| Objective:(1). To find new methods for the isolation, cultivation, and identification of endothelial progenitor cells (EPCs) from the marrow of minipig;(2). To set up an acute myocardial infarction model in minipig and observe the protective effect of Marrow-Derived EPCs in autograft transplantation;(3) To examine the ability of translocation, survival, and immigration of EPCs in vivo and in vitro using SPIO-labelling technology.Methods:(1). EPC culture in vitro: Collect the marrow from minipig and isolate the MNCs by Ficoll density-gradient centrifugation technology. The isolated cells were incubated with EPC culture medium containing recombinant VEGF and bFGF. Proliferated cells were labeled antibodies for the detection of CD31, CD133, Flk-1, and VIII factor by using flow cytometry (FACS); The isolated EPCs were identified by using Western blot analysis, confocal microscopy, and transpective? EM technologies.(2). SPIO-labeling in vitro: EPCs were labeled with different concentrations of SPIO. Find an optional incubation time and concentration for SPIO labeling; observe the labeling rate in EPCs as well as the effect of SPIO on the cell growth; determine the optional labeling time for MR image.(3). Set up the acute myocardial infarction model in minipig: 27 Chinese minipig (30-35 kg) were prepared for interventional operation. Sacculus was used to block the left anterior descending artery for 90 min to make acute myocardial ischemia/reperfusion injury. ECG was monitored during the operation and infarct area and heart functions were determined by 2-D ultrasound before and after the surgery.(4). Interventional autograft transplantation of EPCs through coronary artery: 15 minipig with myocardial ischemia/reperfusion injury was randomly divided into 3 groups: control group (A group, n=3), EPCs group B1 (EPCs treatment on the 1h of surgery, n=6), and EPCs group B2 (EPCs treatment on the 2w of surgery, n=6). Proliferated EPCs in vitro (labeled with SPIO) was sent to infarct area through coronary artery.(5). Detection of cellular edema and Fe ion with Clinical high field density 3T MR machine and specific MR sequences (T2-weighted TrueFISP Imaging of Myocardium and CV-3DRAD-400C, respectively, provided by Simon Company): The image from this MR machine is specific for the determination of myocardial infarct area and stem cells containing Fe ion. Animals in control group were scanned by 3T MR machine at the same day, 1 week, 2 week, 3 week, and 4 week of the surgery. The animals in EPCs groups were scanned at the same day, 1 week, 2 week, 3 week, and 4 week of the surgery or EPCs autograft transplantation. The left heart function was examined by ultrasound before and after EPCs autograft transplantation. The results from three groups were compared and infarct area and heart functions were monitored at different time points. The translocation, survival, and immigration of transplanted EPCs were analyzed from the scanned images. According to MR images and ultrasound results, the protective effect of transplantation was evaluated.(6) Biochemical parameter determination: serum myocardial enzymes and cardiac troponin I were monitored before the artery was blocked by sacculus or 30 min, 1 h, 2 h, and 4 h after the artery was blocked. The correlation between biochemical parameters and results from EEG, ultrasound, and 3T MR machine was analyzed.(7). Histological examination: the survival and immigration of SPIO-labeled cells were examined by the heart tissue sections.Results:(1). The cultured cells were identified as EPCs from bone marrow by multiple methods (morphology, protein expression, and functions). Weibel-Palade bodies were observed under transpective EM. Cell surface protein Flk-1 reached peak concentration at 14 day after induction by Western blot analysis. The results from flow cytometry demonstrated that the percentages of cells expressing CD31, CD133, and Flk-1 were 83.86%, 85.34%, 83.52%. The percentages were higher under confocal microscopy (about 95%-98%).(2). The optional concentration of SPIO for EPC labeling was 20～50μgFe/ml. Biological activities of EPCs were affected when SPIO concentration was higher than 50μgFe/ml. The optional incubation time was 24 h for SPIO and EPCs. The labeling effect can keep more than 4 weeks.(3). Eleven minipig died during or after the surgery because of heart failure in 26 animals. Ultrasound results demonstrated that EPCs transplantation improved the heart functions after 4 weeks of transplantation compared control group. However, there is no difference between two EPCs groups (B1 and B2, P>0.05).(4). MR image scanning indicated that EPCs transplantation effectively restored the heart functions and reduced infarct area by using the specific MR sequences.(5). After the left anterior descending artery was blocked by sacculus, typical myocardial infarction changes were observed in ECG . Serum myocardial enzymes and cardiac troponin I levels began to increase at 1 h after the ischemia and reach the peak at 4 h. MR images were more sensitive to myocardial infarction than serum myocardial enzymes. In addition, MR images were stable and clear compared with Ultrasonic cardiography(UCG).(6). Pathology examination indicated that EPC transplantation improved the heart function, reduced the infact volume, due to the formation of new vessels.Conclusion:(1). Combination of density gradient centrifugation and adherence screening methods can make pure EPCs isolation easier. Those cells that did not adhere to walls after culture for 24 h can be induced into pure mature functional EPCs.(2). SPIO can effectively label the transplanted EPCs.(3). MR machine and its specific sequences can clearly demonstrate myocardial infarct area and SPIO-labeled EPCs in the heart and trace the location, suvival, and immigration of EPCs.(4). EPC transplantation can contribute to the formation of new vessels in the ischemia area, reduce the infarct volume, and improve heart functions and prognosis. |
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