| Cardiovascular disease is a disease that seriously endangers human health,among which the middle-aged and elderly people have the higest incidence.It easily leads to stroke,disability or even death.It is a disease that medical workers all over the world urgently want to solve.According to statistics,more than 17.9 million people died of cardiovascular diseases all over the world in 2016,ranking first among all causes of death.Even with the most advanced treatment,such disease cannot be completely overcome.As we all know,the function of vascular endothelial cells（ECs）locating in the inner wall of blood vessels plays a vital role in the pathogenesis of cardio-cerebrovascular diseases.ECs are in direct contact with blood and affected by fluid shear stress（FSS）.Abnormal FSS is not only the key stimulating factor in the pathogenesis of atherosclerotic（AS）,but also the main cause of focal AS.The magnitude and flow field of local shear stress affect the biological processes such as development,proliferation and apoptosis of ECs,which lead to abnormal function of vascular ECs and promote the occurrence of cardiovascular disease including AS.Although many mechanical sensitive genes have been screened and studied,it is not enough to explain the changes of ECs function in abnormal mechanical environment.Previous studies used ECs,cultured in vitro to screen differentially expressed genes（DEGs）as mechanical sensitive factors by applying different mechanical treatments,but this study noted that zebrafish embryos developed in vitro produce blood flow at 24-26 hour-post-fertilization（hpf）.Based on this,this study put forward a scientific hypothesis that"The expression of mechanical sensitive genes will change significantly in ECs during the development of zebrafish from no blood flow to blood flow".In this study,the transcriptome and gene chip data of zebrafish embryos before and after 24 hpf were analyzed and mined by means of bioinformatics,and the corresponding key node genes were expected to be found.Furthermore,through the analysis of these key genes and their upstream and downstream pathways,it could be useful to better understand the pathogenesis of cardiovascular disease and provide a theoretical basis for clinical treatment of cardiovascular disease.Here are the main contents and conclusions of our study.Part One:Screening of DEGs in zebrafish ECs under FSSDuring the development of zebrafish,blood flow appears at 24 hpf.Using this characteristic,the high-throughput data of zebrafish ECs before and after 24 hpf were selected from the GEO database for data mining and analysis,and finally we found two sets of data sets（GSE126617 and GSE20707）into our study.Then,the"edge R"package in R and the GEO2R online analysis tool were used to screen the DEGs in ECs of zebrafish before and after 24 hpf.It was found that there were 6 common DEGs（SRGN,SLC12A3,SLC25A4,PVALB1,ITGAE.2,zgc:198419）responding to FSS in the two sets of data sets.Furthermore,through the comparison of the homology of these genes between zebrafish and humans,and to ensure the consistency of the results of the two sets of data,four differential genes（SRGN,SLC12A3,SLC25A4,PVALB1）were finally identified for further verification.Part Two:Verification of SRGN as a mechanical sensitive gene in vitro.（1）Human umbilical vein endothelial cells（HUVECs）were loaded with 5dyne/cm2 shear stress in vitro for 6 h using the parallel plate flow chamber mechanical loading system.The total RNA,of HUVEC was extracted and the expression of 4differential genes was verified by fluorescence quantitative PCR.The results showed that the expression of these genes have significant differences under low shear stress（LSS）.Among them,SRGN was found to be mainly expressed in hematopoietic cells,endothelial cells and embryonic stem cells.It was considered to be closely related to cardiovascular health,so SRGN was selected for further research.（2）Promo online analysis software was used to predict transcription factor binding sites in the upstream promoter region of SRGN,and it was found that there was a CREB binding site in the upstream 123bp.Previous studies have also proved that cAMP/PKA/CREB signal pathway can transmit mechanical signals to ECs and regulate gene expression.Therefore,we used PKA inhibitor（H89）to treat HUVECs,and applied LSS to cells.After detecting the expression of SRGN by fluorescence quantitative PCR,immunofluorescence and ELISA,we found that H89 could significantly inhibit the response of SRGN to FSS.Part Three:Study on the relationship between SRGN and AS（1）By using immunofluorescence technique and Griess method to detect the proportion of cells with high expression of SRGN in Ki-67 positive cells and the content of NO in ECs after mechanical loading,we found that SRGN played a role in promoting ECs proliferation,while the effect of SRGN was significantly inhibited after H89treatment.（2）Local ligation of carotid artery was performed in ApoE-/-mice to establish shear-stress-induced AS model.After the operation,the mice were fed with high-fat diet for 4 weeks,and then the left and right carotid arteries of mice were taken for frozen sections.Using immunofluorescence technique,it was found that SRGN was mainly expressed in the ECs of the left common carotid artery（LCA）with obvious plaques,but almost no expression in the right common carotid artery（RCA）.Meanwhile,we also found that SRGN was mainly expressed in the areas of suspected angiogenesis in AS plaques,suggesting that SRGN might affect angiogenesis in AS plaques by promoting ECs proliferation,promote the formation of vulnerable plaques and induce secondary thrombosis to harm human health. |
