| Background Study shows that vascular endothelial cells play an important role in the development and progression of ischemia/reperfusion injury.Mature endothelial cell has a low proliferation rate and lacks the ability of differentiation and cannot repair damaged vascular endothelium.The study found that EPCs is not only involved in embryonic angiogenesis in postnatal angiogenesis but also in th repairing process after endothelial injury.EPCs cells can be added in vitro and can be induced to differentiate into mature endothelial cells.The number and quality of EPCs obtained in bone marrow is limited,and the number of EPCs in peripheral blood is also small.Vascular endothelial growth factor(VEGF)is an important cytokine that regulates the differentiation of EPCs into endothelial cells,and VEGF165,as a very important specificity cytokine,can effectively mobilize EPCs and increase the number and function of EPCs.VEGF,because of its powerful role in promoting the formation of new blood vessels,has been a hot spot of research.And animal model has been confirmed,because sake of effective carrier,VEGF gene therapy can only repair a small amount of vascular endothelial cells.The EPCs was modified by means of transgenic VEGF gene,gene therapy and stem cell therapy were combined in treatment of vascular endothelial injuring,can promote angiogenesis and improve the vascular endothelial repairing.Objective Constructed and identified recombinant adenovirus vector Ad-hVEGF165-GFP carrying green fluorescent protein-labeled human VEGF 165 gene,then transfected rat EPCs with Ad-hVEGF165-GFP,and explored the method and effect of hVEGF 165 gene transfection of EPCs.The hVEGF 165 gene transfected with EPCs was transplanted into myocardial ischemia/reperfusion injury rats.The changes of myocardial histopathology,myocardial structure and infarct size and immunohistochemical staining of myocardium were observed.At the same time,A and NF-KBp65 and TLR-4 in myocardium,and to explore the protective effect of hVEGF 166 transfected EPCs on myocardium of myocardial ischemia/reperfusion injury and its possible mechanism.Methods 1,hVEGF 165 gene recombinant adenovirus vector construction.The recombinant plasmid pAV-MCMV-HA-P2A-GFP was transformed into DH5a competent cells.The transformants were identified and sequenced by PCR cloning.The recombinant plasmids were transected into HEK293 cells with virus Adax adenovirus packaging and amplification system.The HEK293 cells were observed under microscope and the adenovirus titer was determined.Western Blot was used to detect the expression of recombinant adenovirus.2,hVEGF 165 gene recombinant adenovirus vector was transfected into rat vascular endothelial progenitor cells.Ad-hVEGF165 was transfected into EPCs by transfection of bone marrow mononuclear cells and bone marrow endothelial progenitor cells.The cell growth was observed under fluorescence microscopy and the VEGF protein was detected with Western blot.3,Protective effects of Ad-hVEGF 165 transfected EPCs on myocardial ischemia-reperfusion injury in rats and its mechanism.48 rats were randomly divided into sham operation group,myocardial ischemia reperfusion group,myocardial ischemia-reperfusion Ad-hVEGF165 transfected EPCs group(transfection group),and EPCs group,12 rats in each group.The ischemia-reperfusion group was established by using the left anterior descending coronary artery ligation/opening method.Release left anterior descending coronary artery after 30 minutes ligation and then reperfusion 2 hours.In the transfection group,EPCs cells cultured in the first 7 days were injected into the animals through the tail vein.After 2 hours of reperfusion,the ischemic area and infarct area were observed.The myocardium was fixed,embedded and sliced.The pathological changes were observed after HE staining.The expression of TLR4 mRNA in myocardium was detected by immunofluorescence staining and reverse transcription polymerase chain reaction(RT-PCR).The expression of NF-?Bp65 protein in myocardium was detected by imunohistochemistry.Blood samples were collected from the right carotid artery and the level of plasma cytokine TNF-a was detected by ELISA.Results 1.Construction of hVEGF 165 gene recombinant adenovirus:vector hVEGF was successfully constructed 165 recombinant adenovirus transfected HEK 293 cells showed significant cytopathic effect,titer of hVEGF 165 gene recombinant adenovirus was 1.106 x 108(ifu/ml),Western Blot detection in 25KD.2.hVEGF 165 gene recombinant adenovirus vector was transfected into rat vascular endothelial progenitor cells:hVEGF165 recombinant adenovirus transfected EPCs,24 h,VEGF protein expression was detected.The results showed that the expression of hVEGF protein was mostly in EPCs,and the infection efficiency was about 80%.Compared with before transfection,the expression of 24 h hVEGF protein increased significantly with transfection time.3.The protective effect and mechanism of rats:Ad-hVEGF165 transfection of EPCs transplantation on myocardial ischemia reperfusion injury,myocardial infarct size:compared with ischemia/reperfusion group 47.72 ±9.77%,EPCs group of myocardial cells transfected Ad-hVEGF165 32.26 ? 10.44%infarct area was significantly reduced,the difference was statistically significant(P<0.01);EPCs group(infarction area of 44.19±8.15%),compared with the ischemia/reperfusion group,there is no significant difference(P>0.05),compared with Ad-hVEGF165 transfected EPCs group,there is significant differences,larger infarction area,with statistical significance(P<0.05).The pathological changes of myocardium in 2 and 2 hours:multiple necrosis foci were observed under the microscope in the ischemia/reperfusion group,the original myocardial structure of the necrosis site disappeared,and a large number of necrotic myocardial cells were scattered around.Ad-hVEGF165 transfected EPCs group showed some scattered necrosis,no large myocardial cell necrosis,inflammatory cell infiltration decreased;single injection of EPCs group,compared with the the ischemia/reperfusion group,infarction area slightly smaller,the degree of inflammatory cell infiltration was less,with no statistical significance.The myocardial vascular immunofluorescent staining:ischemia reperfusion group(vascular area density 0.67±0.15)visible vascular distribution is sparse,Ad-hVEGF165 transfection group EPCs(vascular area density 2.76 ±0.14)significantly increased the number of blood vessels,and there is also significant difference with EPCs group(vascular area density 1.39 ±0.30).The plasma TNF-? level:2 h plasma level of ischemia/reperfusion group rats(518.09±79.24 pg· ml-1)was significantly higher than the sham operation group(103.23±12.33 pg ·ml-1),and the difference was statistically significant(P<0.01).The 2 h plasma TNF-a level of Ad-hVEGF165 transfected EPCs group(256.18±27.26 pg· ml-1)was significantly lower than that of ischemia/reperfusion group,there was significant difference(P<0.01);and also' significantly lower than EPCs group(P<0.05).The myocardial tissue TLR4 mRNA content:the TLR4 mRNA integral absorbance ratio of the ischemia/reperfusion group myocardial tissue(0.737±0.025),significantly higher than the control group(0.268±0.006),the difference was statistically significant(P<0.01),the TLR4 mRNA integral absorbance ratio of Ad-hVEGF165 EPCs transfection group integral absorbance ratio(0.347±0.071)was significantly lower than the ischemia/reperfusion group,also significantly lower than the EPCs group(P<0.05),and there is no statistical significance with EPCs group and ischemia reperfusion group.The myocardial tissue NF-?B p65 expression levels:the myocardial tissue NF-?B p65 expression integral value of ischemia/reperfusion group(5.33±0.61),significantly higher than the control group(0.67±0.82),the difference was statistically significant(P<0.01),the myocardial tissue NF-?B p65 expression integral value of Ad-hVEGF165 transfected EPCs group was 2.75±0.42,which was significantly lower than that in ischemia/reperfusion group,and the difference was statistically significant(P<0.01).EPCs group(integral value of 4.38±0.29),compared with Ad-hVEGF165 transfected EPCs group was significantly higher,the difference was statistically significant,and there is no statistically significant difference compared with ischemia reperfusion group(P>0.05).Conclusion Recombinant adenovirus Ad-hVEGF165-GFP was successfully obtained and transfected into rat EPCs.Ad-hVEGF165 transfection of EPCs transplantation on myocardial ischemia-reperfusion injury has a significant protective effect,can significantly promote angiogenesis,reduce myocardial cell necrosis,and reduce myocardial infarct size.The mechanism may be to inhibit the expression of TNF-a and other inflammatory factors,reduce the inflammatory response,and its upstream regulatory gene NF-?B p65 and signaling pathway molecules TLR4 which were also inhibited may play a very important role. |
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