同种异体手移植术后患者的早期免疫学状态

背景:同种异体肢体移植在动物实验中已有大量免疫学方面的研究,但由于临床只是早期的探索,对其临床免疫学研究需要进一步积累。目的:动态分析异体手移植患者术后早期免疫学状态的变化。设计:对照实验。单位:解放军广州军区广州总医院骨科,解放军第一军医大学南方医院创伤骨科和检验科。对象:实验组纳入解放军广州军区广州总医院骨科两例单侧异体手移植的患者,观察时间1999-09/2000-03。健康对照组为同期本院体检者20人,男12人,女8人,年龄20～45岁,均为自愿者,无活动性免疫性和感染性疾病。方法:2例手移植患者手术前1和3d各采外周血1次,术后第1周采血1次/d,2～4周采血3次/周,5～8周采血2次/周,9～16周采血1次/周,5～6个月采血2次/月。①运用流式细胞仪检测外周血T细胞亚群(CD3 ,CD4 ,CD8 T细胞),用ELISA法检测血清群体反应性抗体,免疫透射比浊法测定血清C反应蛋白,酶动力法检测血清肌酸肌酶。②混合淋巴细胞反应:以丝裂霉素C处理的供体外周血淋巴细胞作刺激细胞,3H-TDR掺入法检测患者外周血淋巴细胞对供体移植抗原的增生反应(阴性:刺激指数的均值与1无显著性差异,反之为阳性)。以自体相同处理过的外周血淋巴细胞代替供体刺激细胞作为对照,计算每一标本的刺激指数(刺激指数=实验cpm/对照cpm)作为比较指标。健康对照组20人均采外周血检测外周血T细胞亚群(CD3 ,CD4 ,CD8 T细胞)、血清群体反应性抗体、C反应蛋白和肌酸肌酶;20人随机分为10组,互为供受体,行外周血混合淋巴细胞反应。主要观察指标:①外周血T细胞亚群(CD3 ,CD4 ,CD8 T细胞)。②血清群体反应性抗体。③C反应蛋白。④肌酸肌酶。⑤混合淋巴细胞反应。结果:①术后1周内CD3 ,CD4 ,CD8 T细胞明显下降,1周后除CD8 细胞比术前升高外均逐渐恢复至术前水平[CD3 :(66.43±4.56);CD4 :(30.55±3.94);CD8 :(33.45±2.69)]。实验组与健康对照组差异无显著性。②血清群体反应性抗体为0～10%,与对照组差异无显著性。③血清C-反应蛋白为0～0.359mg/L,与对照组差异无显著性。④血清肌酸肌酶肌酸肌酶为25～170mmol/L,与对照组差异无显著性。⑤移植后混合淋巴细胞反应为阴性,5个月后转为阳性。对照组均为阳性。结论:T细胞亚群的短期的变化与长期的再分布与免疫抑制剂密切相关,说明免疫抑制剂对异体手移植术后T细胞亚群有明显的影响。免疫诱导方案使患者处于免疫抑制状态,有效地避免了早期排斥反应的发生,但患者还未达到特异性耐受,还需要免疫抑制剂的抑制。

Early immunologic status of patients following hand allotransplantation

BACKGROUND: There are a lot of immunologic studies about limb allotransplantation in animal experiment. But, it is only early investigation in clinic; its clinical immunologic study needs further accumulation.OBJECTIVE: To dynamically analyze the early immunologic state change in patients following hand allotransplantation.DESIGN: Controlled trial.SETTING: Department of Orthopaedics, Guangzhou General Hospital,Guangzhou Military Area Command of Chinese PLA; Department of Traumatic Orthopaedics and Department of Laboratory Medicine, Nanfang Hospital, First Military Medical University of Chinese PLA.PARTICIPANTS: Two patients who underwent unilateral hand allotransplantation in the Department of Orthopaedics, Guangzhou General Hospital,Guangzhou Military Area Command of Chinese PLA were enrolled, serving as experimental group. The observation was between September 1999 and March 2000. Twenty persons, including 12 male and 8 female, who homochronously received health examination, aged 20 to 45 years, were enrolled, serving as healthy control group. They all had no reactive immune and infectious diseases, and voluntarily participated in the trial.METHODS: Peripheral blood was collected from 2 patients who underwent hand allotransplantation once respectively at pre-operative 1 day and 3 days. Blood collecting was performed once per day at post-operative 1 week, three times per week at post-operative 2 to 4 weeks, twice per week at 5 to 8 weeks post-operation, once per week at 9 to 16 weeks post-operation, twice per month at 5 to 6 months post-operation. ① Peripheral blood T cell subgroups (CD3+,CD4+,CD8+T cells)were detected by flow cytometer,serum panel reactive antibody (PRA) by ELISA method, serum C-reactive protein by turbidimetric immanoassay (TIA), serum creatine kinase (CK) by enzyme dynamics method. ②Mixed lymphocyte reaction(MLR): mitomycin C-treated donor peripheral blood lymphocytes were used as stimulator, and proliferative reaction of peripheral blood lymphocyte of patients to donor transplanted antigen was detected with the incorporation of 3H-TDR method (Negative: There was no significant difference between the mean value of stimulation index and 1, conversely positive). Autogenic peripheral blood lymphocytes treated with the same way replaced donor stimulator, serving as control. Stimulation index of each specimen was calculated (Stimulation index=Experiment cmp/controlcpm), serving as control index. Peripheral blood T-cell subgroups (CD3+,CD4+,CD8+T cell), serum PRA, C-reactive protein and CK were detected in 20 persons in healthy control group;Twenty persons were randomly divided into 10 groups. Two persons in each group were used as donor and recipient mutually and performed MLR.MAIN OUTCOME MEASURES: ① Peripheral blood T cell subgrpups (CD3+,CD4+,CD8+T cell). ②PRA. ③ C-reactive protein. ④CK. ⑤MLR.RESULTS: ①CD3+,CD4+,CD8+T cell levels were obviously decreased within one week after operation. CD3+ and CD4+T cell levels both recovered to be the pre-operative levels, but CD8+ level exceeded pre-operative level significantly [CD3+: (66.43±4.56); CD4+: (30.55±3.94); CD8 +:(33.45 ±2.69)]. There was no significant difference between experiment group and control group. ②Serum PRA was 0 to 10%, there was no significant difference as compared with control group. ③ Serum C-reactive protein was 0 to 0.359 mg/L, there was no significant difference as compared with control group. ④ Serum CK was 25 to 170 mmol/L, and there was no significant difference as compared with control group. ⑤ MLR after transplantation was negative, and it turned into be positive 5 months later.They were all positive in control group.CONCLUSION: Short-term change and long-term redistribution of T cell subgroups are closely related to immunosuppressive agent, suggesting that immunosuppressive agent has obvious effect on T-cell subgroup following hand allotransplantation. Immuno-induction schedule make patients be in immune suppression state, which effectively avoid early rejection. But patients cannot bear specificity yet; they need the inhibition of immunosuppressive agents.