| Objective: To investigate the effects of bone marrow mesenchymal stem cells(BMSCs) modified by antiapoptosis gene(Bcl-2) transplantation on cardiocyte apoptosis, angiogenesis and cardiac function in ischemic cardiac dysfunction rabbits and discuss the possible mechanisms.Methods: The isolation, culture, purification of BMSCs: The rabbit bone marrow which contained BMSCs was obtained by puncture, and plenty of purified BMSCs were acquired by density gradient centrifugation and adherent in vitro. The construction of recombinant adenovirus vectors: The target gene(Bcl-2) from the original plasmid was obtained by enzyme digestion method, and then it was connected to the p Shuttle-CMV-EGFP recombinant shuttle vector. The p Shuttle-CMV-EGFP-Bcl-2 was transfered to the p Adeno carrier, and the recombinant adenovirus plasmid was obtained which contained bcl-2 gene. Then we used the recombinant plasmid to transfect the 293 cells, collected the virus and amplified them. After repeated freezing and thawing, the cells were stored at-80 ℃ for later use. Before cell transfection, the recombinant adenovirus were thawed, resuscitated and then the titer of recombinant adenovirus was calculated. Cell transfection: The BMSCs which had a good growing status were selected, and then they were transfected with adenovirus or adenovirus-Bcl-2 respectively, both of the adenovirus vectors were labeled with enhanced green fluorescent protein(EGFP). Models preparation and cell transplantation:Totally twenty-eight New Zealand rabbits were randomly divided into the following three groups: MI+BMSCs+Bcl-2 group, MI+BMSCs group and MI+DMEM group. The rabbit myocardial infarction(MI) model was established by the ligation of left anterior descending artery(LAD). Two weeks later, the successful rabbit models were screened out by echocardiograph(standard for the cardiac insufficiency: LVEF≤50%). Two weeks after ligation, the rabbits were injected with Ad-Bcl-2-BMSCs(MI+BMSCs+Bcl-2 group), Ad-BMSCs(MI+BMSCs group) and DMEM(MI+DMEM group) in infarction marginal zone respectively. Indicators testing:The cardiac function was measured by echocardiography four weeks after cell transplantation:including left ventricle ejection fraction, left ventricle fractional shortening, left ventricle end- diastolic diameter and left ventricle end-systolic diameter. Myocardial tissue of in both normal and infarction marginal zone was cut into small pieces, then paraffin and frozen sections were made. The survival and distribution of the transplanted cells were observed by fluorescence microscope. The myocardial pathological changes were assessed by the H-E staining, the apoptosis of cardiocyte was measured by TUNEL, the expression of CD31 was examined by immunohistochemical staining, and newly born capillaries were counted, the gene expression level of VEGF was detected by RT-PCR. The correlation of the above values with cardiac function was analyzed respectively.Results:1 The isolation and culture of BMSCs: The bone marrow liquid was separated by density gradient centrifugation in percoll, and then the lacte floccule layer was visible. After repeatly changing the liquid and the adherent culture, the purified BMSCs were obtained. After culturing for twenty-four to forty-eight hours, the spindle powder BMSCs were observed in the wall. Seven to ten days later, the morphology of BMSCs was like a long shuttle, tubers could be seen between cells, the nuclei were in the middle and the arrangement of cells was loosely. Eighteen to twenty-one days latter, the fusion of bunchy adherent monolayer cell were visibled, with closely packed as shape of spiral or radial. BMSCs were digested by trypsin, and then were subcultured with the proportion of 1:2. Cells were completely adherent after twenty-four hours, and the adherent cells were grown into 80% confluence after three to five days. The growth speed of the cells from the tenth generation was slowed down gradually.2 A large number of recombinant adenovirus vectors were constructedsuccessfully in our experiment. The 293 cells were infected by differentdiluted gradient adenovirus respectively, after observing the rate of cellchanges, eventually the titer of virus was 2.5×1010 pfu/ml.3 Cells status after transfection: We selected the ninth generation of BMSCs which had a good growing status. Twenty-four to forty-eight hours after virus transfection, the BMSCs with green fluorescent protein markers(EGFP) could be observed by fluorescence microscope, and the cells were arranged orderly and the morphy was uniform as long fusiform, according to the result of preliminary experiments, the transfection efficiency was best when the MOI was 500.4 Model establishment and animal survival: A total of twenty-eight New Zealand rabbits were achived the surgery, and twenty-four rabbits were survived, and the LVEF were≤ 50% mearsured by echocardiography. Among them there were eight rabbits alived and one death in MI+BMSCs+Bcl-2 group; and eight rabbits were survived, two were died in MI+BMSCs group; while eight rabbits were survived, one was died in MI+DMEM group. Therefore there are four rabbits were died totally, among them, two rabbits were died for ventricular fibrillation in the process of anterior descending ligation, one was died for respiratory arrest after operation, only one was died within forty-eight hours after secondary thoracotomy for cell transplantation.5 The survival situation of BMSCs was observed by fluorescence microscope: Four weeks after BMSCs transplantation, the green fluorescent could be observed in the cells of both MI+BMSCs+Bcl-2 group and MI+BMSCs group by the fluorescence microscope, but not in the MI+DMEM group.6 The myocardial pathological changes were observed by microscope: The morphology of normal myocardial cell was neat and uniform. The cytoplasme was stained into light blue evenly, the form of nuclears was consistent with the shape of round or oval, and located in the central of cytoplasm. While the arrangement of cardiocytes in the edge area was disordered, the myocardial fiber was ruptured partly, and many kinds of inflammatory cells were infiltrated in the intercellular space. The normal morphology of cardiocyte in infarcted area was disappeared which were replaced by fibroblasts and thick collagen bundles. The arrangement of them was loosely, and no inflammatory cell was infiltrated. Compared the myocardial tissue in the edge area of infarction with the normal area, we chould found that the pathological changes in the BMSCs+MI+Bcl-2 group were slighter, the MI+BMSCs group were more seriously, and the MI+DMEM group were the worst.7 The level of cardiocyte apoptosis rate, neovascular density and VEGF m RNA gene expression quantity in infarction marginal zone: The apoptosis rate in the MI+BMSCs+Bcl-2 group and the MI+BMSCs group were lower than the MI+DMEM group, while the former group decreased more significantly[(30.75±5.65)%,(44.14±5.99)%,(63.38±8.52)%,F=46.00,P<0.05]; Both the capillary density and VEGF m RNA expression level in MI+BMSCs+Bcl-2 group were higher than MI+BMSCs group, and the latter group was significantly higher than the MI+DMEM group(capillary density :24.25±3.46,14.38±2.92,6.88±2.03,F=74.07,P<0.05;RQ:1.64±0.29,0.99±0.17,0.64±0.19,F=40.56,P<0.05).8 Cardiac function parameters were measured by echocardiography four weeks after transplantation: Compared with the MI+DMEM group, the LVEF and LVFS in both the MI+BMSCs+Bcl-2 group and the MI+BMSCs group were higher, and the LVEDD and LVESD were lower, among them the former g r o u p c h a n g e d m o r e o b v i o u s l y [ LV E F :( 6 6. 6 3 ± 2. 6 7) %,( 6 0. 5 0 ± 3.16)%,(55.25±2.60)%,F=32.532,P<0.05;LVFS:(40.50±1.60)%,(34.63 ±2.33)%,(27.25±2.05)%, F=86.736,P<0.05; LVEDD:(10.13±0.64)mm,(12.07±0.59)mm,(13.93±0.51)mm,F=85.225,P<0.05;LVESD(6.61 ±0.35)mm,(7.02±0.26)mm,(8.20±0.49)mm,F=37.697,P<0.05].9 The results of correlation analysis:The LVEF had a negative correlation with the cardiocyte apoptosis rate(r=-0.769,P<0.01,Y=76.091- 0.36X), at the same time it had a positive correlation with VEGF m RNA expression level and the capillary density(r=0.85,P<0.01,Y=47.726+0.776 X, r =0.741, P<0.01, Y=47.484+11.047X).Conclusion: The transplantation of BMSCs modified by Bcl-2 gene can significantly reduce the cardiocyte apoptosis, promote angiogenesis, improve cardiac function of the rabbits. The mechanism may be related to the antiapoptosis effect of Bcl-2 gene and paracrine effect of stem cells which induce to the improving in microcirculation. |
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