Experimental Research On Promoting The Homing Of Bmscs To Infarcted Myocardium By Diagnostic Ultrasound-Mediated Microbubble Destruction Combined With Intracoronary Transplantation

BMSCs are cell subsets which exist in marrow stroma and source fromnon-hematopoietic cells in mesoderm. They can expand in vitro and have thepotential of multi-directional differentiation. Having been induced in vitro, theycan eventually differentiate into myocardial cells, osteoblasts, chondrocytes, andso on. As a kind of adult stem cells, compared with the stem cells from othersources, they have the following advantages:①It's easy relatively to obtainmaterials. And it's harmless to the body.②Autologous cell transplantation doesnot involve social and ethical problems.③BMSCs can divide and differentiateboth in vivo and in vitro,and proliferation in vitro is very fast. Large BMSCsamplification can be obstained within a short period of time.④The program ofimmunogic rejection does not exist. And it has the effect of immunologicalregulation.⑤The methods of transplantation are easy and various.⑥It is easy toconduct gene engineering technology process.⑦The myocardial cellsdifferentiated from transplanted BMSCs can conduct efficient electro-mechanicalcoupling with peripheral myocardial cells. Based on the above advantages,inrecent years,BMSCs transplantation are largely used to cure myocardial infarctionin experimental and clinical study.Both domestic and foreign animal experiments show thatultrasound-mediated microbubble destruction can increase the homing quantity ofthe replanted stem cells in the ischemic areas, enhance arteriolar formation andthe rebirth of blood vessels, improve partial blood flow of the ischemicmyocardial, recover the function of myocardial contractile, and have no adverse effect to the proliferation and the apoptosis, and cell cycle of replanted BMSCs.The first purpose of the research is to isolate, culture and identify BMSCs, toobtain enough BMSCs which are in good condition, and to decide that theobtained cells are the BMSCs needed by detecting cell surface molecular markersby immunohistochemical method, and to label BMSCs in vitro with BrdU.The second purpose of this research is to establish MI model by pluggingcoronary artery with OTW balloon.The third purpose of this research is to promote BMSCs homing to infarctedmyocardium through diagnostic ultrasound-mediated microbubble destructioncombined intracoronary transplantation, and explore a new method for curingmyocardial infarction clinically.Experiment1: The isolation, culture, identification and markers in vitro of thebone marrow mesenchymal stem cells. The method is to pierce canine,sPhumeruswith an injector, extract10ml bone marrow, and to mix it with equal volumetricheparin, Separate the bone marrow liquid with density gradient centrifugation.Remove the cells which do not stuck with internal surface of the tube, such asakaryocytes, and macrophages, etc. And then the bone marrow mesenchymal stemcells can be obtained. Vaccinate and cultivate those cells. The cells begin topassage when they grow up to70%. The result is, after1w, as many as1×107BMSCs are obtained. After3w, as many as24×107BMSCs are obtained.Immunohistochemical identification shows that CD34is negative, and CD44ispositive. The analysis to the growth curvature of MTT cells shows that thegrowing status of BMSCs is very good. Mix Brdu24hours before coronary arterytransplantation. Immunohistochemical markers in vitro show that BrdU hasmixed into the caryon.Experiment2: The building of canine MI model. The method is to push in a6F OTW balloon catheter, and make it arrive at the position which is betweenthe first septal branch and second septal branch. Inject the right amount ofcontrast medium where it is at0.5-1.0cm away from LAD and the left circumflex artery bifurcation. After3to5times of ischemic preconditioning(balloon filling20s each time, interval time3-5minutes), inject the right amountof contrast medium again. Fill the balloon at4～6amt and plug the anteriordescending coronary artery in order to block the blood. Make the balloon stay atanterior descending branch. Four hours after building the model, remove theballoon. The model is built. The result shows that electrocardiogram of themodel experiences characteristic and dynamic changes of AMI. Coronaryangiography video and pictures show that the obstructive site is between the firstseptal branch and second interval. Beyond the obstructive site, there is nocontrast medium. The blood flow breaks off4hours. The heart Colour DopplerUltrasound displays heart function fall compared with before. MCE displaysDA%rise.24experimental canines are all survived.Experiment3: Promoting of homing of BMSCs to infarcted myocardium bydiagnostic ultrasound-mediated microbubble destruction combined withintracoronary transplantation. The method is to divide the experimental caninesinto different groups randomly. That is, PBS group(1st group),ultrasound+microbubble+PBS group(2nd group), BMSCs group(3rd group) andultrasound+microbubble+BMSCs group(4th group),4groups in total, six caninesin each group. After models are made7d, each canine in the4th group isconducted diagnostic ultrasound-mediated microbubble destruction, and theninjected2×107BMSCs, which are the third generating and are marked BrdU, intocoronary artery through the OTW balloon centering catheter cavity respectively.The the3rd group is not given diagnostic ultrasound-mediated microbubbledestruction, only injected the same amount of BMSCs in the same way. The the2nd group is conducted diagnostic ultrasound-mediated microbubble destruction,and then injected2ml PBS. The the1st group is not given diagnosticultrasound-mediated microbubble destruction, only injected2ml PBS in the sameway. The results shows:both ultrasound+microbubble+BMSCs group and BMSCsgroup can reduce the size of myocardial infarct and perfusion defect, improve heart function, promote the surviving of BMSCs in myocardium,have thecharacteristic of α-actin. The former effect is more obvious. The experimentalso shows the positive correlation between DA%which reflects the leftventricular perfusion defect area and pathological indexes of the rate ofmyocardial infraction area to the total section surface area of myocardium whichreflects area of myocardial infarction. r=0.97.Conclusion:First, BMSCs can be separated and cultivated by adopting the density gradientcentrifugation and adherent culture method.Second, the stable canine MI model can be successfully established byplugging coronary artery with OTW balloon.Third, both ultrasound+microbubbles+coronary artery transplantationBMSCs and coronary artery transplantation BMSCs can reduce the size ofmyocardial infarct and perfusion defect, improve heart function, promote thesurviving of BMSCs in myocardium.The former effect is more obvious.They bothhave cells of the characteristic of α-actin,and the former have more.It providesexperience for the treatment of myocardial infarct clinically.Fourth, it shows the positive correlation between DA%which reflects the leftventricular perfusion defect area and pathological indexes of the rate ofmyocardial infraction area to the total section surface area of myocardium whichreflects area of myocardial infarction. r=0.97, which indicates that DA%andpathological indexes of NBT staining (%) have the equal value in reflecting themyocardial infarction area.