The Expression And Interference Effect Of Cyclooxygenase-2 In Rabbit Autogenous Vein Graft Model: An Experimental Study

Objective:Coronary heart disease(CHD) is a serious hazard that does harm to human's health. With the change of people's living style and eating habits,its morbidity and the mortality has increased year by year.Coronary artery bypass graft(CABG) is the main surgical method to treat the coronary heart disease,which can improve the patient's quality of life and prolong their life-span significantly.Internal mammary artery and saphenous vein is the main material of CABG.Although the saphenous vein is the mostly common used,it could not maintain long-term patency effectively after the surgery.Some research shows that there is 12 to 27%of the vein graft being occluded a year after the CABG surgery. This increase to 25-31%five years later with the occlusion rate grows 2 to 4%annually, and 48-66%of the vein graft is absolutely occluded ten years later.And there's about 10% of the patients have to have re-operation.Therefore,the method to avoid the vein grafts' restenosis has become a hotspot.Cyclooxygenase(COX),also known as prostaglandin-endoperoxide synthase,is a membrane-bound protein,have the function of both cyclooxygenase and peroxidase synthetic enzyme,and it is the key enzyme to convert arachidonic acid into the prostaglandin.The study of COX medically is originated from the research of pain and inflammation.COX is the key enzyme in the process of prostaglandin's(PG) biosynthesis which is an important medium of inflammation.It is researched that there are two isomers COX,COX-1 and COX-2,the former is type of structure with the main distribution in endoplasmic reticulum,and it is involved in the body's normal physiological process and functions,and the latter is inducible enzyme with the distributed in endoplasmic reticulum and karyotheca,express very low in the normal conditions,but this circumstance is doubled with the stimulation of inflammation.Since the discovery of COX inhibitor,that is, aspirin could prevent and cure atherosclerosis(AS),COX and isozyme COX-2 has become the focus.The pathophysiological process of AS is also the process of chronic inflammatory reaction with the essence of inflammatory diseases has been proved by many years of research.Systematic monitoring of inflammatory markers' change has become a progress of AS-related disease's prevention and cure in modern times.At present,the study has found the evidence that COX-2 taken part in atherosclerosis. And by reverse transcriptase enzyme and poly chain reaction in human AS plaque molecular biology study found that AS sites of vascular COX-2 mRNA expression is 4.8 times to a normal vascular,and the expression of COX-1 mRNA is only 1.1 times to normal.By immunohistochemical staining,people has found all ingredients of AS lesions human cells have a wealth of COX-2 protein expression,which could not express in the normal vascular wall without AS lesions.At the same time,it is found that COX-2 is not only involved in the formation of AS,but also may have an important influence,in its occurrence,development and the vesting process.It was found in rabbit artery balloon dilation injury model that damaged arterial's smooth muscle cells in the COX-2 mRNA expression levels were significantly higher than the normal control group,and selective COX-2 inhibitors can inhibit the expression of it;and the selective COX-2 inhibitors also can decrease the inflammatory response and intimal hyperplasia in the rabbit balloon injury model.After the Coronary artery bypass grafting(CABG),the vein grafts restenosis performs as the proliferation of vein graft vascular endothelium and smooth muscle,and atherosclerosis in advanced period.Accordingly,we assume that,COX-2 in the vein graft restenosis model will be significantly increased in expression,and COX-2 expression related to the process of restenosis.According to the assumptions,we conducted this study on the subject.OBJECTIVE:①Take rabbit as an experimental animal to establish a stable autogenous vein graft model to confirm that COX-2 in the vein graft will be obvious in the increased expression;②Culturing primary rabbit vein endothelial and smooth muscle cells,and continuous passage to meet the needs of experiment in vitro;③Through the use of specific COX-2 inhibitor,in vitro experiments to confirm that the COX-2 expression relates to the rabbit vein endothelial and smooth muscle cell proliferation;④Establishing an effective RNA interference system,which can be efficiently specific inhibition of rabbit vein cells COX-2 expression in cell experiment;⑤Use RNAi technology in the cell experiment to confirm that COX-2 expression and rabbit vein endothelial and smooth muscle cell proliferation are associated;⑥Through the use of COX-2 specific inhibitor to confirm the intervention of COX-2 expression can influence the hyperplasia,a narrow process in the vein graft in animal experiments.Methods: 1,The study of COX-2 expression in rabbits autogenous vein graft wallTaking 24 New Zealand white rabbits(weight 2.2 to 2.5 kg),of either sex,were randomly divided into three groups,each with eight.Ear vein injection of sodium pentobarbital 30mg/kg of narcotic,"Cuff" casing law firms unilateral external jugular vein -carotid artery autologous transplant.After the operation inject penicillin 200,000 U intramuscularly,and subcutaneous injection of heparin 1mg/kg once,every 12 hours for three days.Three groups of rabbits shall be respectively taken vein graft and the contralateral external jugular vein samples for self-comparison after 2,8 and 12 weeks. Each sample is divided into two parts,half immunohistochemical stained;half stored in liquid nitrogen to be used in quantitative PCR.Research observation:①Observed the difference of immunohistochemical stained ones in vein slice;②Taking the total RNA in vein samples,later,do a real-time quantitative RT-PCR analysis,quantitative COX-2 expression.2,Culture rabbit venous endothelium,smooth muscle cellsTaking the New Zealand white rabbits' inferior vena cava as a cell culture organizations after anesthesia.Strip the outer membrane when separate,use the warm isotonic Na chloride to rinse the blood after harvesting the blood vessels,moving it to sterile console.The intended cultured vein in smooth muscle cells,first overturn the vein and incubate it in 0.1%collagenase typeⅠ37℃for 20 minutes,fully rinse the endothelial cells,cut the veins into about 1 to 2 mm,then vertically cut them in half, expanding them and culturing them using explant attached method with M199 medium (containing 20%fetal calf serum,penicillin 100 U/ml,streptomycin 100μg/ml).The veins cultured with vascular endothelial cells,directly cut them about 1 to 2 mm above,then vertically cut them in half,expanding them and culturing them using explant attached method with the RPMI-1640 medium(containing 20%fetal calf serum,penicillin 100 U/ml,streptomycin 100μg/ml,heparin 100μg/ml,insulin 10μg/ml,hydrocortisone 10μg/ml).After the cells cultured,observe as endothelial cells or smooth muscle cells,then respectively identify them throughⅧfactor and a-SMA(a-smooth muscle actin) immunohistochemical method.3,The impact of RNAi and COX-2 inhibitors in vitro on the growth of rabbit venous endothelium and smooth muscle cells This section is divided into two steps:First,use EGFP genes in vitro screening to interfere more effective siRNA fragments in rabbit COX-2;then use the elected siRNA fragment and NS-398 to intervene the rabbit's vein endothelial and smooth muscle cells in vitro,and examine their effect on cell proliferation.Indicators of detection:①Immunohistochemical staining to observe difference of positive staining degree in different groups;②MTT measured the ratio of brightness;③Cell cycle measured by flow cytometry;④Rate of apoptosis measured by flow cytometry;⑤Real-time quantitative PCR to test COX-2 mRNA expression;⑥Westernblot to test the expression of COX-2, Bcl-2,Caspase-2 portein.4,The impact of COX-2 inhibitors on proliferation of the autogenous graft vein's wallTake 48 New Zealand white rabbits(weight 2.2 to 2.5 kg),of either sex,randomly divided into three groups,each with 16.Each group is randomly divided into the experimental group and the contral group,each group with eight.Both do the external jugular vein-carotid artery transplant.After the operation inject penicillin 200,000U intramuscularly,and subcutaneous injection of heparin 1mg/kg once,every 12 hours for three days.The experimental group was given a daily COX-2 inhibitor of celecoxib at a dose of 5 mg/kg.Three groups respectively harvested vein grafts after 2,8 and 12 weeks. Each sample is divided into two parts,half for immunohistochemical stained;half stored in liquid nitrogen,using in real-time quantitative PCR.Indicators of detection:①HE,VB staining,measure the thickness of intima and tunica media of the vascular wall under light microscopy;②Immunohistochemical staining to observe difference of positive staining degree in different groups;③Taking the total RNA in vein samples,reverse transcription, do a real-time quantitative RT-PCR analysis,quantitative COX-2 expression.Results:1,Confirmed that COX-2 expression in the vein graft was higherIt is found under microscopy that immunohistochemistry biopsy specimens,the normal venous has no obvious positive staining,while the specimens at three time points showed strong positive staining.Real-time quantitative PCR reacted that the COX-2 mRNA expression in the graft vein increased significantly.2,Successfully cultured a rabbit venous endothelium,smooth muscle cells and they can continuous passage.First,digest the vein endothelial cells,use explant attached method with M199 medium(containing 20%fetal calf serum,penicillin 100U/ml,streptomycin 100μg/ml) to culture,about five days later spindle cells climbed out around the organizations,nine days-about they growth,a single Bunch,close.About 40 minutes after passage,the cells were adherent,3 to 5 days can cover 70～80%of a dish,their morphology is basically the same as the original one,re-generation.The Cell's morphology is same as the vascular smooth muscle cell morphology reported in the literature,cell sustainable more than 10 passages.Immunohistochemical stained with a-SMA(a-smooth muscle actin),it was positive,confirmed as smooth muscle cells.The vein organization cultured in RPMI-1640 culture medium(containing 20%fetal calf serum,penicillin 100U/ml,streptomycin 100μg/ml,heparin 100μg/ml,insulin 10μg/ml and hydrocortisone 10μg/ml),after 4 to 7 days were around with the block polygon, oval-shaped cells,about 9 to 11 days cell growth,arrange as paving stones,a small amount of overlap,to subculture.About one hour,the cells were adherent,3 days can cover 70～80%of a dish,with the morphological characteristics are basically the same as the original, re-generation.Cell's morphology is same as the vascular endothelial cell morphology reported in the literature,cell sustainable more than 10 passages.Immunohistochemical stained withⅧfactor,it was positive,confirmed as endothelial cells.3,Confirmed that the use of RNAi and COX-2 inhibitors in vitro can inhibit rabbit vein endothelial and smooth muscle cell proliferation.We successfully screened effective rabbit COX-2 siRNA site.Immunohistochemical staining showed that the use of RNAi and COX-2 inhibitors can inhibit the expression of COX-2 in vein cells.MTT assay showed that the use of RNAi and COX-2 inhibitors can inhibit the vein cells' proliferation.Flow cytometry analysis showed that the cell cycle,compared with the blank group, stayed cells proportion in G1 phase rised,and the proportion of cells in S phase decreased, showed that cell proliferation was inhibited,the difference has statistical significant.Flow cytometry analysis showed that the rate of apoptosis increased markedly in COX-2 inhibitor group;the rate in RNAi group slightly higher than the control group;but there was no significant difference between RNAi and RNAi control group.Real-time quantitative PCR showed that the use of RNAi and COX-2 inhibitors can inhibit the COX-2 mRNA expression in vein cells.Westernblot test showed that the use of RNAi and COX-2 inhibitors can inhibit the expression of COX-2 protein;the use of COX-2 inhititor can inhibit the expression of Bcl-2 protein and increase the expression of Caspase-3 protein;the later did not appear in the RNAi and RNAi control group.4,Confirmed that the use of COX-2 inhibitors can influence the autologous vein graft of rabbit blood wall proliferation processTake each time point of graft vein's specimens,HE,VB staining,measure the intima and media thickness of the vascular wall under the light microscopy,it is showed that:two weeks after transplanting,the intima and media obviously thickening in two groups,and the control group is more severe than the experimental group,the difference is significant; the specimens which transplanted after eight weeks can obviously seen hyperplasia in vein graft,and obviously more in the control group;samples after 12 weeks' transplantation,the intima thickness decreased to the 8 weeks's,the media continue to thicken,and the degree of hyperplasia in intima and media was more heavy in the control group.Conclusion:1.COX-2 in the normal vein has low expression while in the vein graft has significantly high expression.2.Establish a simple and effective method to culture rabbit vein endothelial and smooth muscle cells,and its cells can consecutive passages to meet the experimental requirements.3.Applicating EGFP reported gene can screen siRNA site which interfered rabbit COX-2 effectively.4.The use of RNAi and COX-2 inhibitors in vitro can inhibit rabbit vein endothelial and smooth muscle cells expression of COX-2 mRNA,as well as inhibit cell proliferation.5.Using of COX-2 inhibitor could increase the rate of apoptosis of vein cell in vitro, while using RNAi did not show this effect.6.In rabbit autogenous vein graft model,use specific COX-2 inhibitor can inhibit the graft vein's COX-2 expression,and decreasing the proliferation of the grafted vein's wall. In short,COX-2 expression increased in the grafted vein significantly,its expression related to the process of proliferation of the wall of grafted vein,the inhibition of COX-2 expression can affect hyperplasia of vein graft in intima and in tunica media,can slow down the process of vein graft stenosis.