抗淋巴细胞血清联合他克莫司诱导肾移植免疫耐受的实验研究
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摘要
目的:
目前,器官移植已经成为终末期器官功能衰竭患者的有效治疗手段,但长期使用免疫抑制剂可引起许多不良反应,而且免疫抑制剂无助于移植慢性排斥反应和移植物的长期存活。器官移植后最理想的措施是针对供体移植物建立特异性免疫耐受,因此,如何诱导供体特异性移植耐受成为解决器官移植排斥反应的最佳手段,也成为临床器官移植免疫学研究的热点领域及主要目标之一。本研究通过大鼠肾脏移植受体输注抗淋巴细胞血清后对移植肾的影响,进行抗淋巴细胞血清联合免疫抑制剂诱导免疫耐受的研究。
方法一:
建立一种新的简便的大鼠肾脏移植模型:采用支架导管连接固定代替血管吻合和输尿管膀胱瓣皮肤造瘘的方法(修剪两段长约0.8cm的3F导管作为支架,分别将其置入供受体肾动静脉断端后固定,行输尿管膀胱瓣皮肤造瘘)。
结果一:
共施行大鼠肾移植30例,1例因麻醉过量呼吸抑制于术中死亡,2例因游离受体肾静脉时损伤血管导致大出血死亡,3例因动脉吻合口血栓形成导致手术失败,2例因静脉吻合口血栓形成导致手术失败,剩余22例术后5～10d死亡(存活5d以上视为成功),手术成功率为73.3%。
方法二:
联合应用抗淋巴细胞血清(ALS)与他克莫斯(FK506)诱导免疫耐受:受体Wistar大鼠共32只分为4组,各组样本量8只。A:正常对照组(仅行肾移植);B:ALS+肾移植;C:FK506+肾移植组;D:ALS+FK506+肾移植。(ALS组):移植手术前1天腹腔注射ALS,剂量1ml/kg,以后每天1次,连续应用至术后第10天;(FK506组):移植手术前1天开始应用FK506,剂量0.5mg/kg,每天1次,连续应用至术后第10天;(ALS+FK506组):联合使用ALS与FK506,方法同前。观察各组大鼠的存活时间,监测血清肌酐浓度,并进行移植肾彩色多普勒超声。移植物病理学检查,在光镜下观察各组移植物淋巴细胞浸润和组织结构的变化。采用迟发性超敏反应(DTH)法检测存活大鼠的免疫耐受状态。
结果二:
1.受者存活时间:D组平均存活时间为37d,与A组比较,差异有统计学意义(P＜0.01)。D组平均存活时间与B组及C组比较,差异均有统计学意义(P＜0.01)。
2.移植肾功能测定:A组术后3d切除自体肾脏后,血清肌酐浓度迅速上升;B组及C组血清肌酐浓度呈现缓慢上升趋势,D组移植肾功能基本保持稳定,术后30天出现缓慢上升。
3.移植肾病理学观察结果:术后1周正常对照组移植肾肾间质水肿,并有淋巴细胞浸润,肾小管上皮细胞变性坏死、脱落,血管内皮细胞肿胀,并可见微血栓形成。术后10天抗淋巴细胞血清(ALS)+肾移植组和FK506+肾移植组未见急性排斥反应,见少量淋巴细胞浸润,血管内皮细胞肿胀不明显。同期ALS+FK506+肾移植组未见明显淋巴细胞浸润,无急性排斥反应改变,肾小管上皮细胞未见变性坏死、脱落。ALS+FK506+肾移植组在移植后25天进行移植肾病理检查,发现肾间质有少量淋巴细胞浸润,肾小球基底膜稍增厚,但无微血栓形成,无明显急性排斥反应表现。
4.移植肾彩色多普勒超声检测:术后1周检查A组移植肾血供差,平均阻力指数0.80±0.06;B组和C组移植肾血供良好,平均阻力指数分别为0.80±0.06、0.6 3±0.08;D组移植肾血供良好,平均阻力指数0.61±0.04。A组与D组相比较,P＜0.05,差别明显。
术后20天检查D组移植肾血供良好,平均阻力指数0.62±0.03;术后35天检查D组移植肾血供差,平均阻力指数0.78±0.07。D组术后1周与术后35天比较,P＜0.05,差别明显。
5.迟发型超敏反应(DTH):A组Wistar大鼠对SD大鼠脾细胞抗原的DTH非常明显,5只实验小鼠的右后足掌发红及肿胀明显,以两足掌厚度之差(mm)表示的DTH指数为0.44±0.05。第2、第3组和第4组8只Wistar大鼠DTH反应引起的足掌肿胀明显减轻,其DTH指数分别为0.24±0.05、0.25±0.06和0.22±0.05,对三组均数进行t检验,与第1组比较差异均有显著性。
结论:
1.采用支架导管连接固定代替血管吻合和输尿管膀胱瓣皮肤造瘘的方法所建立的大鼠肾移植模型可靠、简便、易掌握、手术时间短。
2.联合应用ALS与FK506能够延长受者存活时间,移植肾功能保持稳定,停药后短期内未见排斥反应,ALS联合FK506能诱导出短期的免疫耐受。
Objectives:
To induce immune tolerance in recipient rats of allogenic kidney transplantation with antilymphocyte serum combined Tacrolimus. Methods:
A model of renal transplantation was established.Receptors were fed Tacrolimus,then ALS were injected.Renal transplantation was performed within the same day.The survival days of receptor rats were observed.Serum creatinine was measured and color Doppler flow imaging was checked.The immunologic tolerance status of rat renal allografts was detected by DTH.
Results:
The survival days of receptor,the levels of Serum creatinine,the color Doppler flow imaging and the DTH showed that the difference was significant between case group and other three control groups.
Conclusion:The antilymphocyte serum combined Tacrolimus can induce immune tolerance from allograft.
目前,器官移植已经成为终末期器官功能衰竭患者的有效治疗手段,但长期使用免疫抑制剂可引起许多不良反应,而且免疫抑制剂无助于移植慢性排斥反应和移植物的长期存活。器官移植后最理想的措施是针对供体移植物建立特异性免疫耐受,因此,如何诱导供体特异性移植耐受成为解决器官移植排斥反应的最佳手段,也成为临床器官移植免疫学研究的热点领域及主要目标之一。本研究通过大鼠肾脏移植受体输注抗淋巴细胞血清后对移植肾的影响,进行抗淋巴细胞血清联合免疫抑制剂诱导免疫耐受的研究。
方法一:
建立一种新的简便的大鼠肾脏移植模型:采用支架导管连接固定代替血管吻合和输尿管膀胱瓣皮肤造瘘的方法(修剪两段长约0.8cm的3F导管作为支架,分别将其置入供受体肾动静脉断端后固定,行输尿管膀胱瓣皮肤造瘘)。
结果一:
共施行大鼠肾移植30例,1例因麻醉过量呼吸抑制于术中死亡,2例因游离受体肾静脉时损伤血管导致大出血死亡,3例因动脉吻合口血栓形成导致手术失败,2例因静脉吻合口血栓形成导致手术失败,剩余22例术后5～10d死亡(存活5d以上视为成功),手术成功率为73.3%。
方法二:
联合应用抗淋巴细胞血清(ALS)与他克莫斯(FK506)诱导免疫耐受:受体Wistar大鼠共32只分为4组,各组样本量8只。A:正常对照组(仅行肾移植);B:ALS+肾移植;C:FK506+肾移植组;D:ALS+FK506+肾移植。(ALS组):移植手术前1天腹腔注射ALS,剂量1ml/kg,以后每天1次,连续应用至术后第10天;(FK506组):移植手术前1天开始应用FK506,剂量0.5mg/kg,每天1次,连续应用至术后第10天;(ALS+FK506组):联合使用ALS与FK506,方法同前。观察各组大鼠的存活时间,监测血清肌酐浓度,并进行移植肾彩色多普勒超声。移植物病理学检查,在光镜下观察各组移植物淋巴细胞浸润和组织结构的变化。采用迟发性超敏反应(DTH)法检测存活大鼠的免疫耐受状态。
结果二:
1.受者存活时间:D组平均存活时间为37d,与A组比较,差异有统计学意义(P＜0.01)。D组平均存活时间与B组及C组比较,差异均有统计学意义(P＜0.01)。
2.移植肾功能测定:A组术后3d切除自体肾脏后,血清肌酐浓度迅速上升;B组及C组血清肌酐浓度呈现缓慢上升趋势,D组移植肾功能基本保持稳定,术后30天出现缓慢上升。
3.移植肾病理学观察结果:术后1周正常对照组移植肾肾间质水肿,并有淋巴细胞浸润,肾小管上皮细胞变性坏死、脱落,血管内皮细胞肿胀,并可见微血栓形成。术后10天抗淋巴细胞血清(ALS)+肾移植组和FK506+肾移植组未见急性排斥反应,见少量淋巴细胞浸润,血管内皮细胞肿胀不明显。同期ALS+FK506+肾移植组未见明显淋巴细胞浸润,无急性排斥反应改变,肾小管上皮细胞未见变性坏死、脱落。ALS+FK506+肾移植组在移植后25天进行移植肾病理检查,发现肾间质有少量淋巴细胞浸润,肾小球基底膜稍增厚,但无微血栓形成,无明显急性排斥反应表现。
4.移植肾彩色多普勒超声检测:术后1周检查A组移植肾血供差,平均阻力指数0.80±0.06;B组和C组移植肾血供良好,平均阻力指数分别为0.80±0.06、0.6 3±0.08;D组移植肾血供良好,平均阻力指数0.61±0.04。A组与D组相比较,P＜0.05,差别明显。
术后20天检查D组移植肾血供良好,平均阻力指数0.62±0.03;术后35天检查D组移植肾血供差,平均阻力指数0.78±0.07。D组术后1周与术后35天比较,P＜0.05,差别明显。
5.迟发型超敏反应(DTH):A组Wistar大鼠对SD大鼠脾细胞抗原的DTH非常明显,5只实验小鼠的右后足掌发红及肿胀明显,以两足掌厚度之差(mm)表示的DTH指数为0.44±0.05。第2、第3组和第4组8只Wistar大鼠DTH反应引起的足掌肿胀明显减轻,其DTH指数分别为0.24±0.05、0.25±0.06和0.22±0.05,对三组均数进行t检验,与第1组比较差异均有显著性。
结论:
1.采用支架导管连接固定代替血管吻合和输尿管膀胱瓣皮肤造瘘的方法所建立的大鼠肾移植模型可靠、简便、易掌握、手术时间短。
2.联合应用ALS与FK506能够延长受者存活时间,移植肾功能保持稳定,停药后短期内未见排斥反应,ALS联合FK506能诱导出短期的免疫耐受。
Objectives:
To induce immune tolerance in recipient rats of allogenic kidney transplantation with antilymphocyte serum combined Tacrolimus. Methods:
A model of renal transplantation was established.Receptors were fed Tacrolimus,then ALS were injected.Renal transplantation was performed within the same day.The survival days of receptor rats were observed.Serum creatinine was measured and color Doppler flow imaging was checked.The immunologic tolerance status of rat renal allografts was detected by DTH.
Results:
The survival days of receptor,the levels of Serum creatinine,the color Doppler flow imaging and the DTH showed that the difference was significant between case group and other three control groups.
Conclusion:The antilymphocyte serum combined Tacrolimus can induce immune tolerance from allograft.
引文
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