The Experimental Study Of Ultrasound-mediated Microbubble Destruction Combined With Bone Marrow Mesenchymal Stem Cell For Rebuilding Functional Cardiac Muscle

Objectives:1. To compare the isolating cultural method and biological features ofmesenchymal stem cells (MSCs) derived from bone marrow (BM) andperipheral blood (PB), and to explore the PB-derived MSCs mobilizationresponse by recombinant human granulocyte colony-stimulating factor(rhG-CSF).2.To investigate whether low-intensity pulsed ultrasound (LIPUS)irradiation directly combined SonoVue microbubbles could enhance5-azacytine (5-aza)induced cardiomyogenic differentiation of human bonemarrow mesenchymal stem cell(HMSC-BM) in vitro, initially explore theoptimal irradiation condition,and to provide experimental basis forultrasound-mediated microbubble destruction (UTMD) application in MSCstransplantation for acute myocardial infarction.Methods:1. Fifteen healthy male C57BL/6mice were divided into3groups: group1:splenectomy+rhG-CSFsubcutaneous injection for5days;group2:splenectomy+rhG-CSFsubcutaneous injection for12daysgroup；3：shamoperation+physiological saline injection. Six hours after the last injection, theblood samples of splenectomized mice were collected from tail for leukocytecount and from abdominal aorta for flow cytometry analysis ofCD34-CD45-CD29+CD44+MSCs. 2. Forty healthy male C57BL/6mice were divided into2groups：group1：PB-derived MSCs culture；BM--derived MSCs culture. Two weeks aftersplenectomy，the rhG-CSF (350ug/kg.d）was injected to the mice of group1subcutaneously for5days，then the peripheral blood were collected fromabdominal aorta and the PB-derived MSCs were separated and cultured bydensity gradient centrifugation combined with adherence culture. TheBM-derived MSCs were isolated from the femur and shin marrow of the miceof group2and cultured by complete bone marrow adherent culture. Thencompare the morphological features and growth condition of the two sourcesof MSCs.3. In order to screen out the appropriate ultrasound irradiation intensityand time without conspicuous harm to HMSC-BM, stem cells were exposedto different ultrasound intensity irradiation(0.11w,0.33w,0.55w,0.77w,0.99w)and time(0,30,60,90s) and then cell proliferation determined by methylthiazolyl tetrazolium (MTT).In order to screen out the optimalmicrobubble-to-cell ratio without conspicuous harm to HMSC-BM, stem cellsof different microbubble-to-cell ratios were exposed above the screenedultrasound irradiation intensity and time and then cell proliferationdetermined by MTT.4. HMSC-BM were divided into four groups and inducedcardiomyogenic differentiation by different inducing systems in vitro asfollows: no-treatment group；5-aza inducing group；5-aza inducing withmicrobubble and ultrasound group；5-aza inducing with microbubble andultrasound group， followed by four weeks of further culture. Themorphological changes were studied daily under phase-contrast microscope,then the expression of cardiac troponin-T was detected by immunocytochemistry method to analyze and compare differentiation rate ofcardiomyocyte-like cells in each group.Results:1. The blood samples of splenectomized mice were collected from tailfor leukocyte count and from abdominal aorta for flow cytometry analysis,and it demonstrated that leukocyte and CD34-CD45-CD29+CD44+MSCs ofthe peripheral bloodincreased after rhG-CSF-mobilized-bone marrow andthose of the injection of5-day group was higher than12-day group.2. The mononuclear cells from BM can cultivate MSCs-like colony,whereas those of PB failed.3. The direct ultrasound irradiation condition of no conspicuous harm toHMSC-BM, but with cavitation was intensityof0.55W/cm2, exposure time of30s and microbubble-to-cell ratio of50.4.At28days after induction,the stem cells of5-aza inducing group and5-aza inducing with microbubble and ultrasound group expressed cardiactroponin-T and the positive rate of the latter was higher than the former byimmunocytochemistry（13.69vs34.13%，P <0.05）. This study confirmed thefeasibility of direct ultrasound exposure of LIPUS on MSCs in vitro.Conclusions:1. Mouse BM-derived MSCs can be mobilized effectively by rhG-CSFinto the peripheral blood, for a limited time. It was more difficult to cultureand isolate MSCs from PB than that of BM, it concerned with a small numberof MSCs in peripheral blood both under the physiological conditionsandrhG-CSF-mobilized bone marrow.2. The appropriate ultrasound direct irradiationconditions was intensityof0.55W/cm2, exposure time of30s and microbubble-to-cell ratio of50. Appropriate conditions of LIPUS could enhance5-aza inducedcardiomyogenic differentiation of HMSC-BM in vitro. This study confirmedthe feasibility of direct ultrasound exposure of LIPUS on MSCs in vitro.