大鼠小肠移植早期应用趋化因子受体拮抗剂抑制移植物急性排斥反应

背景:排斥反应的发生是导致小肠移植失败的主要原因,趋化因子RANTES及其受体介导的细胞免疫在急性排斥反应中具有重要作用。目的:观察小肠移植术后早期应用趋化因子受体拮抗剂Met-RANTES对排斥反应的免疫抑制作用及其与他克莫司的协同效应。设计:随机化完全区组设计,观察对照动物实验。单位:解放军第四五一医院普通外科、解放军第四军医大学西京医院胃肠外科实验室及解放军第四军医大学基础部电子显微镜中心。材料:实验于2003-09/2005-03在解放军第四军医大学西京医院胃肠外科实验室完成。以96只SD大鼠为供者,96只Wistar大鼠为受者,施行异基因节段性异位小肠移植。方法:移植后的大鼠以随机化完全区组设计分为4组(n=24):对照组、Met-RANTES组(200μg/d)、他克莫司组(0.5mg/(kg·d))及Met-RANTES 他克莫司组(Met-RANTES200μg/d 他克莫司0.5mg/(kg·d))。后3组均于移植术后腹腔注射给药,共7d;对照组移植前后不作任何处理。术后观察移植大鼠的一般状况、存活时间以及免疫细胞浸润情况,并于移植术后3,5,7d分别取各组大鼠移植肠标本(n=6)进行组织病理学检查,采用免疫荧光染色和激光扫描共聚焦显微镜技术对移植肠RANTES和CD4 ,CD8 ,CD25 T淋巴细胞的表达进行连续定量测定。每组剩余6只大鼠作存活时间观察,观察期限为5周。主要观察指标:①各组大鼠移植术后存活时间。②各组大鼠移植肠的病理改变。③各组大鼠移植肠RANTES及T淋巴细胞亚群的表达。结果:移植术后96只Wistar受体大鼠全部进入结果分析。①Met-RANTES组、他克莫司组和Met-RANTES 他克莫司组大鼠平均存活时间显著长于对照组(P<0.01);Met-RANTES 他克莫司组大鼠存活时间最长,与Met-RANTES组、他克莫司组差异均有显著性意义(P<0.01)。②对照组全部死于急性排斥反应及感染,组织病理学检查显示移植后第3,5,7天分别符合轻、中、重度排斥反应。Met-RANTES组、他克莫司组和Met-RANTES 他克莫司组病理学检查无明显排斥反应征象。③对照组大鼠的移植肠RANTES表达在术后各时段均显著高于其他3组(P<0.01),其动态变化与急性排斥反应的进程成正相关;Met-RANTES组和Met-RANTES 他克莫司组大鼠移植肠RANTES、CD4 、CD8 和CD25 T细胞的表达均低于对照组(P<0.01)。结论:Met-RANTES能明显抑制小肠移植急性排斥反应,有效保护移植肠功能,显著延长移植物的存活时间,并可增强小剂量(0.5mg/(kg·d))他克莫司的免疫抑制作用。

Met-RANTES, a chemokine receptor antagonist, is used to suppress acute rejection at early stage following small bowel allografting in rats

BACKGROUND: Rejection is the main cause of the failure in small bowel transplant. Chemotatic factor RANTES and receptor mediated cellular immunity are very important in acute rejection.OBJECTIVE: To explore the immunosuppressive effect of early adopting chemokine receptor antagonist, Met-RANTES after small bowel transplant on acute allograft rejection and its coordinative effect with Tacrolimus (FK506).DESIGN: Randomized complete-block design, controlled animal experiment.SETTING: Department of General Surgery, the 451 Hospital of Chinese PLA; Laboratory of Department of Gastrointestinal Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA; Electronic Microscope Center, School of Basic Medicine, Fourth Military Medical University of Chinese PLA.MATERIALS: This study was carried out in the Laboratory of Department of Gastrointestinal Surgery, Xijing Hospital,Fourth Military Medical University of Chinese PLA from September 2003 to March 2005. Totally 192 animals including 96 SD rats (donors) and 96 Wistar rats (recipients) were involved in this study. Heterotopic segmental small bowel transplantation was performed.METHODS: The transplant rats were divided into 4 groups averagely by the randomized complete block design: control group (allogeneic small bowel transplant untreated group), Met-RANTES group(200 μg/d, 0-7 days, i.p.), FK506 group 0.5 mg/(kg·d) ,0-7 days,i.p.], Met-RANTES + FK506 group Met-RANTES, 200 μg/d,0-7 days,i.p.+ FK506 0.5 mg/(kg ·d),0-7 days, i.p.]. Rats in the latter 3 groups were intraperitoneally administrated after transplant within 7 days successively.Rats in the control group were not given any treatments before and after transplant. Postoperatively, gross status,survival time and immunocyte infiltration were observed. Pathological examination was conducted in 6 rats of each group on postoperative days 3, 5 and 7. Fluorescent staining and successive quantitative measurement were conducted to detect the expressions of intragraft RANTES, CD4+, CD8+ and CD25+ T lymphocyte. Survival duration of the rest 6 rats of each group was observed for 5 weeks.MAIN OUTCOME MEASURES: ① Survival time of rats in each group following transplant. ② Pathological changes of small bowel intragraft of rats in each group. ③ RANTES and T lymphocyte expressions of rats in each group.RESULTS: Following transplantation, 96 Wistar rats (recipient) were all involved in the final analysis. ①Compared with control group, the survival time of rats in Met-RANTES group, FK506 group, Met-RANTES + FK506 group was significantly longer (P ＜ 0.01). In addition, rats in Met-RANTES + FK506 group survived the longest. There were significant differences in survival rate as compared with Met-RANTES group and FK506 group (P ＜ 0.01). ②All rats in the control group died of acute rejection and infection. Histopathologic examination showed mild, moderate and severe rejection on the postoperative days 3,5 and 7, respectively. No obvious rejection was found in the rats in the Met-RANTES group, FK56 group and Met-RANTES+FK506 group on the postoperative days 3,5 and 7. ③Postoperatively, intragraft RANTES expression of rats was significantly higher in each time period in control group than in the other 3 groups (P ＜ 0.01), and its dynamic change was positively correlated with the process of acute rejection; The expression of intragraft RANTES, CD4+, CD8+ and CD25+ T lymphocytes of rats was significantly lower, respectively, in the Met-RANTES group and Met-RANTES+FK56 group than in the control group (P ＜ 0.01).CONCLUSION: Met-RANTES may obviously suppress acute allograft rejection in small bowel transplant, effectively protect the function of grafts, and significantly prolong the survival time of the recipients. In addition, Met-RANTES may enhance the immunosuppressive function of small dose of FK5060.5 mg/(kg · d)].