Enhanced Homing Of Bone-marrow Derived Mesenchymal Stem Cells To The Ischemic Myocardium By Ultrasound-targeted Microbubble Destruction

PART I ISOLATION, CULTIVATION AND IDENTIFICATION OF CANINE BONE MARROW MESENCHYMAL STEM CELLS IN VITROObjective: To explore the approaches of isolation, culturvation and identification the canine bone marrow mesenchymal stem cells (BMMSCs) in vitro.Methods: Bone marrow mononuclearcells were extracted from canine bone marrow and purified via a density gradientcentrifugation on Percoll (density 1.073g/ml), then proliferated in vitro. Cellular immunohis- tochemistry study and flow cytometry detection were performed identify the cells and determine the cycle for surface marker protein of canine BMMSCs.Results: Immunohistochemistry detection showed the expression of CD29 and CD44 was positive, and the expression of CD31, CD34, CD45 and vWF was negative. 88.88% cells were in the G0/G1 period. The cells were identified to BMMSCs which population via 3 generations may increase to 107, which purity may reach above 99%, so as to provide adequacy canine BMMSCs for therapy of ischemic heart disease by stem cellular transplant.Conclusions: By use of density gradient centrifugation on separating medium, 1.073g/ml percoll, combinding with the method of adherent purity can gain MSCs on high purity. Cultured cells through 3 generations within 2 weeks can expand to 107 orders of magnitude, which is more corresponding to the requirements to construct the seed cells for tissue engineering.PART II EXPERIMENTAL STUDY ON BIOEFFECTS OF CANINE MYOCARDIUM BY MICROBUBBLES DESTRUCTION VIA DIVERSITY OF ULTRASONIC INTENSIONObjective: To explore the bio-effects of canine myocardium by microbubble destruction to optimization ultrasonic intension for experiments.Methods: Nine mongrel dogs were randomly divided into 3 groups. Ultrasound (1 MHz) in diversity of intension (0.5 W/cm2, 1.0 W/cm2, 2.0 W/cm2) was applied to expose canine myocardium after intravenous injection microbubbles of 2.0 ml. All the dogs were killed after being treatment for 5 minutes. The myocardium was harvested for HE staining and observed with Transmission Electron Microscope to observe the tissue microstructures.Results: The myocardium of hyperemia, disfiguration and necrosis were observed in all groups. Myocardial edema but not hemorrhage appeared with 0.5 W/cm2, mild myocardial hemorrhage and slight inflammatory cell infiltration happened with 1.0 W/cm2, whereas obvious hemorrhage an certain degree of inflammatory cell infiltration occurred with intension of 2.0 W/cm2. With the augmentation of ultrasonic intension, myocardium trend to aggravate.Conclusion: Ultrasound of diversity intension can induce different bioeffects of canine myocardium. Ultrasound mediated microbubble destruction with the intensity of 1.0~2.0 W/cm2 can provoke a certain degree of inflammatory reaction with mild myocardial damage. PART III ENHANCED HOMING OF BONE-MARROW DERIVED MESENCHYMAL STEM CELLS TO ISCHEMIC MYOCARDIUM BY ULTRASOUND TARGETED MICROBUBBLE DESTRUCTION IMPROVED MYOCARDIAL MICROENVIRONMENT CHANGESSECTION I THE CHANGES OF MYOCARDIAL MICROENVIRONMET INDUCED BY ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION IN A CANINE MYOCARDIAL INFARCTION MODLEObjective: To investigate the changes of myocardial microenviron- ment after MI induced by ultrasound-targeted microbubble destruction.Methods: Ten dogs were randomly divided into treatment group and control group after the establishment models of myocardial infarction. One week later, dogs in the treatment group were treated with an ultrasound at a frequency of 1 MHz and an intensity of 1.0 W/cm2 media microbubble destruction for three times in one week; there was no treatment for dogs in the control group. Levels of vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), vascular cell adhesion molecule-1 (VCAM-1) and interleukin-1β(IL-1β) in the myocardium were measured by RTQ-PCR and western blot analyses in half of the dogs in each group after 2 weeks.Results: Ultrasound at a frequency of 1 MHz and an intensity of 1.0 W/cm2 provoked inflammatory reactions with mild myocardial damage. The expression of VEGF, SDF-1, VCAM-1 and IL-1βin the treatment group was much higher than the control group.Conclusion the expression of VEGF, SDF-1, VCAM-1 and IL-1βcaused by UTMD may change the local myocardial microenvironment.SECTION II ENHANCED HOMING OF MESENCHYMAL STEM CELLS TO THE ISCHEMIC MYOCARDIUM BY ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTIONObjective We tested the effects of myocardial micro-environment changes induced by UTMD on promoting the homing of mesenchymal stem cells (MSCs) to the ischemic myocardium.Methods Ten dogs were randomly divided into treatment group and control group after the establishment models of myocardial infarction. Dogs in treatment group were treated by UTMD with a frequency of 1 MHz and an intensity of 1.0 W/cm2 ultrasound for three times in one week; no UTMD in the control group. All dogs were transplanted with DAPI-labeled MSCs via coronary injection. Five days later, the myocardium was harvested for Confocal laser scanning microscopy (CLSM) observation.Results: Ultrasound at a frequency of 1 MHz and an intensity of 1.0 W/cm2 provoked inflammatory reactions with mild myocardial damage. CLSM revealed that the distribution of MSCs in the pretreated myocardium was significantly higher than the control group (66.8±5.03 vs 14.6±3.76, P<0.05).Conclusion Myocardial microenvironment changes caused by UTMD may promote the homing of MSCs to the ischemic myocardium. This non-invasive technique may be a promising method for cardiac cell transplantation therapy.