Study Of Treating Chronic Heart Failure By Transplanting Bone Marrow Mesenchymal Stem Cells Transferred With HO-1Gene

ObjectiveBone marrow mesenchymal stem cell transplantation in the treatment of cardiovasculardisease mainly in acute myocardial lesions and myocardial local lesion, and on the diffusedilated heart disease research is very little. This study use of adriamycin construction of ratmodel of chronic heart failure with dilated heart disease, isolated from bone marrowmesenchymal stem cells and the use of5-aza were induced to differentiate intocardiomyocyte-like cells, using a recombinant adenoviral vector HO-1gene transfection toinduce cell and expression of transfected HO-1gene, stem cell transplantation into heartfailure rat model, to observe the treatment of rats with chronic heart failure effect, and themechanism was discussed.Method1.The adriamycin rat dilated cardiomyopathy heart failure model preparationExperimental rats tail vein injection of doxorubicin hydrochloride solution,1mg/kg,weekly one time injection1times, for a total of6weeks; the normal control group tail veininjection of equal volumes of saline, the same time weekly injections of1, a total of6weeks.In the last2weeks after drug withdrawal after the injection, the rats' general physiologicalstatus and circumstances of death. The use of ultrasound in detecting left ventricular endsystolic diameter (LVDs), left ventricular end diastolic diameter and ejection fraction(LVDd), fractional shortening of FS EF. The polygraph record left ventricular systolicpressure (LVSP), left ventricular end diastolic pressure (LVEDP) and left ventricularpressure rising and falling speed (DP/dtmax). In experiment and the day of injection drugsfor two weeks after the beginning of heavy weight, respectively, and end weight.Measurement of ventricular mass (VW), and VW/BW ratio calculation. Myocardialpathology.2. Bone marrow mesenchymal stem cell separation, culture and induced to differentiateCharge and mass of stem cells and subculture density gradient separation method ofbone marrow to draw the three generations of MSCs growth curve immunohistochemistyevaluation,5-aza induction of the third generation of bone marrow between the charge and quality of stem cell differentiation into cardiomyocyte-like cells, the differentiation ofcardiomyocyte-like cells identification and calculation of the cardiomyocyte-like cellsconversion rate.3.Recombinant pcDNA3.1-of HO-1plasmid and its expression in MSCsDigested fragment of the GFP and the plasmid pcDNA3.1-HO-1connection, and toconstruct the recombinant plasmid pcDNA3.1-GFP-HO-1HO-1eukaryotic cell expression,the use of a single enzyme digestion of BamH Ⅰ and Hind Ⅲrestriction endonuclease thesize and the direction of the transferred gene, and amplification and purification. Theminimum lethal dose of G418for bone marrow mesenchymal stem cells were measuredusing lipofectamine plasmids were transformed into the spinal cord-derived bone marrowmesenchymal stem cells, the use of fluorescent protein expression by RT-PCR, Westernblotting detection of HO-1expression.4.Experimental study of transfer of HO-1gene, transplantation of MSCs in the treatment ofchronic heart failureRat model of heart failure were randomly divided into3groups of10. Will be inducedcardiomyocyte-like cells were labeled with BrdU, syringe-induced expression of MSCssuspension2:00injected into the left ventricular wall myocardium. Four weeks later, eachgroup were sacrificed5rats, ultrasound detection of left ventricular end systolic diameter,left ventricular end-diastolic diameter, left ventricular ejection fraction, and determination ofthe maximum pressure change rate and other hemodynamic parameters. Heart tissueembedded sections Brdu immunohistochemical staining.Result1. Adriamycin rat model of heart failure of dilated cardiomyopathy PreparationRat mortality rate was26.7%, while control animals did not kill. Compared with normalrats of CHF experimental rats at12days poisoning phenomenon. Significant increase in thenormal control group, body weight (BW), BW was significantly lower in the experimentalgroup, P <0.05, two significant differences in ventricular weight (VW) compared the twogroups was no significant difference, while the experimental group, VW/BW increasedsignificantly, and The normal control group, P <0.05, two significant differences. Andnormal control group compared to the experimental group of rat cardiac systolic functionand diastolic function was significantly impaired, were significantly lower heart rate, LVSPwas significantly lower LVEDP was significantly elevated±dp/dt max significantlydecreased (P <0.05), have different sex. Experimental myocardial tissue damage, myocardial fibers arranged in a wavy, myocardial cells showed a pathological kind of change. Electronmicroscope observation of the visible swelling of mitochondria in the rat model of heartfailure intracellular vacuoles, steep to reduce some of the myocardial fibers breakage,myocardial fibrosis.2. Bone marrow mesenchymal stem cell separation, culture and induced to differentiatePrimary cultured cells adherent for12h, the first exchange of medium for24h,adherent cells increased significantly after48h, and stretch for long spindle shape, about7dclose covered,3~4d can be passaged. Generation of MSCs is similar to the growth curve.MSCs cell specific markers CD44, cell surface antigen CD34negative.5-aza added to theculture medium to about8d about MSCs by flat polygonal into long fusiform, arrangedbetween the colony after15days with a directional, to differentiate into cardiomyocyte-likecells. Immunocytochemical staining show part culture cell TroponinT positive expression.5-aza-induced MSCs conversion of27%conversion rate of myocardial cells.3.Recombinant pcDNA3.1-of HO-1plasmid and its expression in MSCsBamH Ⅰ digested plasmid pcDNA3.1-GFP-HO-1is linearized; digested with Hind Ⅲdigestion under2998bp,5256bp two fragments, demonstrate the direction of the plasmidsand the correct size. G418selection by preliminary experiments to determine the screeningconcentration of0.4μg/ml. The use of fluorescence microscopy to observe GFP expression inGFP expression started24h after lipofection gradually weakened, show that HO-1also has acertain level of expression. After RT-PCR, and transfected with plasmidpcDNA3.1-GFP-HO-1available to a length of about809bp of the bands and a length tomatch the target gene. Western blotting analysis of transfected cells HO-1protein expressionwas positive.4.Experimental study of transfer of HO-1gene, transplantation of MSCs in the treatment ofchronic heart failureMSCs in BrdU incorporation24h after immunohistochemical staining, the nucleistained for the tan to prove the incorporation of BrdU in the nucleus, the labeling efficiencyof more than80%. After four weeks, four weeks after surgery, compared with the controlgroup, MSCs transplantation group, left ventricular end-systolic pressure, left ventricularejection fraction and left ventricular pressure, maximum rate of change were significantlyhigher (P <0.05), left ventricular diastolic the end pressure was significantly lower (P <0.01)compared with MSCs group, transfection of HO-1MSCs group left ventricular end systolicpressure, left ventricular ejection fraction and left ventricular pressure, maximum rate of change were significantly higher (P <0.05), left ventricular end diastolic pressure wassignificantly lower (P <0.01). The remaining indicators were not statistically significant (P>0.05). Four weeks CHF after transplant team can detect Brdu labeling-positive cells, controlthe group brdu immunization combined test results were negative.Conclusion:HO-1gene transfected MSCs in the treatment of chronic heart failure can improvecardiac function and provides a new method for the treatment of chronic heart failure.