Bioinformatics Analysis Of Drug-induced Osteoporosis Susceptibility Genes And Validation Of Zebrafish Model

Drug-induced osteoporosis is a common bone disease at present,which is caused by long-term and extensive use of drugs that affect bone metabolism.At present,some studies have confirmed the mechanism of drug-induced osteoporosis,but no researchers have explored the individual differences of drug-induced osteoporosis,and the genetic susceptibility of the population has not yet been reflected.Genes have important clinical significance for the prevention and treatment of primary diseases and drug-induced osteoporosis.In this project,the bioinformatics analysis of gene chip was used to screen the core genes related to drug-induced osteoporosis,and to establish a zebrafish model for verification.Objective:To screen out the susceptible population of drug-induced osteoporosis at the genetic level,and provide a theoretical basis for reducing the occurrence of drug-induced osteoporosis.Method:1.Use the GEO gene database to screen normal and osteoporosis-related core genes,apply computer tools to find differential genes,use the stitch tool to analyze the action pathway of each drug that is likely to lead to osteoporosis,and intersect the differential genes.species to pick out susceptibility genes.2.Select the most commonly used drug of each type,select wild AB-type zebrafish,start administration from the 4th dpf,adopt the method of exposure administration,culture until the 9th dpf,use 0.2%calcein solution to avoid light staining,use positive The 3-7 vertebrae were observed under a fluorescence microscope,and the statistical data were recorded by taking pictures.3.Use CRISPR/Cas9 gene editing technology to construct gene mutant zebrafish embryos,give different kinds of drugs,observe the degree of osteoporosis after the drugs,and compare them with normal zebrafish to observe the changes in body phenotype and bone calcification of the larvae.degree.Result:1.Download the gene chip GSE35956 from the GEO database,including 5 human mesenchymal stem cell(MSC)gene chips from osteoporosis patients and 5 normal people.The limma package of R language was called to find a total of 9292 differential genes.Query the 10 main action pathways of various drugs on the stitch website,and intersect the differential genes to obtain the candidate susceptibility genes for each type of drug.2.Dexamethasone,rosiglitazone,warfarin,rabeprazole and sodium valproate were selected as representative drugs for modeling.Compared with the negative control group,the fluorescence area of zebrafish vertebrae in the 25μM concentration group of dexamethasone group was significantly different(P<0.1).The two concentrations of 200 μM and 400 μM in the warfarin group were successfully constructed(P<0.1);the low-concentration group of rosiglitazone had no obvious osteoporosis,and the high-concentration group had higher mortality;the rabeprazole group and sodium valproate There was no significant difference between the saturated concentration group and the control group,so they were discarded.3.After reviewing the data,the differential genes were finally screened:CDK1 and PER2 in the dexamethasone group,and GGCX and NQO1 in the warfarin group.Using CRISPR/Cas9 gene editing technology,a zebrafish model with loss of CDK1 gene expression was successfully constructed.Compared with normal zebrafish,after administration of dexamethasone,the body shape was significantly deformed,and calcein staining showed that there was almost no bone growth and calcification.status.Conclusion:CDK1 gene has obvious effects on bone development and may be an important gene for glucocorticoid-induced osteoporosis,which provides a reference for future precision medicine and individualized drug delivery.To sum up,this topic screened out a susceptibility gene CDK1 that may be related to drug-induced osteoporosis through a new strategy combining bioinformatics and zebrafish experiments.The relationship between this gene and drug-induced osteoporosis Rarely reported in previous studies,we demonstrated for the first time that it affects the occurrence of osteoporosis in zebrafish.However,if we want to clarify the relationship between CDK1 and this disease and the confirmation of the related mechanism,further research through genomics and cytomolecular methods is needed in the future.