| BackgroundBig organ transplantation is a national level of organ transplantation,The heart transplant is the crystallization of a number of highly precise technology collaboration medical field research, is also an important measure of a country’s level of organ transplantation. Someone predict, to the middle of the twentieth century, half of the surgery will be organ replacement. More than ten thousand people around the world have received heart transplant, and the number is rising at an annual rate of more than5000cases. Different individuals supply and receptor have histocompatibility of differences and the rejection is not the same after organ transplantation. The ideal immune inhibitor application strategy should be after organ transplantation according to different individuals "individualized use" rejection level of immunosuppressant, in small doses of immunosuppressive achieve satisfactory result of anti rejection, in order to reduce the side effects of drugs, to reduce the economic burden. There is no simple and noninvasive, high efficient and real-time method to monitoring of rejection, immunosuppressant is therefore difficult to do "individualized use", in order to achieve high efficiency, low toxic side effects of ideal state. Acute rejection is still one of the biggest obstacle to successful organ transplantation, reduce patients’ check of the pain after organ transplantation, reduce medical costs, reduce the side effects of drugs, improve the quality of survival is an important issue which we unable to avoid.Explore the molecular mechanisms of graft rejection reaction, and looking for efficient, rapid, specific and sensitive rejection monitoring method is a gre at significance. Homotransplant rejection is mediated by the activation of T cel Is immune response, intracellular calcium and relay information in T cell activ ation, proliferation and differentiation playing a key role. To explore T lympho cytes activated during the intracellular calcium and other related information of internal relations, And their correlation and heart transplant rejection, to furthe r clarify the molecular mechanism of acute rejection, to prove the new molec ular level monitoring of acute rejection monitoring method, for the same transp lant rejection of monitoring and find a new breakthrough in the prevention and control.Purpose1. Modified Ono’s classical abdominal heterotopic heart transplantation model, doing some improvement, and further explore the ideal model of heart transplantation, should be used for acute and chronic rejection mechanism, immune tolerance, immune rejection monitoring, resistance to heart transplant rejection drug screening of clinical and basic experiment research.2. This project from the CD8+CD28+T cells biological characteristics and the signal transduction direction to study the pathogenesis of immune rejection and T cell activation during the change of intracellular calcium ion concentration, extracellular radioactive calcium (45ca2+) associated with acute rejection (AR) of heart transplant, May is organ transplantation research new breakthrough. 3. From the molecular level study of acute rejection after heart transplantation, maybe find more, better and more clinical value of new drugs to treat the acute rejection; and can be prove acute rejection from the level of cell and molecular mechanism, looking for the early diagnosis of acute rejection for clinical find more simple and effective method.MethodThe abdominal heterotopic heart transplantation model in ratsUsing improved Ono’s classic abdominal heterotopic heart transplantation in rats model method, do preliminary experimental group40, redo80groups of formal experiment. Mainly includes:The donor and receptor anesthesiaâ†'Preoperative preparation of a receptorâ†'To get the transplant heartâ†'heart transplantationâ†'Postoperative observation.1. Heart transplant acute rejection and calcium ion concentration changes of CD8+CD28+T correlation studies1.1experimental group:T1group (normal group) in rats:SD rats (n=24), and then divided into four groups, each group of6; T2group (control group):SD rats (except no heart transplantation, other operating procedures are the same)(n=24) animal model, and then divided into four groups, each group of6; T3group (treatment):the same group, heart transplant (WISTAR-> SD rats) animal model (n=24), and then divided into four groups, each group of six.1.2T1, T2and T3groups were divided into postoperative day1,3,5,7,4groups, each group of six. Take blood groups of abdominal aorta in rats, and to be put to death in the rat, a transplant do pathological section, according to the AR classification standard (Stanfort standard), for grading. Specimens were numbered cells after the analysis and so on.1.3Comparative research of T1, T2and T3group rats before and after transplantation of CD8+CD28+T intracellular calcium ion concentration change rule, and these changes and acute rejection if there is a certain amount of activity relationship.1.4Statistical methods:all data are expressed as a mean+standard deviation (I+s), were applied spssl3.0statistical software for the control group, control group and model group variance analysis, such as the homogeneity of variance, analysis of variance, the variance is not neat, the line of nonparametric test. Heart transplantation group of CD8+CD28+T lymphocytes calcium content and AR nonparametric Spearman rank correlation analysis. Statistically significant different (P<0.05).2. Heart transplant acute rejection and CD8+CD28+T cell45Ca2+uptake rate the relevance of research2.1experimental group:T1group (normal SD rats group)24, then divided into4groups, each group of6; T2group (the same heart transplantation group, WISTAR-> SD rats)24, immediately after the success of the building is divided into four groups, each group of six.2.2CD8+CD28+T cells get:after aseptic conditions dao spleen, by immune magnetic beads points plus or minus two step separation to obtain CD8+CD28+T cells.2.3Determination of CD8+CD28+T cells:45Ca2+uptake rate by using glass fiber filter paper collection of CD8+CD28+T cells, after drying with liquid ShanYi determination of number of pulses per minute (CPM)2.4Statistical methods:the experimental group and control group the Mann-Whitney U test, inspection, the experimental group of CD8+CD28+T grading of extracellular calcium intake rate and AR rank correlation analysis. All data is to mean+/-standard deviation (X±SD) said, using SPSS13.0statistical software for analysis. Statistically significant different (P<0.05).Result1.The abdominal heterotopic heart transplantation in rats experiment40cases,21cases, survival rate of52.5%. Is the main cause of death in traumatic big, separate vessels operating rough to the great vessels rupture caused by shock of rats can’t survive, harvesting time is long for the heart and vascular anastomosis after long time heart cannot jump, shock rats can’t survive. After completion of preliminary experiment, summing up experience and lessons, and earnestly strengthen the perioperative treatment, formal experiment again, significantly reduce the surgical complications.80live70formal experiment,87.5%survival rate. And preliminary experimental contrast, formal experimental cold, warm ischemia time, heart jump time, abdominal aorta block time significantly reduced, considerably increase the success rate of surgery, the difference statistically significant (P<0.05).2.New heart pathological grading (see above, HE×400). AR classification with reference to the Stanford grading standard for AR. Level0:no rejection, myocardial tissue is normal, no myocardial interstitial mononuclear cell infiltration. Level1:is there a focal outside the blood vessels and myocardial interstitial mononuclear cell infiltrates, not with myocardial cell damage. Level2:single focal mononuclear cell infiltrates, yeah, myocardial cell damage. Level3:diffuse mononuclear cell infiltrates with damaged or myocardial necrosis. Level4:diffuse multi-type cell infiltration, obvious myocardial interstitial edema, hemorrhage and necrosis. Performed by pathology teaching and research section, southern medical university.3.1. The control group found a small amount of inflammatory cells infiltration,5days after operation was found myocardial edema, necrosis and acute rejection performance. Model group on postoperative day1found only focal inflammatory cells infiltration and yeah, myocardial cell necrosis,3days most rejection, visible diffuse multi-type cell infiltration, obvious myocardial interstitial edema, hemorrhage and necrosis, day5times, but still visible inflammatory cell infiltration, myocardial cell necrosis, reduce7days. Each time period in the model group group of CD8+CD28+T cells in calcium ion concentration were compared with control group, control group increased significantly (P<0.01), control group and the control group of CD8+CD28+T intracellular calcium ion concentration difference is no statistical significance (P>0.05). That postoperative CD8+CD28+T lymphocytes in calcium ion concentration is associated with postoperative acute heart transplant rejection, has nothing to do with surgical trauma. Spearman correlation analysis showed that CD8+CD28+T cells in the severity of the calcium ion concentration and AR into positive correlation, the correlation coefficient is0.358, significantly (P<0.05).4.Group3,5days postoperatively calcium intake rate [CR:T M M phi phi (+)/T (-)] is higher than control group, the difference was statistically significant (P <0.05).1,7days no statistically significant difference (P>0.05). Spearman correlation analysis showed that CD8+CD28+T cells, calcium intake rate and the severity of the AR into positive correlation, the correlation coefficient is0.428, significantly (P<0.05)Conclusion1.Ono abdominal heterotopic heart transplantation in rats method is a basic model of heart transplant, a heart transplant model to an ideal operation method is simple, short for heart ischemia time and postoperative stability conducive to long-term experimental observation for the standard model. Model of heterotopic heart transplantation in rats after so many years of practice and explore, already had a big improvement and innovation, but a lot of skills and the improvement of the theory still has great space. 2.Ca2+in the human body not only has important physiological functions, pathological reaction when its concentration in tissues or cells also can reflect the degree of the pathological changes of organism or cell, Ca2+cells as an important information material, has the function of regulating cell, participate in the signal across the membrane transfer and mediating cell an important role in response to external stimuli.3.Homo transplant rejection is the cell of T cell-mediated immune response, through activation of T cells by corresponding antigen in graft and release the cell active factor to produce the immune damage. T lymphocytes has a variety of regulating function, namely the auxiliary function, function and effect function, the function of normal play to depend on T cells from static state to activate the state. In the process of homograft acute rejection, activated T lymphocytes mediated cell immune response is cause damage to the immune rejection and lead to graft function key.4.This study found that heart transplantation in rats with acute rejection severity and CD8+CD28+T cells increased expression are positively related. CD8+CD28+T cells in the calcium ion concentration increased significantly with acute rejection after heart transplantation and increase has a close relationship, and increased with the increase of acute rejection. Detection of peripheral blood CD8+CD28+T cells within the calcium ion concentration, helps to make a diagnosis of acute rejection.5.This study found that the severity of heart transplantation in rats with acute rejection and extracellular calcium intake into positive correlation, CD8+CD28+T cells calcium intake rate by liquid scintillation counting instrument accurately, so this method is expected to become a clinical diagnosis of acute rejection is simple, effective method. |
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