Combination Transplantation Of Ad.HGF Transfected MSCs In The Treatment Of Chronic Ischemic Heart Disease In Pigs

Chronic ischemic heart disease is the one of the most common species disease to result in death, Although early surgical treatment within a timely and effective to save the lives of many patients with acute myocardial infarction, but there are still about20%of the development of heart failure. Studies found that the main reason is due to local myocardial ischemia, hypoxia occurred a large number of irreversible necrosis. To compensate this ability to repair damaged heart, A scientis try to transplanting different types of exogenous cells into damaged myocardium, desire to increasing the number of myocardial cells. At the same time transplanted cells secrete angiogenic growth factor to promote myocardial angiogenesis and improve myocardial perfusion, and restore to normal physiological function.In this study, we designed to contribution the porcine ischemic heart model. Then via directed injection MSCsAd.HGF transplantation for myocardial infarction region. Research to both the promote angiogenesis, inhibition apoptosis and fibrosis, inhibition of ventricular remodeling and improved cardiac function。Part1The study HGF affect to biological characteristics of MSCsObjective:The study HGF affect to biological characteristics of MSCs Proliferation, differentiation, survival, etc.Methods:pig iliac bone marrow were separated by density gradient and adherence separation; sterile culture, ingredient identification (CD71, CD34and CD44and VIII of factor and desmin, and cell surface markers); to Ad.HGF transfection of MSCs, test purposes HGF expression and secretion of the protein; observe the proliferation and differentiation of MSCs and MSCs apoptosis under hypoxic environment; to detect the synthesis and secretion of type Ⅲ collagen fibers.Results 1、inoculation when cells are spherical suspended in culture medium,48h basic adherent,3-5d was clone-like growth,10-12days cells reached90%confluence. Immunohistochemical staining indicated that BMSCs of3rd generation were positive for CD71,CD44and negative for,CD34、Ⅷ facto and rDesmin2、Ad.HGF significantly promote MSCs growth, show the MSCs high expression of HGF protein; the group MSCsAd.HGF PCNA expression was significantly higher than group MSCsAdNull; hypoxia of group MSCsAd.HGF apoptosis rate (3.5±0.5%) of group MSCsAdNull (5.8±0.9%); HGF can effectively reduce the secretion of type Ⅲ collagen fibers.■ConclutionsA percoll density gradient and adherence combination of detachable high purity,acticity,vigorous growth of MSCs.HGF can be safe and effective transfection of MSCs carrying the HGF gene will be eligible for a higher level of expression both in vitro and in vivo.HGF to promote the role of MSCs proliferation and differentiation with a very clear.HGF in vitro to significantly inhibit the MSCs cell apoptosis inhibition of transplanted MSCs cell type III collagen secretion, oxygen conditionsPart2Eveluation to transplantation of MSCsAd.HGF in a porcine ischemic heart model thrugh direct injectionObjective:To study of MSCsAd.HGF combination transplantation for myocardial revascularization to improve the microenvironment of ischemic myocardium to promote the survival of the transplanted cells, inhibition of cardiomyocyte apoptosis and fibrosis, inhibition of left ventricular remodeling, protect and improve heart function.Methods:(1) Etabilish of pig modelThe40mini pigs, Left lateral chest incision into the chest, the left coronary circumflex artery(LCX) free trunk put into the ring Ameroid. Evaluation of this pig model by electrocardiogram, coronary angiography, color Doppler ultrasound;(2)Eperiment group 4weeks later, randomly divided into5groups(n=8),the multi-point injection of the ischemic myocardial regions:group MSCsAd.HGF injected5×108/ml MSCs200μl; group AdHGF4×109pfu200μl AdHGF; group MSCs5X108/ml MSCs200μl; group AdNull4×109pfu and200μl AdNull; group control lml Saline. Conventional closed chest again after feeding for4weeks.(3) Clinical eveluation4weeks later, the detection of cardiac function by ultrasoundcardiogram, included left ventricular systolic volume (LVEDV)、left ventricular ejection fraction (LVEF)、 fractional shortening (FS); Rentorp method to evaluate the extent of collateral formation, SPECT myocardial perfusion test. Execution model removed the heart, after the slices for pathological daignosis, count the number of factor Ⅷ-positive blood vessels, HGF gene expression, myocardial TUNEL staining, etc,■Results:1. All animals successfully constructed model of chronic ischemic heart disease, put after ring the troponin blood was significantly higher (P<0.05),ECG ST-T segment changes significantly. DSA examination showed most of LCX occlusion, there have been a remote myocardial infarction; ultrasound confirmed the left ventricular wall modeled after the myocardial infarction zone can be seen, significantly lower degree pulse, ventricular wall thinning, LVEF, FS significantly increased;4. Treatment after4weeks echocardiography showed:group MSCsAd.HGF of cardiac function before transplantation significantly improved ventricular wall thickness increased, the ventricular chamber decreases, the LVEDV significantly narrowed,LVEF, FS, than increased significantly (P<0.05); SPECT myocardial perfusion:perfusion of group MSCsAd.HGF ischemic area improve, increased myocardial thickness, sports enhancement (P<0.05); DSA score Rentorp:that of group MSCsAd.HGF collateral circulation compared with the previous formation was significantly increased (P<0.05); myocardial HGF protein expression:of group MSCsAd.HGF protein HGF to obtain higher expression (P<0.0.5); fluorescence microscopy:group MSCsAd.HGF to see the CM-DiI labeled transplanted cells (P<0.0.5); vessel count:of group MSCsAd.HGF infarct marginal region of obvious angiogenesis, marginal zone than the infarcted area (P <0.05); factor Ⅷ of detection:group MSCsAd.HGF factor Ⅷ-positive blood vessels, positive vascular rate significantly higher than group AdNull group (P<0.05); TUNEL staining:group MSCsAd.HGF fewer cells are stained brown positive rate of apoptotic cells was lower than AdNull group (P<0.05).■Conclutions1Confirmed MSCsAd.HGF transplantation in myocardial ischemic region, this could be improve the microenvironment significantly and the MSCs more colonization and survival;2MSCsAd.HGF implantation effectively inhibit cardiomyocyte apoptosis and left ventricular remodeling, development, protection and improvement in cardiac function.3The implantation of MSCsAd.HGF treatment of ischemic heart disease effective more pronounced compared with alone transplantation Ad.HGF or MSCs, the more obviously improvement of cardiac function and inhibition of left ventricular remodeling.