| Objective1 To establish of renal transplantation acute rejection rat model for studying that detects acute rejection exactly and promptly by non-invasive methods.2 To research the pathology feature of the rat transplantation renal,the expression of CD44 in a transplantation renal and the the expression of sCD44 in peripheral blood of the renal transplantation rat model.3 To research the expression of CD44 in a human transplantation renal and the expression of sCD44 in peripheral blood of the human renal transplantation.Methods1 To establish of renal transplantation acute rejection rat modeladult SPF male Wistar rats and male or female SD rats,weigh 300-350g, were used in this study.kidney transplantation was performed using Wistar donors and SD recipients. The weight of recipient was little than donor's. Wistar rat was anesthetized and entered abdominal cavity, Resected donor organs were right and left kidneys,part of abdominal aorta and postcaval vein, left ureter and vesica urinaria. Right kidney was resected after ligated left kidney vessel,Organs were clipped, recipient rat was anesthetized and entered abdominal cavity, abdominal aorta and postcaval vein of donor and recipient were sutured by end-to-side respectively. Recipient rat was setting in constant temperature cabinet 2h,and then ws setted in clearing cage by oneself, administrated 5%glyco-salt water. to observe transplanted rat characteristics of spirit, activity, drink and food and body weight.2 The expression of CD44 in the renal transplantation rat modelThe rat model anmals were divided into three groups,each of which contents 10 rats,the first group was acute rejection group(AR),in this group Wistar and male SD rats were used as donor and recipient respectively, the second group was non-rejection group(control), in this group Wistar and male Wistar rats were used as donor and recipient respectively.the third group was normal group consisted of Wistar rats. The graft specimens were harvested on 3,5,7,9and 14 days postoperatively. They were examined histologieally after sectioning and staining with hematoxylin and eosin, Masson's trichrome, periodic aeid-Sehiff reagent and periodic acidsilver metheramine. Acute rejeetion was diagnosed according to the Banff 2005 classification.This rejeetion score was prepared semiquantltatively Into seven grades from 0 to 6 according to the methods Of Banff2005 classification. The expression of CD44 in the allograft was detected by immunohistochemistry.Rat peripheral blood specimens were taken on3,5,7,9and 14 days postoperatively. sCD44 and hyaluronic acid were detected by ELISA.3 The expression of CD44 in the human renal transplantationThe first part patients,28 renal transplantation patients were dignosised AR by needle biopsy of graft,28 renal transplantation patients were dignosised CR(chronic rejection) by needle biopsy of graft,and 28 normal patients. The expression of CD44 in these allografts or kidneys was detected by immunohistochemistry.The second part patients,13 renal transplantation patients were dignosised AR by needle biopsy of graft,13 renal transplantation patients were stable post-operation as stable control. Blood samples of these patients were obtained before transplantation and on day 3,5,7,9and 14 after transplantation.one part blood samples were centrifuged within 2 hours and plasma was separated from cells.Human sCD44 instant ELISA kits were used to measure serum levels of sCD44. the other part blood samples were used as detecting the expression of CD44 on T lymphocyte with flow cytometry.The third part patients,15 renal transplantation follow-up patients were dignosised AR by needle biopsy of graft,15 renal transplantation patients were stable post-operation as stable control.One part blood samples were centrifuged within 2 hours and plasma was separated from cells.Human sCD44 instant ELISA kits were used to measure serum levels of sCD44. the other part blood samples were used as detecting the expression of CD44 on T lymphocyte with flow cytometry.Results1 The establishing of rat kidney transplant modelThe first 3 months we performed 50 rats kidney transplantations, this period we were in order to practice segregating blood vessel, perfusing donor kidney and suturing blood vesse. The death rate was high in this period, we performed 50 rats kidney transplantations in later period, Of 50 rat kidney transplants performed,30 surived morn than 2 days,the suceess rate was60%.The mean operation time was(130±20)min, the mean time of sutureing vein was (25±5)min. the mean time of sutureing artery was(20±5) min, the mean time of resectting donor organs was (40±5)min. The complications consisted of shock, stoma hemorrhage, stoma stenosis, Lymphorrhagia, urnary leak.2 the pathology feature of the rat transplantation renalThe 3rd day, part of rats AR transplantation renal interstitial were infiltrated by lymphocyte, the pathology scores were 2,part of control group transplantation renal interstitial were also infiltrated by lymphocyte, the pathology scores were 1.From 3 to 14 day, the quantity of rats AR transplantation renal interstitial infiltrated lymphocyte increased gradually, artery occurred intimitis and even intimitis past through artery wall.The control group artery did not occure intimitis. the pathology scores of AR group were significantly higher than those of control group. The pathology scores in 5,7,9and 14 days were significantly higher than those in 3 days.3 the expression of CD44 in rat transplantation renalOn 3rd day after renal transplantation,of 10 AR rats,4 rats transplantation renal expressed CD44, positive rate was 40%,CD44 expressed in renal tubules and interstitial. Of 10 control group rats,3 rats transplantation renal expressed CD44, positive rate was 30%. On 14th day after renal transplantation, of 10 AR rats,9 rats transplantation renal expressed CD44, positive rate was 90%,Of 10 control group rats,the highest positive rate was 60%, On 14th day after renal transplantation, the positive rate descreased to 20% again,Of 10 normal group rats,2 rats transplantation renal expressed CD44, positive rate was 20%.4 The expression of sCD44,HA in peripheral blood in the renal transplantation rat model.From 3 to 14 day, The expression of sCD44,HA in peripheral blood increased gradually in AR, The expression of sCD44,HA in peripheral blood increased lightly gradually in control group, The expression of sCD44,HA in peripheral blood had no this regularity in normal rats. The expression of sCD44,HA in 5,7,9and 14 days were significantly higher than those in 3 days in AR.5 the expression of CD44 in patient transplantation renalOf 28 AR patients,21 humans transplantation renal expressed CD44, positive rate was 75%,CD44 expressed in renal tubules and interstitial. Of 28 chronic rejection group patients,13 patients transplantation renal expressed CD44, positive rate was 46.43%. Of 28 normal grouphumans,9 humans transplantation renal expressed CD44, positive rate was 32.14%. The differences of the expression of CD44 in human renal were significantly between these three groups.6 The expression of sCD44,HA,Cr in peripheral blood in pre-transplant patientThe expression of sCD44 was 597.69±24.62 ng/ml in AR pre-transplant patien-ts, The expression of sCD44 was 573.08±13.03ng/ml in stable group pre-transplant patients, The expression of sCD44 was 269.31±50.86ng/ml in normal human; To analyze these three datas with one-way ANOVA, The differences of the expression of CD44 were significantly between these three groups. The expression of HA was428.30±23.03ng/ml in AR pre-transplant patients, The expression of HA was 420.62±13.87ng/ml in stable group pre-transplant patients, The expression of HA was 66.77±9.10ng/ml in normal huaman; To analyze these datas with one-way ANOVA, The differences of the expression of HA were not significantly between AR and stable groups. The expression of Cr was 854.77±89.28μmol/L in AR pre-transplant patients, The expression of Cr was 838.15±66.07μmol/L in stable group pre-transplant patients, The expression of Cr was 65.08±15.67μmol/L in normal huaman;To analyze these datas with one-way ANOVA, The differences of the expression of Cr were not significantly between AR and stable groups.7 The expression of sCD44,HA,Cr in peripheral blood in post-transplant patientOn the first three day after renal transplantation,of 13 AR patients, The expressions of sCD44 in peripheral blood in post-transplant patient did not decrease obviously, The expressions of sCD44 in peripheral blood increased quickly after renal transplantation and to 844.23±41.68 ng/ml on 7th day, The expressions of sCD44 in peripheral blood decreased quickly after acute rejection turning round and to 201.02±11.41 ng/ml on 14th day. The differences of the expression of sCD44 in peripheral blood were significantly between two groups. The differences of the expression of sCD44 in3,5,7,9 and 14 days were significantly AR. The expressions of HA,Cr in peripheral blood was similar to sCD44. To analyze these datas of AR with correlation analysis, The expression of sCD44 was positive correlation with HA and Cr.8 The expression of sCD44,HA,Cr in peripheral blood in follow-up patientThe expression of sCD44 was 841.85±43.71ng/ml when acute rejection group was occurring acute rejection, The expression of sCD44 descreased to 354.12,58.33ng/ml after post-treatment. The expression of sCD44 was 224.57,17.60 ng/ml in stable group. The AR prior treatment and post-treatment expressions of HA,Cr in peripheral blood was similar to sCD44. To analyze datas with t test, The differences of the expression of sCD44,HA,Cr were significantly between prior treatment and post-treatment in AR.9 The expression of CD44 in peripheral blood T lymphocyte in pre-transplant patientThe percentage of CD8+CD44+T lymphocyte was 8.75±1.01 in AR group,The percentage of CD8+CD44+T lymphocyte was 9.73±0.61 in stable group. To analyze datas with t test, The differences of the expression of CD8+CD44+T lymphocyte were significantly between AR group and stable group. The percentage of CD44+T lymphocyte was 19.34±2.14 in AR group,The percentage of CD44+T lymphocyte was 21.55±1.94 in stable group. To analyze datas with t test, The differences of the expression of CD44+T lymphocyte were significantly between AR group and stable group. To analyze these datas of AR with correlation analysis, The expression of sCD44 pre-transplant patient was positive correlation with The percentage of CD8+CD44+T lymphocyte in AR group.10 The percentage of CD8+CD44+T lymphocyte in follow-up patientThe percentage of CD8+CD44+T was 8.47±0.46 when acute rejection group was occurring acute rejection. The percentage of CD8+CD44+T was 9.77±0.60 after post-treated. The percentage of CD8+CD44+T was 9.78±0.61in stable group. To analyze datas with t test, The differences of The percentage of CD8+CD44+T were significantly between prior treatment and post-treatment in AR.The percentage of CD8+ T was 30.46±1.64 when acute rejection group was occurring acute rejection.The percentage of CD8+T was 29.06±1.62 after post-treated. The percentage of CD8+T was 29.78±1.62 in stable group. To analyze datas with t test, The differences of The percentage of CD8+T were not significantly between prior treatment and post-treatment in AR.conclusion1 This experiment constructed renal transplantation acute rejection rat model for next research.2 This experiment proved that the rat combination of Wistar→SD is fit for construction renal transplantation acute rejection rat model. The rat combination of Wistar→Wistar is fit for construction renal transplantation non-rejection rat model.3 the expression of CD44 in renal transplantation acute rejection rat model increased, The differences of the expression of CD44 were significantly between AR and control group.4 The expression of sCD44,HA in peripheral blood in the renal transplantation rat model was higher than control group.5 the expression of CD44 in AR patient transplantation renal was higher than that in CR.6 The expression of sCD44 in peripheral blood in pre-transplant patient in AR group was higher than that in stable group. The percentage of CD8+CD44+T and CD44+Tin AR group was lower than that in stable group 7 The expression of sCD44 increased when acute rejection group was occurring acute rejection,The expression of sCD44 descreased after post-treatment.8 The expression of the percentage of CD8+CD44+T decreased when acute rejection group was occurring acute rejection, The percentage of CD8+CD44+T increased after post-treatment.9 CD44 and HA interaction participated in the pathology of acute rejection.
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