| Purpose: In this study,the rat model of acute myocardial ischemia was taken as the research object.The intervention method was acupuncture combined with bone marrow mesenchymal mtem mells(BM-MSCs)through vein transplantation.The aim of this study was to investigate the effect of acupuncture combined with BM-MSCs through vein transplantation on acute myocardial ischemia(AMI)in rats,and to explore the molecular biological mechanism of acupuncture combined with BM-MSCs through vein transplantation in improving AMI of rats from three aspects of inflammatory response,apoptosis and growth factor change.Material and method:1.Isolation,culture,passage,identification and labeling of BM-MSCs: three healthy male SD rats(SPF grade,4 weeks old,weight 150-160g)were anesthetized by intraperitoneal injection of 7% chloral hydrate(5ml/kg),soaked in 75% alcohol and sterilized for 5min.Tibia and femur were taken out under sterile conditions.PBS buffer was used to wash out bone marrow and made into a single cell suspension.After centrifugation,they were inoculated in a culture bottle and placed in a culture box for collection.The whole bone marrow adherent method was used for culture,and the culture medium was changed in time.When the cells were spread over the bottom of the culture bottle and fused into a single layer,they were digested with 0.25% trypsin,then cultured in 1:2 passages.The morphological changes of the cells were observed under the microscope.The third generation cells were detected by flow cytometry for cell surface markers(CD29,CD73,CD90,CD44,CD45),and the third generation cells were labeled by Brd U.The positive cell labeling rate was observed by fluorescence microscope.2.Preparation of AMI rat model: 60 healthy male SD rats were intraperitoneally injected with7% chloral hydrate(5 Ml/kg)anesthesi.The AMI rat model was made by ligating the left anterior descending branch of the coronary artery,and the surviving rats were randomly divided into model group,stem cell group,acupuncture group,acupuncture combined with stem cell group(10 rats in each group).At the same time,another 15 rats only punctured thesurgical suture in the same position without ligation,and 10 rats survived after the operation were randomly selected as the sham operation group.3.Acupuncture combined with BM-MSCs vein transplantation in the treatment of AMI rats: 1hour after the end of modeling,rats in the stem cell group and acupuncture combined with stem cell group were injected 0.5ml BM-MSCs(2×106/ml)via caudal vein,while rats in the other groups were injected with the same dose of normal saline.The rats in the acupuncture group and the acupuncture combined with stem cell group were treated with electroacupuncture at Neiguan and Xinshu points on both sides,with a depth of 2-3 mm,density wave,frequency of 2 Hz and intensity of 2 m A.The rats in the acupuncture group and the acupuncture combined with stem cell group were treated with mild tremor and contraction of local muscles for 30 minutes and 24 hours respectively for 10 times.The rats in the other groups were not treated with acupuncture,but were grasped and fixed in the same way at the same time.The body weight,hair,body shape,diet,defecation and exercise of rats in each group were observed.At the end of the treatment,ECG and echocardiography were detected in each group,and then rats in each group were killed,the whole blood of abdominal aorta was taken,and haemorrhage was separated for use.At the same time,the heart was taken out quickly and washed with normal saline.One heart sample was randomly selected from each group and five slices were cut in the ischemic area for the determination of myocardial ischemic area in rats.Other heart samples were frozen in the ultra-low temperature refrigerator at 80? for standby.This paper is divided into two parts to discuss the protective effect of acupuncture combined with BM-MSCs through vein transplantation on myocardial injury in rats with acute myocardial ischemia and its molecular biological mechanism.(1)Effect of acupuncture combined with bone marrow mesenchymal stem cell transplantation on AMI in rats.To observe the morphological characteristics and changes of BM-MSCs in subculture,analyze the identification and labeling results of BM-MSCs,collect and analyze the ECG and echocardiography data of rats,and determine the myocardial ischemic area of rats in each group after AMI by TTC staining.(2)Study on the molecular mechanism of acupuncture combined with bone marrow mesenchymal stem cell transplantation in improving AMI in rats.HE staining was used to detect the morphology and inflammatory infiltration of myocardial cells in each group afterAMI.Western blot technique(WB)was used to detect the relative expression of Bcl-2,Bax,Caspase-3,NF-?B,VEGF,b-FGF and TGF-? protein in myocardial tissue of rats.Enzyme linked immunosorbent assay(ELISA)was used to detect the relative expression of TNF-a,IL-1,IL-6,IL-8 and IL-10 protein in rat myocardium.Results:1.Compared with sham operation group,LVDd and LVDs in model group were significantly higher(P < 0.01),while LVEF and LVFS were significantly reduced(P < 0.01).Compared with the model group,the LVDd and LVDs of the rats in the stem cell group,acupuncture group and acupuncture combined stem cell group decreased significantly(P < 0.01),while the LVEF and LVFS increased significantly(P < 0.01).Compared with stem cell group,LVDd,LVDs,LVEF and LVFS in acupuncture group were not significantly different(P > 0.05).Compared with acupuncture group,LVDd and LVDs in acupuncture combined with stem cell group were significantly reduced(P < 0.01),while LVEF and LVFS were significantly increased(P < 0.01).2.There was no ischemic myocardium in the sham operated group.Compared with the sham operated group,the ischemic area in the model group was significantly increased(P < 0.01),accounting for 18.39% of the total area of the left ventricle.Compared with the model group,the proportion of myocardial ischemic area in the stem cell group,acupuncture group and acupuncture combined with stem cell group was significantly reduced(P < 0.01).Compared with the stem cell group,the myocardial ischemic area in the acupuncture group was significantly reduced(P < 0.01).Compared with the acupuncture group,the ischemic area of rats in the acupuncture plus stem cells group was significantly higher than that in the acupuncture plus stem cells group(P < 0.01).3.Compared with the sham operation group,the relative expression of NF-?B,TNF-?,IL-1,IL-6,IL-8 protein in the model group was significantly increased(P < 0.01).Compared with the model group,the relative expression of NF-?B,TNF-?,IL-1,IL-6,IL-8 protein in the stem cell group,acupuncture group and acupuncture combined with stem cell group was significantly reduced(P < 0.01).Compared with the stem cell group,the relative expression of NF-?B,TNF-?,IL-1,IL-6 and IL-8 protein in the acupuncture group was significantlyreduced(P < 0.01).The relative amounts of NF-?B,TNF-?,IL-1,IL-6,IL-8 protein in the myocardium of the acupuncture combined with stem cell group were significantly reduced(P< 0.01 or P < 0.05).4.Compared with the sham operation group,the relative expression of IL-10 protein in the myocardial tissue of the model group significantly decreased(P < 0.01).Compared with the model group,the relative expression of IL-10 protein in the myocardial tissue of the rats in the stem cell group,acupuncture group and acupuncture combined with stem cell group significantly increased(P < 0.01).Compared with the stem cell group,the relative expression of IL-10 protein in the myocardial tissue of the rats in the acupuncture group had no significant change.Compared with the acupuncture group,the relative expression of IL-10 protein in the myocardium of the acupuncture combined with stem cell group significantly increased(P < 0.01).5.Compared with the sham operation group,the relative expression of Bax and caspase-3protein in the model group was significantly higher(P < 0.01).Compared with the model group,the relative expression of Bax and caspase-3 protein in the stem cell group,the acupuncture group and acupuncture combined with stem cell group was significantly reduced(P < 0.01).The relative expression of Bax and caspase-3 protein in the acupuncture combined with stem cell group was significantly reduced(P < 0.01).The amount was significantly lower than that of acupuncture group and stem cell group(P < 0.01).6.Compared with the sham operation group,the relative expression of Bcl-2 protein in the myocardial tissue of the model group was significantly reduced(P < 0.01).Compared with the model group,the relative expression of Bcl-2 protein in the myocardial tissue of the stem cell group,acupuncture group and acupuncture combined with stem cell group was significantly increased(P < 0.01).Compared with the stem cell group,the relative expression of Bcl-2 protein in the myocardial tissue of the acupuncture group had no significant change(P > 0.05),the relative amount of Bcl-2 protein in myocardial tissue of rats in acupuncture plus stem cells group was significantly higher(P < 0.01).Compared with acupuncture group,the relative amount of Bcl-2 protein in myocardial tissue of rats in acupuncture plus stem cells group was significantly increased(P < 0.01).7.Compared with the sham operation group,the relative expression of VEGF,b-FGF andTGF-? protein in the myocardial tissue of the model group was significantly increased(P <0.01).Compared with the model group,the relative expression of VEGF,b-FGF and TGF-?protein in the myocardial tissue of the stem cell group,acupuncture group and acupuncture combined with stem cell group was significantly increased(P < 0.01).Compared with the stem cell group,the VEGF,b-FGF and TGF-? in the myocardial tissue of the acupuncture group and acupuncture combined with stem cell group was significantly increased(P < 0.01).Compared with the acupuncture group,the relative expression of VEGF,b-FGF and TGF-?protein in the myocardium of the acupuncture combined with stem cell group was significantly increased(P < 0.01).Conclusion:1.Acupuncture combined with BM-MSCs transplantation can significantly improve the changes of ECG and echocardiography in AMI rats,reduce the area of left ventricular ischemia in AMI rats,and improve the cardiac function of AMI rats,which provides a new idea for clinical treatment of this disease.2.Acupuncture combined with BM-MSCs transplantation can reduce the expression of NF-? B in AMI rats,the level of TNF-a,IL-1,IL-6,IL-8 and the level of IL-10,reduce the over expression of inflammatory response after AMI,reduce the necrosis of cardiomyocytes;regulate the expression of Bcl-2,Bax,Caspase-3,reduce the apoptosis of cardiomyocytes after AMI;regulate the expression of growth factor VEGF,the expression of,b-FGF and TGF-?;induce the formation of endothelial cells,so as to improve the myocardial blood supply and protect the ischemic myocardial cell injury. |
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