Preliminary Exploration Of Murine Atherosclerosis Vulnerable Plaque Model

ObjectiveAtherosclerosis is a common cardiovascular disease that poses a serious threat to human health.In the process of atherosclerotic lesions,rupture of vulnerable plaque can cause acute blockage of the brain or heart arteries,which is the leading cause of death.Studies have shown that foam cell apoptosis(especially macrophage apoptosis)in atherosclerotic plaques caused by various causes is an important cause of vulnerable plaque formation.The study of the formation mechanism of vulnerable plaque is very important,but the traditional atherosclerotic animal model is difficult to establish a vulnerable plaque model,which makes many studies still stay in the correlation discovery,and can not carry out more in-depth exploration.Escherichia coli purine nucleotide phosphorylase(ePNP)is a prodrug-converting enzyme suicide gene that converts the adenine analog Fludarabine into a toxic substance that kills cells.Scavenger receptor(SR)A is a glycoprotein on the surface of macrophages,and studies have shown that part of its promoter region has macrophage specificity.In this study,the SR-A promoter core region and enhancer region were linked to the suicide gene ePNP,an overexpression vector was constructed,and a macrophage-specific SR-A-ePNP transgenic mouse model was established,and then SR-A-ePNP transgenic mice were crossed with ApoE-/-mice,and SR-A-ePNP-ApoE-/-homozygous mice were identified through several generations of breeding,and plaques were formed in the blood vessels by high-fat feeding.The drug Fludarabine induced different degrees of apoptosis in macrophage foam cells in plaques,and a vulnerable plaque model was established.However,some studies have suggested that STAT1 is a junction between atherosclerotic IFN-?,TLR4 and IL-6 activation pathways,which has amplifying proinflammatory signaling,so its inhibitor Fludarabine develops atherosclerosis.It is likely to have an effect.In this study,the specific expression plasmid was improved.The study proved that diphtheria toxin could not affect the mouse cells.In this study,a macrophage-specific vector expressing human diphtheria toxin receptor SR-HBEGF was constructed.After transfecting macrophage foam cells,it induced specific apoptosis of macrophage foam cells through diphtheria toxin,which laid a foundation for the establishment of a new vulnerable plaque model.Methods? Preliminary establishment of vulnerable plaque model in SR-A-ePNP-ApoE-/-transgenic miceThe SR-A-ePNP overexpression vector was constructed,transgenic mice were established,SR-A-ePNP positive mice were propagated and identified,and SR-A-ePNP transgenic mice were crossed with ApoE-/-mice to progeny,and PCR reactions were identified.SR-A-ePNP-ApoE-/-homozygous mice,which formed plaques by high-fat feeding,induced apoptosis of macrophage foam cells in plaques under the action of prodrug Fludarabine,and identified tissue structure by HE staining,ANNEXIN V-FITC/PI Apoptosis assay and TUNEL reaction for apoptosis identification.? Improvement of SR-A-ePNP to SR-A-HBEGF overexpression plasmid The SR-A enhancer and promoter region were amplified by SR-A-ePNP as a template,and the pUCm-T vector was ligated;the diphtheria toxin receptor gene HBEGF was inserted into the pcDNA3.1 vector to amplify the HBEGF and BGHpolyA tail fragments;The HBEGF and BGHpolyA tail fragments were ligated to the promoter region of the SR-A enhancer in the pUCm-T vector,and the SR-A-HBEGF overexpression plasmid was constructed.The plasmids were transfected into macrophages and mast cells,respectively,and their expressions were identified by mRNA and Western blotting,immunohistochemistry,etc.at mRNA and protein levels,respectively.Foamed macrophages were transfected and induced to undergo apoptosis by diphtheria toxin,and their function was identified by TUNEL staining.Results? Preliminary establishment of vulnerable plaque model in SR-A-ePNP-ApoE-/-transgenic mice1.Positive SR-A-ePNP transgenic mice were identified by ordinary PCR.At present,four strains of Z1,Z2,Z3 and Z7 SR-A-ePNP transgenic mice have been passaged to 7-8 generations,and the expression of gene ePNP is stable.2.The positive SR-A-ePNP transgenic mouse FO was hybridized with ApoE-/-mouse FO to obtain F1 mice,and the F1 mice were further hybridized,and SR-A-ePNP-ApoE-was identified by PCR./-Homozygous mice.At present,the hybrid progeny of Z2 mice have been passed to the sixth generation,and the hybrid mice of other lines have passed to the third generation.The genetic inheritance is stable,and only the required SR-A-ePNP-ApoE-/-pure can be selected by identification.Zygote mice.3.Using the prodrug Fludarabine to inject the peritoneal cavity of SR-A-ePNP-ApoE-/-transgenic mice,extract the peritoneal macrophages,and detect the apoptosis of macrophages by flow cytometry.And the apoptotic rate is positively correlated with the drug concentration.4.After injection of SR-A-ePNP-ApoE-/-mice with different concentrations of prodrug Fludarabine,the liver,spleen and other tissues of the mice were taken.TUNEL staining showed that Fludarabine would not contain the content within a certain dose range.Other organs of macrophages cause serious damage.5.High-fat-fed SR-A-ePNP-ApoE-/-mice formed plaques,and the mice were injected with different concentrations of prodrug Fludarabine.The vascular tissues containing plaques were taken for TUNEL staining to identify giant plaques.Different degrees of apoptosis occur in the foaming cells.The higher the dose,the more apoptotic cells are in the plaque,and the vulnerable plaque model is initially established.? Improvement of SR-A-ePNP to SR-A-HBEGF plasmid1.The SR-A enhancer and promoter regions were amplified using SR-A-ePNP as a template.2.Construct plasmid pUCm-T-SR-A.3.Amplify the human diphtheria toxin receptor gene HBEGF using human cDNA as a template.4.Construct plasmid pcDNA3.1-HBEGF.5.Amplify HBEGF and BGHpolyA fragments.6.Construction of the pUCm-T-SR-A-HBEGF plasmid with macrophage specificity was completed.7.Ordinary PCR identified macrophages and mast cells transfected with the plasmid,respectively,and found that only the transfected macrophage group was able to amplify the band of the human diphtheria toxin gene.8.Quantitative PCR identified the expression level of the target gene in the transfected macrophage group was higher than other groups.9.Wester blot and immunohistochemistry identified the transfected macrophage group to express the target gene.10.Foaming of macrophages was induced by different concentrations of oxLDL,and macrophage foaming was identified by oil red O staining.11.After transfecting the plasmid into macrophages,the oxLDL foaming was used to induce apoptosis by diphtheria toxin,and the human diphtheria toxin receptor expressed by the transfected group was identified as normal by TUNEL staining.Conclusion? Establishment of a vulnerable plaque model for SR-A-ePNP-ApoE-/-transgenic mice In this study,a SR-A-ePNP-ApoE-/-transgenic mouse model was established.After the high-fat feeding,the prodrug Fludarabine can induce different degrees of apoptosis in the macrophage foam cells in the plaque,thus completing the new type of easy.The damaged plaque model is established.? Improvement of plasmid SR-A-ePNP to plasmid SR-A-HBEGF In this study,the plasmid SR-A-HBEGF,which is specifically expressed by macrophage,was reconstituted on the basis of plasmid SR-A-ePNP.It was identified to be expressed in macrophages,and it can induce macrophage foam cells to apoptosis through diphtheria toxin.