The Effect Of Lipa Gene Deletion On Foam Cell Formation In Atherosclerosis

BackgroundIn recent years,the incidence of cardiovascular and cerebrovascular diseases has increased in China,and the mortality rate of these diseases ranks the first among all kinds,among which Atherosclerosis(AS)accounts for a large proportion.Therefore,a special prevention and treatment research cooperation area has been established in China,and atherosclerosis has become a key part of medical research with a number of preliminary results that have been obtained in its diagnosis and prevention.In the Genome wide association study(GWAS),Lipa--lysosomal acid type(Lipase A)was found to be closely associated with the formation of atherosclerosis,but the influence of foam cell formation in atherosclerosis was not elucidated.ObjectivePopulation genetics studies have found that Lipa is related to the pathogenesis of atherosclerosis,but the mechanism is not clear.In this study,the effects of Lipa gene on the formation of foam cells in the development of atherosclerosis were explored by knockout and knockout of Lipa gene in RAW264.7 macrophages and phenotype analysis of the key pathological process of atherosclerosis.MethodsThrough literature analysis,based on the genetic results of whole genome association analysis(GWAS)population of atherosclerosis patients,candidate genes are screened and Lipa gene is identified as the research object through tissue expression analysis.Specific transcription sequences of target genes are downloaded and labeled in SnapGene software,and specific sites are designed for target shooting.Mouse RAW264.7 macrophages have been widely used in studies on the mechanism of atherosclerosis and the formation of foam cells,and Lipa gene is highly expressed in macrophages.Using the CRISPR/Cas9 gene editing technique,the transfection plasmid of RAW264.7 macrophage was genetically modified in this cell line.Primers are designed to detect the knockout of the target genes at the gene level by PCR,and monoclonal cells of the appropriate gene modified cells are selected.Flow cytometry is used to detect the expression of key scavenger receptor CD36 in RAW264.7 cells and wild-type RAW264.7 cells after gene knockout.Flow cytometry is used to detect the uptake levels of Ox-LDL labeled by Dil red fluorescence in knockout cell lines and wild-type RAW264.7 cells.Using the CRISPR/Cas9 gene editing technology,the monoclonal knockout cells are designed for gene re-knockout,and protein labels such as FLAG are added at both ends of the knockout genes.Flow cytometry is used to separate the cells that were successfully knocked in.Western blotting and immunoprecipitation are used to detect the protein knocked in.Flow cytometry is used to detect the differences in atherosclerotic phenotypes between Lipa gene knockout cells,RAW264.7 cells,and wild-type control RAW264.7 cells.The contents included the expression of CD36,a key scavenger receptor in foam cell formation,and the level of Ox-LDL uptake by macrophages to Dil red fluorescent marker.ResultsBased on the summarization of AS-GWAS data,the study on the effect of Lipa gene on the formation of foam cells in atherosclerosis is carried out,and the target specific knockout plasmid of its important exon sequence is designed.Primers are designed and detected for Lipa gene knockout.PCR is used to detect Lipa gene knockout cells,and a number of Lipa gene knockout monoclonal cell lines are obtained.The design of primers to detect the deletion are completed.PCR is used to detect the Lipa knockout cells and a mass of monoclonal Lipa knockout cells are obtained.The corresponding Lipa gene is extracted and the monoclonal cell DNA is knocked out.Gene expression in the process of the three codes corresponding to a single amino acid protein,due to the found targets between exons bases with fewer Numbers are not multiples of three,namely the codon frameshift mutations appeared in the process of translation protein,further analysis due to single gene knockout cloned cell lines than normal gene sequences appear early termination codon,lead to amino acid amount is greatly reduced,translation will cause the protein translation failure results.The gene is successfully knocked out by the differences in the levels of both the base sequence and the amino acid sequence.Flow cytometry also showed that the uptake ability of Dil-Ox-LDL in Lipa single gene knockout RAW264.7 cells is significantly reduced compared with wild-type RAW264.7 cells.OST-Lipa-FLAG is successfully knocked in into the obtained monoclonal Lipa single gene knockout cells.Fluorescence microscopy and flow cytometry are used to confirm the presence of a large number of BFP-positive cells in the knocked cells,and the BFP-positive cells are successfully sorted by flow cytometry and cultured then.Western blot is used to verify the success of gene knockout at the protein level by using OST beads binding specificity and FLAG antibody.Flow cytometry is used to find that the expression of CD36 in gene return of single gene knockout RAW264.7 cells is back compared with that of wild-type 264.7 cells.The expressions of CD36 each cell type is in the same condition.Flow cytometry is used to detect the expression levels of gene return of single gene knockout RAW264.7 cells is back compared with that of wild-type 264.7 cells.The uptake ability of Dil-Ox-LDL is the same condition.ConclusionsLipa gene deletion can significantly reduce the ability of macrophages to absorb lipids and transmit liposuction signals,and affect the formation of foam cells.Lipa may be a new target for the study on the formation of foam cells in atherosclerosis,and provide support for the diagnosis,treatment and prevention of atherosclerosis.