| Acute myocardial infarction, because of massive myocardial necrosis are not renewable, can lead to left ventricular remodeling and expansion. Eventually lead to heart failure, serious harm to people's lives. Is coronary reperfusion therapy (including emergency thrombolysis and intervention), although able to save the ischemic myocardium, necrotic myocardium but can not be added. while if myocardial infarction are happened,local heart muscle suffer ischemic and hypoxia,much heart muscle cells occurre cellular mecrosis or apoptosis,the abnormal heart muscle are taken place by fibrosis tissue,then the cardiac ventricle are remodelled.When the heart muscle are remodeled,the heart muscle will be thinner than before,the relaxation pressure of the left ventricle will be increased,the volume of the left ventrile will be enlarged,then the heart failure is happened.Stem cells are a kind of undifferentiated, can be infinite or longer-term proliferation, self-renewal, and under appropriate conditions can differentiate into a variety of functional cells or tissues and organs of the original cells. MSCs transplantation is currently considered the main mechanism of MI in the following areas (1) Differentiation of cardiac contractile function with like cells, expressed cardiac-specific contractile proteins, the formation of myocardial cells with normal intercalated disc connections, synchronous contraction in the host myocardium, reduce scar area. (2) Their differentiation into vascular endothelial cells or through paracrine secretion of various cytokines mechanism.There are two types of cytokines that angiogenesis and inflammation factors. (3) Increased cardiac expression of mucin and atrial sympathetic nerve sprouting and the over-distribution. (4) By regulating the extracellular matrix, ease the infarct wall thinning and left ventricular cavity to expand and improve regional wall motion, improves ventricular remodeling.Studies have shown that the decoction of ginseng Can protect the endothelial function, Narrowing after coronary recanalization AMI no-reflow area , Improve myocardial reperfusion and improve cardiac function, anti-apoptosis, promote angiogenesis and other effects. Differentiate into cardiomyocytes and endothelial cells, is ideal for cardiovascular regenerative medicine of new cells, and MSCs confirmed the effectiveness and feasibility of treatment of AMI. Studies have shown that Ginseng alone can protect the endothelial function, reduced post-AMI coronary reperfusion no-reflow area, improve myocardial reperfusion and improve cardiac function, anti-apoptosis, promote angiogenesis and other effects. Traditional Chinese medicine joint MSCs, MSCs can improve myocardial infarction survival and differentiation in the efficiency and significantly improve cardiac function and reduce the perfusion defect area may be independent Chinese Ginseng improved "soil " (myocardial injury in the environment), is conducive to "seed "(transplanted stem cells) in the presence, Differentiation and to be effective. we can promote the neovascularization and improve the blood supply to the infarctus tissues,encourage the heart muscle's funcation and the function of the heart,then reduce the remodeling of the left ventricle.Purpose:1 Bone marrow mesenchymal stem cells isolated, amplified and training.2 With ligation of anterior descending coronary artery (LAD) method, the establishment of acute myocardial infarction.3 Clear bone marrow mesenchymal stem cells (MSCs) combined decoction of ginseng The direct injection of cardiac transplantation can improve cardiac function, prevention and treatment of ventricular remodeling.4 Observed in rat MSCs combined decoction of ginseng apoptosis after acute myocardial infarction and apoptosis-related proteins caspase-3 expression.Method:1 Bone marrow mesenchymal stem cells isolated, amplified and culture; take Wistar male rats, the use of adherence screening method combined with density gradient centrifugation method of purification, Wistar male rats were each taking a cut off were sacrificed, soaked in 75% ethanol volume fraction of 10min, removed under sterile conditions, femur and tibia, to sterile balanced salt PBS buffer repeatedly washed bone marrow cavity. Collection of bone marrow fluid in the pre-accession 50mL sterile DMEM medium tube to 2000r/min speed centrifugal 20min, draw a single cell layer (low-density cells, located at the two-layered liquid). DMEM, rinse cells twice, 1000r/min speed centrifugal 5min, supernatant obtained cell pellet. Added to cell pellet volume fraction of 15% fetal bovine serum DMEM medium, adjusting the cell density of about 1×10~6/mL, inoculated in 25cm2 cell culture flask. After 48h the medium replacement, removal of adherent cells did not, after the medium was changed every 1 3d. 80% of primary fusion cells to be passaged, and discard the old and medium to sterile balanced salt PBS buffer rinse 3 times, 1000r/min speed centrifugal 10min. Supernatant obtained cell pellet, add volume fraction of 15% fetal bovine serum DMEM medium, according to the proportion of 1:2 passage.in vitro amplification of MSCs. Passage to the 5th generation, balanced salt PBS buffer to prepare single cell suspension for flow cytometry to monitor cell surface markers.2 The use of Olivette way ligation of the left anterior descending coronary artery model of acute myocardial infarction. of anesthesia (35mg/kg), to be fully anesthetized, intubated animals. Connect the small animal respirator, positive pressure ventilation line. After disinfection, skin incision, blunt dissection of pectoralis major, thoracic, fully exposed the heart, with the ophthalmic sutures from the lower edge of the left atrial appendage at about 1-2cm, select the successful ligation of left anterior descending artery, myocardial infarction myocardium darken , the corresponding lead electrocardiogram shows ST-T segment elevation or depression, chest off layer by layer, the insulation after animals well, routine use of penicillin 200 000U daily, continuous application of at least three days, completely healed in the surgical , A separate feed.3 Animal groups: Wistar rats were randomly divided into 4 groups of 40: I set the control group with acute myocardial infarction (AMI, n = 10): rats were injected with equal amount of culture medium after myocardial infarction; II group + stem cells in acute myocardial infarction transplantation group (AMI + BMSCs, n = 10): acute myocardial infarction in rats receiving allogeneic bone marrow mesenchymal stem cell transplantation (2×10~6 BMSCs / 100 u1, divided into 4); III alone acute myocardial infarction + Ginseng Group (AMI + Ginseng alone, n = 10): acute myocardial infarction 2 hours after the independence Ginseng fed, twice daily, sharing the week; IV in acute myocardial infarction + stem cell transplantation group + single Ginseng group ( AMI + BMSCs+ Ginseng, n = 10): post-acute myocardial infarction in allogeneic bone marrow mesenchymal stem cells transplantation (2×10~6 BMSCs / 100 u1, divided into 4 points), 2 hours after gavage of independence Ginseng times a day, sharing a week.4 Cardiac function tests: In bone marrow mesenchymal stem cells 4 weeks after transplantation, the cardiac ultrasound examination. Left ventricular end diastolic diameter (LVDd), left ventricular end systolic diameter (LVDs) and calculated Cardiac output (CO) and fractional shortening (FS).5 Hearts were handled: in bone marrow mesenchymal stem cells after 4 weeks after transplantation, cardiac ultrasound examination, rats were killed by cervical, the hearts were cut into thick slices about 3mm, the line embedded in paraffin, cardiac HE staining.6 Myocardial cell apoptosis: TUNEL labeled apoptotic cells in situ. TUNEL labeled apoptotic cells in situ apoptosis, the chromosomal DNA double-strand breaks or single-strand breaks and produce large amounts of sticky 3 'OH end of a can be terminal deoxynucleotidyl transferase (TdT) the role of , Will deoxyribose nucleotides and fluorescein, peroxidase, alkaline phosphatase or a derivative of biotin tag to the formation of the 3'-OH ends of DNA, allowing for detection of apoptotic cells, these methods call For the terminal deoxynucleotidyl transferase mediated nick end labeling (Terminal deoxyribonucleic transferase mediated d-UTP nick end labeling, TUNEL). As the proliferation of normal cells or are barely breaking DNA, and thus there is no 3'-OH formation, rarely stain. Specific methods according to instructions.7 Caspase-3 activity assay: caspase-3 precursor is activated to show their activity, DEVD-pNA as the substrate hydrolysis by caspase-3 to generate 400-405nm wavelength can absorb the product, according to the product of absorbance Infer the relative level of activity.Results:1 Bone marrow mesenchymal stem cells isolated, amplified and training:Most primary BMSCs adhered to the wall at 2 days after culture, which proliferated faster after passaged, and the 5 passage of cells were mostly purified into BMSCs, spread radially or vortex-likely.?Surface molecule markers of the 5 passage of BMSCs were examined. The isolated cell purities were 88.9% for CD90, 92.4% for CD44, but negative for CD34 and CD45.2 Echocardiography: the parameters of four groups of patients (CO, FS) compared with before surgery were significantly different (P <0.05), cardiac output (CO)and fractional shortening (fractional shortening, FS) were significantly lower, and preoperative parameters between the two groups was no significant difference. Stem cell treatment group, the decoction of ginseng group and decoction of ginseng stem cells + decoction of ginseng alone the parameters of the intervention group compared with postoperative myocardial infarction were increased in control group; intervention group in which stem cells and stem cells + decoction of ginseng alone the parameters of the intervention group was significantly higher than Myocardial infarction control group, and significant differences (P <0.05), but the stem cells + decoction of ginseng intervention group alone after myocardial infarction to improve cardiac pump function best.3 Histopathological Results: After 4 weeks, HE staining of rat hearts were seen extensive myocardial fibrosis, myocardial tissue disorder, associated with adjacent muscle fibers atrophy (or) hypertrophy, myocardial scar tissue between the large number of inclusions, and can be seen Mild infiltration of inflammatory cells mainly lymphocytes, fat-like changes; cell transplantation group hearts hyperplasia, hemangioma did not find the small number of blood vessels are more abundant.4 TUNEL test results: the performance of the nuclei showed apoptotic cells with brown staining , Normal control group of non-apoptotic cells and no or occasional myocardial cells TUNEL-positive cells were stained blue hematoxylin ,nuclear relatively Large, the same shape and size. Myocardial infarction control group I, the highest apoptotic index was significantly higher than II, III, IV group, the difference was significant (P <0.01); IV group control group, the apoptosis index was the lowest and with the group II, III, Than the difference was significant (P <0.01); group II, group III, the difference between groups was not significant (P> 0.05).5 Caspase-3 activity of Results: The activity of caspase-3 between the caspase-3 activity assay showed that IV-myocardial cells with low levels of caspase-3 activity, the highest activity group I(P <0.01), group II, III, significantly after treatment Inhibit its activity increased, the group was better than group IV inhibition II, III.Conclusion:1 using the anterior descending coronary artery ligation (LAD) method, the establishment of acute myocardial infarction. Modeling high success rate, reproducible and strong practicability.2 Bone marrow mesenchymal stem cells in myocardial injection, combined decoction of ginseng Can significantly improve cardiac function after acute myocardial infarction.3 The implantation of MSCs into the infarcted myocardium can improve the supply of blood and oxygen to myocardium.bone marrow mesenchymal stem cells in myocardial injection, combined decoction of ginseng may be significantly reduced myocardial apoptosis. Reduce the expression of caspase-3 activity. |
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