| Objective: Myocardial infarction(MI) pose a serious threat to humanhealth. Ventricular remodeling of MI has seriously affected the quality of lifein patients. The cardiac dysfunction of MI has become the main cause of death.At present, despite the mortality rate of MI is decline, however, the cardiacdysfunction of MI which is a heavy burden on family and society. Cardiacdysfunction is the end stage of various heart diseases. Medication can alleviatethe condition, but cannot make the damaged myocardial cells regeneration. Tomake the myocardial cells regeneration and even to recovery the cardiacfunction remains a huge challenge. Stem cells therapy for end-stage heartimpairment is the most promising treatment. Mesenchymal stem cells (MSCs)exist in a variety of adult tissues, bone marrow is the main source ofMSCs.Bone marrow mesenchymal stem cells (BMSCs) can participate in thereconstruction of damaged myocardium, can increase the left ventricularejection fraction, can decline the left ventricular end-systolic and diastolicvolume,can reduce the infarct size and can improve left ventricularremodeling. The changes of MI microenvironment make the BMSCs in theinfracted myocardium can not be long-term survival which limits the stemcell’s ability to repair. People try to solve this problem by genetic engineeringmethod. Genetic engineering is put the exogenous gene into the target cellswhich have the ability to over express or silence a gene. Modified stem cellshave a better angiogenesis ability, anti-apoptotic, anti-inflammatory andimprove cardiac dysfunction. Exogenous gene depends on the viral ornon-viral vector system to get into the target cells. Non-viral vectors are lowtransduction efficiency. The target gene can only achieve transient expression,easy to trigger an immune response and was cleared by the body. In this study, Adenovirus transfected BMSCs have a highly transfect efficiency andnon-toxic side effects. Bcl-2is a significant apoptosis gene. Bcl-2modifiedBMSCs to be used as a means to improve the survival rate of transplantedcells. The purpose of this study is to observe the effects of transplantation ofallogeneic BMSCs modified by Bcl-2on cardiac dysfunction in rabbits aftermyocardial infarction.Methods: At first, prepared for the original plasmid which includes thetarget gene of Bcl-2. Second, got the target gene from the original plasmidthen connected it to the pShuttle-CMV-EGFP recombinant shuttle vector.Third, we transferred the pShuttle-CMV-EGFP-Bcl-2to the pAdeno carrier.Finally, we got the pAdeno-Bcl-2virus plasmid. We prepared for a lot ofrecombinant plasmid, then transfected to293cells, at last collected virus(passage one of viruses). Used the adenovirus of passage one to infection to293cells, collect ed the virus which repeatedly amplified three generations,used passage four to large amplification of virus. After repeated freezing andthawing, the adenovirus was stored at-80℃for later use. We acquired tibias’bone marrow of a3-month-old rabbit and cultured bone marrowmesenchymal stem cells by density gradient centrifugation and adherent untilto the ninth generation. BMSCs were transfected by empty adenovirus vectorlabeled with enhanced green fluorescent protein (EGFP) or by adenovirusvector labeled with enhanced green fluorescent protein (EGFP) and modifiedBcl-2gene. Cardiac insufficiency models were made by ligating the leftanterior descending artery. Two weeks later the successful rabbit models wereselected according to echocardiography (LVEF≤50%), randomly dividedthem into Ad-EGFP-Bcl-2-BMSCs group (n=6), Ad-EGFP-BMSCs group (n=6), and DMEM group (n=6). Thoracotomy for cell transplantation,Ad-EGFP-Bcl-2group transplanted Ad-EGFP-Bcl-2-BMSCs, Ad-EGFPgroup transplanted Ad-EGFP-BMSCs, DMEM group were injected withDMEM. The cardiac function was detected by echocardiography at four weeksafter cells transplantation, including LVEF, FS, LVEDD and LVESD. Fourweeks after BMSCs transplantation, the mRNA of Bcl-2in the infarct border zone of myocardial was determined by real time quantitative PCR. Weanalyzed the correlation between hearts function and Bcl-2expression.Myocardial pathological changes were assessed by hematoxylin-eosin (H-E)staining. The survival of labeled BMSCs was observed by fluorescencemicroscope.Results:1Titer of Ad-Bcl2-GFP adenovirus was2.5×1010PFU/ml;2BMSCs separated and cultured in vitro successfully by density gradientcentrifugation and adherent, the state of cells of passage twelve was still good;3This experiment used the ninth passage of BMSCs, the best MOI was500,the expression of EGFP was observed after infection about36h, the transfectefficiency of adenovirus was100%;4The cardiac insufficiency of rabbitmodels after acute myocardial infarction was prepared successfully. In total,the models survived21. Two weeks later according to the results ofechocardiography and the criteria of success model (LVEF≤50%), a total ofsuccessful model was18.5Ad-EGFP-Bcl-2-BMSCs group andAd-EGFP-BMSCs group were better than DMEM group; theAd-EGFP-Bcl-2-BMSCs group was the best. EF[（68.222±3.763）%,(57.056±3.555)%,(47.389±5.567)%,P=0.000],EDD[（9.556±0.981）mm，(11.611±0.828)mm,(13.389±1.569)mm, P=0.000],ESD[（5.722±0.647）mm,(7.889±0.544)mm,(9.889±0.749)mm，P=0.000], FS[（39.167±3.817）%,(31.833±2.492)%,(25.778±3.851)%，P=0.000].6Bcl-2mRNA was higher inBcl-2group （1.015±0.115,0.358±0.057,0.090±0.065，P=0.000). All of thedifferences were statistically significant;7EF and Bcl-2expression arepositive correlation, the correlation coefficient (R)was0.940, the coefficientof determination(R2)was0.883, regression equation and all coefficients werestatistically significant, the regression equation wasY=46.664+22.329X;8The survival rate of transplanted stem cells was improved, in the fluorescencemicroscope, the number of BMSCs in Ad-EGFP-Bcl-2group was1.5timesof Ad-EGFP group;9Ad-EGFP-Bcl-2group and Ad-EGFP group arecompared with DMEM group, the pathological changes were lighter, bettereffect in Ad-EGFP-Bcl-2group. Conclusion: Mesenchymal stem cells are easily separaed and massculture, easily transfect by exogenous gene and stable expression,non-immunogenic among the same species. In this study, we use adenovirus,which have a highly transfect efficiency and non-toxic side effects, transfectinto BMSCs and transplantation the Bcl-2modified BMSCs can significantlyimprove cardiac dysfunction in cardiac dysfunction rabbits models. Thepossible mechanisms is to inhibit cells apoptosis and improve the survival rateof transplanted stem cells. |
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