| Cardiovascular disease(CVD)refers to a class of heart and vascular involved disease,among which Coronary artery disease(CAD)is the most common.CAD is a fatal disease with typical symptoms of post-exersice/fatigue chest pain or discomfort which might travel to shoulder,back and jaw showing breath shortness on the basis of damaged blood support to myocardia.Acute or chronic coronary obstruction due to thrombosis or rupture of plaques is the direct cause and anatomical foundation of the damaged blood support.Atherosclerosis(AS)is the histological basis of plaque’s development and progression,pathologically leading to CAD as a major reason.In spite of the wide spectrum of age,CAD commonly aggregates in middle-age and elder population.Because of its high morbidity and mortality,CAD is regarded as one of the top 3 human killer of 21th century together with cancer and cerebrovascular disease.As to its high incidence in material-sufficient circumstances,CAD is threatening almost half of global population,the recidents in emerging countries,along with the social development and coming aging,especially in China.In China,the leading cause of death is being undergone a shift to CAD which would bring tremendous financial burden to ecomony.The origin and mechanism of AS are very complicated,which remain to be illustrated totally.Many studies,however,have proven that macrophage is a central player in the physiopathological process of AS.With damaged homeostasis such as hyperlipemia and inflammation,monocytes could traverse endothelium into the arterial intima and then differentiate into macrophages which can uptake modified lipid,eventrually becoming foam cells.Foam cell can induce the synthesis and release of varieties of inflammatory mediators which result in tropism of secondary monocytes via different pathways,including immunological system.As to this inflammatory cascade effect,the accumulation of foam cells beneath endothelium gets accelerated to form lipid pool,which turns into atherosclerotic plaque.Adverse lifestyle,basic diseases like hypertention and diabetes and unhealthy diet used to be regarded as mainly risk factors of AS.The family history and heritability,however,highlight the genetics issue of AS and CAD.Currently AS and CAD is defined as a polygenic disease which has interactions with the environment as well.In spite of the recent progress on Human Genome Project and Genome-wide association study,the relevant genetic research of AS and CAD remained hard due to long period and high cost of family genomic study as well as ethics and legislation problems.Animal model,especially mice model made a big breaktrough.The high similarity between mice and human genome makes it possible to anticipate the casual genes of human AS with the reference of mice AS genes which are much easier to detect on mice models.By far,many AS genes have been found with the help of mice model.But some questions still remain,such as whether the role of macrophage in AS is relative to genes,whether the lipid phagocytosis and metabolism is regulated by genes,what is the cause gene of lipid metabolism disorder in macrophage.Objective:According to the polygenic characteristic of CAD and AS,we plan to utilize the macrophage lipid metabolism difference between AKR and DBA/2 strain mice whose AS sensitivity are different from each other.We want to use QTL mapping,bioinformatics and genetic engineering to explore the relationship between the lipid metabolism disorder in macrophage and gene control.By this,we want to clarify the genetic basis of macrophage lipid metabolism disorder to enhance the understanding of genetics beneath CAD.We hope to provide new idea and direction for the prevention and therapy of CVD.Methods:1 Build AKR x DBA/2 cross mice group for QTL mappingWe plan to use AKR and DBA/2 strain mice as parental mice to cross and breed AKR x DBA/2 F4 generation mice.By sequencing the gemonic DNA and culturing the bone marrow derived macrophage(BMDM)from F4 mice then testing the lipid metabolism,we plan to acquire the genotype and lipid metabolism phenotype data and then analyze both with R/qrl software.We hope to get the QRLs and gene relavent to macrophage lipid metabolism phenotype,and pick up the candidate gene according to the genetic characteristic.2 Explore the candidate gene Soatl’s differences between the two strains via bioinformaticsBy complimentary DNA(cDNA)PCR test of AKR and DBA/2 Soat1 gene,we plan to find Soat1 gene’s difference on transcription level between two strains,and use the transcription difference to detect the genomic difference between two strains’Soat1 gene with Wellcome Trust Sanger Institue database.3 Use CRIPSR/Cas9 to edit Soat1 gene in DBA/2 Embryonic Stem cellCulture mice pluripotent embryonic stem cell(ES)under aseptic condition,transfect Cas9 plasmid to this ES cell via nucleofection to build Cas9 stably expressed ES cellline.Design doule guide RNA(gRNA)which aim to the ends of AKR Soat1 truncation.Synthesis this double gRNA by plasmid tranformation and in vitro transcription.Carry out CRISPR/Cas9 in ES cell via nucleofection and achieve the deletion of AKR Soat1 trunction in DBA/2 genome.4 Differentiation of gene-edited ES cell to macrophageExecute the differentiation of gene-edited ES cell to macrophage by bioloical signal induction and test the lipid uptaking of ES-derived macrophage(ESDM)to test the function integrity of ESDM.5 Function and lipid metabolism test of gene-edited ESDMTest the lipid metabolism and Soatl expression level of AKR Soat1 truncation deleted macrophage via cholestrol mass assay and qPCR.Find the lipid accumulation profile in gene-edited ESDM by cholesterol staining to explore the association of the gene editting and lipid metabolism in macrophage.Results:1 QTL mapping result:1.1 There are 10 QTLs relevent to the phenotypic difference of BMDM lipid metabolism.And name them sequetailly as Mcmml-10.1.2 We found Mcmml has the strongest associaion with BMDM lipid metabolism difference including free cholesterol(FC)(R2=0.3;β=-23.4),cholesterol ester(CE)(R2=0.33;β=62.9)and FC/CE ratio(R2=0.31;(β=1.37).2 Bioinformatics result:2.1 Mcmml contains 34 genes,in which Soatl gene has the function to esterify FC to CE.So Soatl was chosen as the first candidate gene.2.2 Compared with DBA/2 cDNA.AKR cDNA lacks 118bp sequence containing Exon2.2.3 Compared with DBA/2 genomic DNA,AKR genomic DNA lacks 6818bp section which is located on chrl:158,394,619-158,401,436.2.4 Compared with DBA/2 ACAT1 coding by Soat1 AKR ACAT1 lacks 33 amino acid on the N-terminal.3 Gene editting result:3.1 Western Blot result showed the stably expressed Cas9 protein in Cas9-plasmid transfected and screened ES cell.3.2 gRNA test showed designed sequence was ligased in scaffold successfully.Gel electroporesis result showed designed gRNA was successfully synthesized after in-vitro transcription.3.3 Gene-edited cell pool genomic PCR test showed CRISPR/Cas9 was executed smoothly to delete the target gene section in ES cell as planned.3.4 Colonies’ genomic PCR test showed gene-edited homozygote and heterozyote were acquired,whose target gene was edited as planned.4 ES cell differentiation result:4.1 Observation of DiI-acLDL uptoken macrophage under florescent macroscope showed differentiated cells were fully functional macrophage.4.2 Flow cytometry result of lipid uptoken cells differentiated from different celllines showed 3 gene-edited cell line’s differentiation profile had no difference,which means gene-editting had no influence on differentiation.5 Gene-edited macrophage test result:5.1 qPCR result showed no change on mRNA level in gene-edited macrophage.5.2 Cholesterol mass assay result showed the deletion of AKR Soatl truncation can increase the FC and decrease the CE and FC/CE ratio,P<0.05.5.3 Cholesterol staining result showed the deletion of AKR Soatl truncation can decrease the accumulation of CE in macrophage.Conclusion:1 The lipid metabolism difference between AKR and DBA/2 strain macrophage is associated with the 10 QTL.2 Mcmml from the 10 QTL has the strongest relationship with the difference of macrophage lipid metabolism.3 In Mcmm1,Soat1 gene which codes ACAT1 protein is the first candidate gene related to the macrophage lipid metabolism difference.4 On transcription level,AKR Soatl gene lacks 118bp sequence containing Exon2.On genome level,AKR Soatl gene lacks 6.8kb gene section.On translation level,AKR AC AT1 protein lacks 33 amino acid on N-terminal.5 AKR Soatl truncation is a causal factor of the macrophage lipid metabolism difference. |
