| Carrell and Guthrie performed the first animal experiment of heart transplantation in 1905. The heart beated successfully about 90 minutes after transplantation, and then opened the investigative prologue in heart transplantation. Doctor Bernard of South African performed the first heart transplantation in human, the heart comeback to beat successfully after the operation. The technique of heart transplantation is almost perfect now. Heart transplantation is the most effective method for treatment of severe end-stage heart diseases. With the development of modern medicine, immunosuppressive drugs and operative technology, more and more patients with end-stage heart failure or very complicated congenital heart diseases want to receive heart transplantation in order to improve their own quality of life. However, up to now, there are still many problems such as ischemia /reperfusion injury, acute or chronic graft rejection, cardiac allograft vasculopathy, heart preservation and myocardium protection etc. They need to be further investigated in this field.Professor Kerr put forward a concept of cell apoptosis firstly in 1972. He drew the cell apoptosis that like the leaf or flowerpetals drop. Cell apoptosis is a kind of basic form of cell death. Beranek believed that the cell apoptosis is a main mechanism of cell death in rejection after heart transplantation. It involves the expression of promoting or repressing gene (such as Bax, Bcl-2). More and more experiments proved that myocardial cell apoptosis occured after heart transplantation. The ischemia/reperfusion maybe the main reason for myocardial cell apoptosis. The myocardial cell apoptosis lead to decrease of myocardial cell, and lead to heart function decline. It influences the recipient life quality and survival time too. How to repress the cell apoptosis need to be researched. In the research process of apoptosis mechanism, It is discovered that oxygen free radicals maybe the main factor. Oxygen free radicals lead to cell membrane over oxygenation, and then change the convertibility of cell membrane, make calcium inside the cell overloaded, and result in cell apoptosis.To study the heart transplantation, a model of animal experiment should be established. Based on other kinds of big rats heart transplantation model, we established the heart transplantation experiment model by cuff technique. With this model, we studied the influence factors with heart transplantation, researched the related gene expression in cell apoptosis, and studied the influence of ginko biloba extract (GBE) to the cell apoptosis after heart transplantation. We resected the transplanted heart in different time, and observed histopathological changes. Myocardial cell apoptosis index (AI) is identified with in situ terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Protein products of Bcl-2, Bax, Fas and Fasl were assessed by immunohistochemistry.Material and methodsOne. To establish the transplantation modelA rat heterotopic cervical heart transplatation model is established by cuff technique in this experiment study. Pure grade SD (Sprague- Dawley) rats served as recipient, Wistar rats served as donor, the weight is 250-300 grams. They are provided by Shanghai experimental animal center and Shandong University experimental animal center respectively. The weight of the donor can be lighter than recipient.Preparing recipient firstly and then preparing donor, the heart of donor is transplanted into the neck of recipient. The donor brachiocephalic truck (innominate) is anastomosed to the recipient right common carotid artery, and the donor pulmonary artery is anastomosed to the recipient right external jugular vein.Two. The detection of the myocardial cell apoptosis index and the expression of the related geneSD rats were served as recipient, and Wistar rats were served as donor. The rats were divided into five groups: 1st, 3rd, 5th, 7th, 9th groups. Took off the transplanted heart at the day in 1st, 3rd, 5th, 7th, 9th after heart transplantation, cut off the left ventricle wall tissue, and then dipped it in 10% formalin. Cell apoptosis of donor hearts were identified by TUNEL method. Protein products of Bcl-2, Bax, Fas and Fasl were assessed by immunohistochemistry.Three.The effect of GBE to myocardial cell AIThe SD rats were served as recipient, and the Wistar rats were served as donor. The animals were divided into three groups: control group, acute rejection group and GBE group. GBE group rats were injected GBE into donor aorta before resecting donor heart, and recipients were injected GBE into vein. The control group did notdo heart transplantation. The acute rejection group did not do any treatment before transplantation. To resect the transplanted heart 4 hours after transplantation, to examine the myocardial cell apoptosis index with flow cytmetry (FCM) and detect MDA, SOD. The effect of GBE to protect transplanted heart was investigated.Results1. The situation of heart beating after transplantationThe rate of reverting to beat is 95% after heart transplantation. Heart beating grades are descending gradually along with the increment of days after heart transplantation. Experiment group heart beating grades on 1st, 3rd, 5th, 7th, 9th day are 3. 6 ± 0. 43, 3. 1 10. 51, 2. 6 ±- 0. 33, 2. 1 ± 0. 26, 0. 7 ± 0. 23 respectively.2.The pathologic changing of acute rejected donor heart:We observed that the donor hearts are larger and softer. In the microscopy, the donor myocardium cells are edema slightly. Monocyt can be found in the interstitial. Slight necrosis of myocardium cells, interstitial edema, Lymphocytes and neutrophilic cells infiltrating are found in middle grade injury. Hypertrophy, bleeding, fibrinoid necrosis and myocardial fibers breaking were seen in severe grade injury.The pathologic injury grade of 3rd is worse than the first day, and in the 5th, 9th, it is worse too. Myocardial histological changes were seen on the 1st day after the transplantation and the histological grade of the myocardial injury increased with the time prolongation after transplantation.3. The changing of myocardial cell Apoptosis index in diff irent time after heart transplantationThe AI of Myocardial cell on 1st, 3rd, 5th, 7th, 9th day are 2.39 + 0.53, 5.86 + 1.48, 8.30 + 2.67, 11.75 + 3.76, 9.60 + 3.32respectively. Myocardial cell apoptosis were seen on the 1st day after the transplantation. The AI reached its peak on the 7th day and then began to gradually decrease. The AI of control group is lower than acute rejection group.4. The expression of Bax after heart transplantationThe expression of Bax was observed at different time phases after transplantation. They are located in cell plasma of myocardial cell, blood vessel endothelial cell and smooth muscle cell. The increasing of apoptotic cells was found upon high expression of Bax.5. The expression of bcl-2 after heart transplantationThe positive immunohistochemistry stain of bcl-2 is brownness. They are located in cell plasma of myocardial cell, blood vessel endothelial cell and smooth muscle cell. The expression of bcl-2 is detected at 1st day after transplantation and then keeped a higher level. In the same slices, the expression of bcl-2 is contrast to cell apoptosis.6. The expression of Fas after heart transplantationThe expression of Fas is located in plasma of myocardial cell. It expressed in the normal myocardial cell. Increased expression of fas was observed in transplanted heart and it keeped a higher level in observing period.7. The expression of Fasl after heart transplantationThe expression of Fasl is not found in the normal myocardial cell. It is located in interstitial and infiltrating cell mostly. The expression of Fasl increased and got a higher level along with the increment of day after transplantation and got peak on 5th day. The myocardium pathologic injury and myocardial cell apoptosis are related to the expression of Fasl.8. The content of serum SODThe recipient serum SOD content of control group, acute rejection group, GBE groups are 92. 25 ± 6.86, 57. 37 ± 5.92 . and 86.41 ±6.46 U/L respectively. The content of acute rejection is lower than GBE group and control group (P<0. 05). The control group is as high as GBE group (P>0. 05).9. The content of myocardial MDAMyocardial MDA content of control group, acute rejection group, GBE groups are 48. 52 ± 4. 51, 125. 75 ± 10. 95 and 55. 49 ± 7. 74 nmol/g respectively. The content of acute rejection is higher than GBE group and control group (P<0. 05). The control group is as high as GBE group (P>0. 05).10. Myocardial cell apoptosis indexDonor heart was harvested at 4 hour after transplantation and AI was detected by FCM. The results of control group, acute rejection group, GBE group are 0. 57±0. 14, 6. 21 ±2. 00, 1. 06±0. 28? respectively. The AI of acute rejection group is higher than GBE group and control group (P<0. 05). The AI of GBE group is as high as control group (P>0. 05).Conclusions1. The model of cervical heterotopic heart transplantation has been successfully established with cuff technique.2. After transplantation, significant pathological changes of acute rejection occured in the donor myocardium.3. AI is positively correlated with histological grade of myocardial injury. The AI reached its peak on the 7th day after transplantation and then gradually decreased.4. The expression of Fasl is not deteceted in normal myocardium. It increases after heart transplantation and the positive cells are...
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