evolutionary relationship between experimental animals and human RBPJ gene and the feasibility of RBPJ factor as a model targetMEGA7,BioEdit,Radar,Phyre software and other biological software were used to analyze the chromosomal localization,evolutionary characteristics of RNA and proteins of RBPJ factor in human,non-human primates,rodents,domestic animals and other experimental animals.To evaluate the composition and evolutionary characteristics of RBPJ factor in human and experimental animals,especially in non-human primates,and to analyze the advantages of RBPJ factor as a target for non-human primates cerebrovascular disease model.Further,using 400 cerebrovascular patients and 600 normal control subjects as research materials,and using PCR,case-control,western blot,plink and other methods and software to analyze the relationship between RBPJ factor and cerebrovascular disease.To evaluate the relationship between RBPJ factor and cerebrovascular disease and its feasibility as a model target for non-human primates cerebrovascular disease.The results of RBPJ protein sequence evolution showed that human,chimpanzee and monkey belong to the same big branch,including Rattus,Lagomorpha,cattle and sheep,and their evolutionary distance is relatively far away from that of human beings.RBPJ gene was located on chromosome 4 in human,chimpanzee and Yunnan snub nosed monkey,and on chromosome 5 in macaque,baboon and mouse.The analysis results of amino acid composition,isoelectric point and molecular weight of RBPJ protein showed that RBPJ protein was very conservative in the process of biological evolution;the amino acid composition and tertiary structure of RBPJ protein were very similar in human and non-human primates,rodents,livestock and other experimental animals Protein plays an important role in the normal function of organism.These results suggest that RBPJ gene or factor itself can be used as a target molecule in experimental animal models of cerebrovascular disease.Non-human primates,especially chimpanzees,have the most similar characteristics in the evolution of RNA and amino acids,chromosome distribution,protein molecular weight,isoelectric point,etc.,and non-human primates are ideal experimental animals for cerebrovascular disease models targeting RBPJ gene.2.Based on the relationship between RBPJ factor and cerebrovascular disease pathogenicityGenomic DNA was extracted from 1000 blood samples from 400 patients with cerebrovascular disease and 600 normal controls.The PCR amplification and sequencing of the RBPJ gene showed that rs2871198,rs1397731,rs3822223,rs2077777,rs2270226 and rs2788861 were highly heterozygous.The rs2077777 and rs2270226 mutations in RBPJ gene have great difference in genotype frequency and allele frequency between cerebrovascular disease patients and normal controls.Statistical analysis showed that rs2077777 and rs2270226 variants were significantly associated with the risk of cerebrovascular disease.Further comparison and analysis of Hardy Weinberg equilibrium and Hap-Map CHB database data shows that the experimental data conform to hardy Weinberg equilibrium,and are consistent with the data characteristics in Hap-Map CHB database.From the perspective of trend,dominant and recessive genetic models,rs2077777 and rs2270226 mutations in RBPJ gene were associated with the risk of cerebrovascular disease under the recessive genetic model.Western blot results showed that the protein expression of RBPJ gene was higher in rs2270226t/T group and rs2270226c/C group,but lower in rs2270226t/C group.Rs2270226 and rs2077777 mutations in RBPJ gene were located in the central repressor domain and N-terminal region of RBPJ factor.Based on the above results,The central repressor domain of RBPJ factor,N-terminal region and the expression level of RBPJ gene can be used as the target of establishing animal model of non-human primate cerebrovascular disease;taking the evaluation of experimental animals and human RBPJ factor as the breakthrough point,the effec-tive establishment of non-human primate cerebrovascular disease model is conducive to the mechanism analysis,prevention,treatment and development of targeted drugs.The results of this study show that,RBPJ may be the target gene of RBPJ,which may be the target of RBPJ.It may be used as a target for the establishment of animal models of non-human primate cerebrovascular disease;taking the evaluation of experimental animals and human RBPJ factor as the breakthrough point,the effective establishment of non-human primate cerebrovascular disease model is conducive to the mechanism analysis,prevention,treatment and research and development of targeted drugs.