The Value Of Peripheral Blood Inter-alpha-trypsin Inhibitorsin The Chronic Rejection Of Rat Transplant Kidney

BackgroundAlong with the develop of medical science and technology, the field of organ transplantation, tissue typing technology has madethe leap-forward development and new immunosuppressive agents has been used in clinical,kidney transplantion has became the mainly method in treatment of uremia.Human/Graft survival rates are increasing year by year, the rate of early acute rejection was significantly decreased.and short-term renal allograft survival has been significantly improved, but renal transplantation long-term survival have not been significantly improved.lt contains immune factors and non-immune factors, the immune factors play a very important role in chronic rejection,which affect graft long-term survival, chronic rejection is still a long main problem in the transplant patients.and the balance of donor and kidney recipient is also a perfect goal.So far,Transplant kidney biopsy is still the gold standard for diagnosising chronic rejection.But it belongs to invasive procedures, there are risks of lead to different complications,even affect the transplant kidney function, and the diagnosis of transplant renal biopsy sometimes in error, or lack of procedural renal biopsy.Donor renal transplantation hard to distinguish between chronic rejection and CsA or FK506nephrotoxicity, make the diagnosis of chronic rejection became more complex, and it always take3-5days to diagnosis.Therefore,to investigate of a sensitive and specific non-invasive and objective indicators are great importance to diagnosis and prediction of chronic rejection, in order to intervention in the early of this disease progresses, and develop individualized immunosuppressive program.Inter-alpha-trypsin inhibitors.abbreviated ITI.belongs to a family of structurally related serine protease inhibitors found at relatively high concentrations (400to800mg/liter) in human plasma.ITI plays an important role in inflammation, wound healing and tumor metastasis. The levels of ITI decrease in the plasma of patients with sepsis, necrotizing colitis, and the margin of reducing is relate to the mortality.Renal allograft chronic rejection, whether from the perspective of tissue morphology or cytokine analysis, there are inflammatory cell infiltration, a lot of inflammatory factors release, and to play a very important role in the immune response of chronic rejection.The extensive literature confirmed that ITI can be used to predict the activity strength of the body’s immune, in clinical or animal studies, it has become an important predictor of sepsis, ulcerative colitis, cancer and other diseases, but also a large number of facts to prove it as a treatment of disease, in view of the ITI can predict the strength of in vivo immune response,so we decide to detec the ITI level in plasma of rat chronic rejection, control with normal kidney transplantation group, to investigate the predict effect of ITI in chronic rejection.ObjectiveThe purpose of this experiment is observed inter-alpha trypsin inhibitors dynamic expression in renal transplant recipients with chronic rejection and normal renal transpalnt, and compare to biopsy,to decide the ITI can or not as a forecast index in chronic renal allograft rejection,and make a further study.Methods 1.Modeling the SD-Wistar rat renal transplantationAfter a certain time of the pre-experiment, to familiarize with the microsurgical technique and to establish a stable renal transplant model.In formal experimental stage,we select SD rats and Wistar rats as donor and recipient, the use left kidney of SD transplant to Wistar rat in situ.After anesthesia,separating the left renal artery, vein, part of the abdominal aorta and the ureter, perfusion of the abdominal aorta use4℃C Ringer lactate solution (containing heparin25U/ml),make the color of kidney became homogeneous soilyellow.renal venous effluent was clear, then cut the graft kidney, and save the kidney in4℃C Ringer lactate solution.Disconnecting of recipient left renal artery, vein and ureter.cut down the left kidney from the recipient,end-to-end coincide the artery, renal vein and ureter from donor and recipient.Retention the right kidney,as a control of the transplanted kidney sample.Receptors of the chronic rejection group are given intraperitoneal injection of cyclosporin A micro-emulsifier(2mg/kg, Daily1)three days before transplant to suppress acute rejection, while the normal renal transplantation group were treated with cyclosporine A micro-emulsifier intraperitoneal injection (5mg/kg, daily).Once intramuscular injection of penicillin50mg/kg after surgery to prevent infection. At4weeks,6weeks,8weeks,10weeks,12weeks after transplant,rapid remove the transplanted kidney, fixed with10%neutral formalin,and make HE, PASM and Masson staining,observation of histopathological changes by microscopy, to judgment whether chronic rejection or not.And get blood samples for the next detection at preoperative and4weeks,6weeks,8weeks,10weeks,12weeks after transplant. Randomly selected two groups of rats to detect CsA valley concentration.2.Rat chronic rejection histopathology and detecting the Treg cells.SD rats as donors, Wistar rats as receptors, transplantation the left kidney to Wistar in situ. Retained right kidney as a control for transplant kidney. All receivers are inject penicillin50mg/kg to prevent infection after operation.57of60recipients survived (this number includes the preoperative rats,caused by non-experimental design, the death rats add to60according to the experimental requirements), a total of60renal transplant survival receptor and preoperative rats were randomly divided into normal renal transplantation (control group, n=30), chronic rejection group (experimental group, n=30).Receptors of the chronic rejection group are given intraperitoneal injection of cyclosporin A micro-emulsifier(2mg/kg, Daily l)three days before transplant to suppress acute rejection, while the normal renal transplantation group were treated with cyclosporine A micro-emulsifier intraperitoneal injection (5mg/kg, daily). At4weeks,6weeks,8weeks,10weeks,12weeks after transplant,rapid remove the transplanted kidney, fixed with10%neutral formalin,and make HE,PASM and Masson staining,observation of histopathological changes by microscopy, to judgment whether chronic rejection or not according to Banff.The most significant stage of the histopathological changes, taking the blood samples, and detect the proportion of Treg cells in lymphocytes by flow cytometry, then analyzed statistically between chronic rejection and Treg cells.and make a investigation.3.Detection of ITI and investigate the effect in chronic rejection of rat kidney transplantTaken to blood samples from two groups of rats before surgery and4,6,8,10,12weeks after operation, collected plasma by centrifugation,then preparation the plasma protein, the steps as follows:separation of plastic and laminatedplastic, make offset,Protein denaturation:Prepare the protein sample, diluted with an equal volume of2×Sample Buffer, cooked in boiling water for10min; plus samples:adding20-40μl sample each well; electrophoresis:electrophoresis45min placed in the electrophoresis tank at a constant current of90mA; remove the plastic sheet, and repaired plastic by cutters;immersion the glue on the transfer membrane liquid.Placed these substances as follows:Black face→sponge→filter→plastic→NC membrane→filter (use a straw to remove the air bubbles)→sponge; placed on the transfer film slot, black face to black face, add ice and the transfer membrane liquid.Installed electrodes under constant voltage100V for90min.Stained membrane with Ponceau,washed away Ponceau with TBST, closed:by adding5%nonfat dry milk and TBST.shaked1h at room temperature.recycling blocking solution, adding Anti-Alpha1microglobulin antibody;shaking overnight at4℃; recovery antibody.TBST washing the membrane for10min three times.add HRP goat anti-mouse IgG,shake1h at room temperature.recovery of this antibody,wash the film with TBST for lOmin three times.the membrane soak for about1min in light-emitting liquid,spreaded the membrance in the exposure box.exposure in darkroom,then wash the film.Results1. The CsA concentration of chronic rejection group is101.17±12.17ng/ml, normal renal transplantation group is171.33±18.49ng/ml (F=2.164, P=0.159).Pre-experimental stage had used80rats.SD and Wistar were40rats respectively.Pre-experimental stage focuses on familiar with the microscopy operation and mastery and stable model of rat renal transplantation in the pre-experimental stage, observed for at least12weeks.Rats were recorded survival status and a variety of postoperative complications (Table1-2).Entered the experimental stage after more than two months microscopy skills and modeling training.In order to facilitate grouping and taking into account the rats may be some reasons of the unpredictability result in death, appropriate to expand the sample size (include the death because non-experimental treatment).The total of60pairs in formal experiments.in which the SD, Wistarthe mice were60,recorded survival status as well as a variety of postoperative complications (including mortality in rats represent the rats were not sacrificed according to experimental plan)(Table1-2).In a formal experimental stage,renal warm ischemia time was6S to12S, cold ischemic time was50min to80min, donor surgery taked to1to1.5h, the receptors in kidney transplant time were1.5to2h.2. Normal renal transplantation group:We can saw graft size were appearance and full, tough quality, renal surface were bright red,compare to the right kidney and the graft every time after transplant, the size、shape、texture were no significant difference, except some red cells infiltration but no other obvious change in pathology(Figure2-13).Chronic renal allograft rejection group:along with the time after transplant, chronic rejection changes were gradually presenting, at12weeks after transplant,kidneys were survived, compare to the right kidney, the volume were significantly reduced, color is pale. Further lesions in microscopic, in addition to plasma cells,monocyte,lymphocyte infiltration, the glomerular basement membrane were became thicken, extensive glomerular sclerosis, atrophy and fibrosis, inflammation, atrophy tubular, arterial intimal were thicken, luminal stenosis result in occlusion, they were showed the typical pathological changes of chronic rejection (Figure14-Figure16), especially in small arteries endometrial fibroblast-like thickening,the was characteristic histological change. The right kidney had no any histological change.3.The proportion of CD4+CD25+Treg cells at12weeks after renal transplantation was significant different between normal renal transplantation group and the chronic rejection group(t=6.155, df=8, P=0.000), and CD4+FoxP3+Treg cells in the two groups was also significant differences (t=4.153, df=8, P= 0.003).In short,CD4+CD25+labeled Treg cells and CD4+FoxP3+labeled Treg cells in normal renal transplantation group were higher than the chronic rejection group (Table2-1).4. The plasma ITI level in normal kidney transplantation group and the chronic rejection group was significant different(P=0.040).ITI of two groups before operation was no significant difference (F=2.486, P=0.154), ITI expression4,6,8,10,12weeks after operation were significantly different.ITI level after four weeks, six weeks of normal renal transplantation group was lower than the chronic rejection group (F values were7.942,19.562respectively, P values were0.023,0.002respectively), from8weeks after the operation to12weeks, the normal renal transplantation group were higher than the chronic rejection group (F values were40.004,26.393,27.883respectively, P values were0.000,0.001,0.001respectively),Plasma ITI expression were different at different time points, ITI level of chronic rejection group gradually decreased along with the time after transplantation(P=0.000),The ITI level in normal renal transplantation group4weeks,6weeks,8weeks,10after surgery was different from pre-surgery,12weeks, it meaned that these were lower than the preoperative level, higher than the level after12weeks (P=0.000).ITI between4weeks and6weeks after surgery in Chronic rejection group was no significant difference (P=1.000), no significant difference between8weeks and10weeks after surgery(P=1.000),other two groups were statistically significant,it existed time interaction effect between grouping and time(P=0.000), suggesting that ITI was the lowest at12weeks after transplanting in chronic rejection group.Conclusions1.We could make a stable chronic renal allograft rejection model by this approach in our experiment,It is demonstrated that the pathological of rat chronic renal allograft rejection was most obvious at12weeks after transplant.2.Treg cells of lymphocytes was less than the normal transplant group in the most obvious pathological manifestations of chronic rejection.3.ITI level of chronic renal allograft rejection in plasma gradually decreased along with the passage of time after transplantation, ITI level in plasma within8weeks after transplantation in chronic rejection group were higher than normal kidney transplantation group,but the result is contrary from8weeks to12weeks after operate to former.and plasma ITI of chronic rejection group had reached the lowest level at12weeks after transplant.The ITI level was close relate to chronic rejection of rat kidney transplant.