转化生长因子β_1对坐骨神经移植后免疫耐受的影响

背景:转化生长因子β_1是一种强效细胞生长增殖调节蛋白,在移植免疫的抗排斥反应、移植物血管病发展中扮演重要角色.目的:观察经冷冻处理异体神经移植后,局部注射转化生长因子β_1对移植免疫排斥反应的影响.设计、时间及地点:随机对照动物实验,于2007-06/2008-06在哈尔滨医科大学动物实验中心完成.材料:受体为清洁级SD大鼠60只,分为3组:自体神经移植组、异体神经移植组、转化生长因子β_1质粒+异体神经移植组,每组20只.供体为40只Wistar雄性大鼠.pAdTrack-CMV-TGF-β_1质粒、pAdEasy-1-Bj51833细胞由哈尔滨医科大学附属四院骨科实验室惠赠.方法:取供体大鼠40只作双侧股后外侧纵切口,分离显露坐骨神经,切取双侧整段坐骨神经,置于无菌冷冻管中保存1周,备用.手术显微镜下将受体鼠自骨二头肌与半腱肌和半膜肌间隙剪开结缔组织,显露坐骨神经,从犁状肌孔下0.5 cm处整齐剪下长约1 cm的坐骨神经.自体神经移植组、异体神经移植组选择粗细相等、已预制冷冻的自体及异体神经移植;转化生长因子β_1质粒+异体神经移植组异体神经移植后于大鼠局部肌肉及神经两断端内注射pAdTrack-CMV-TGF-β_1质粒40 μg/只.主要观察指标:术后3,6,9周各组大鼠运动神经传导速度、病理学和轴突计数检查.结果:转化生长因子β_1质粒+异体神经移植组运动神经传导速度高于新鲜异体神经移植组(P < 0.01),与自体神经移植组比较差异无显著性意义.自体神经移植组、转化生长因子β_1质粒+异体神经移植组术后9周轴突计数较新鲜异体神经移植组高(P < 0.01).转化生长因子β_1质粒+异体神经移植组光镜及电镜可见神经纤维走行正常,排列完好,神经纤维可见血管增生,髓鞘结构较好,神经纤维内见有大量再生髓鞘,许旺细胞明显增多,胞质较发达,大量粗面内质网,线粒体结构清晰,再生的轴突内微丝密集排列,与自体神经移植组接近.异体神经移植组光镜及电镜可见神经纤维数量少、排列紊乱,髓鞘轴突变性明显,大部分神经纤维脱髓鞘崩解,轴突消失,未见再生的神经纤维.结论:局部注射转化生长因子β_1质粒联合冷冻处理的冷藏异体神经移植可以协同减轻移植后产生的免疫排斥反应.

Effects of transforming growth factor-beta 1 plasmid on immune tolerance following sciatic nerve transplantation

BACKGROUND: Transforming growth factor (TGF)-β_1, a potent cell growth and proliferation regulatory proteins, plays an important role in development of anti-graft rejection and graft vascular disease. OBJECTIVE: To observe local injection of TGF-β_1 effects on transplant immune rejection following freezing disposal and nerve allograft. DESIGN, TIME AND SETTING: The randomized controlled animal study was performed at the Animal Experimental Center, Harbin Medical University from June 2007 to June 2008. MATERIALS: A total of 60 clean SD rats (recipients) were divided into 3 groups: autogenous nerve graft group, nerve allograft group, TGF-β_1 plasmid + nerve allograft group, 20 in each group. A total of 40 Wistar male rats served as donors. pAdTrack-CMV-TGF-β_1 plasmid, pAdEasy-1-Bj51833 cells were presented by the Orthopedic Laboratory of Fourth Hospital of Harbin Medical University. METHODS: Longitudinal posterolateral incision was made in 40 donor rats to expose sciatic nerve. The whole bilateral sciatic nerve was cut and placed in sterile frozen tubes for 1 week for use. Under the microscope, connective tissue was cut in the biceps muscle and semi-tendon and semi-membrane gap of recipient rats to expose the sciatic nerve. 1-cm sciatic nerve was cut 0.5 cm below the muscle from the plow-shaped hole. Transplantation of frozen autogenous nerve graft and nerve allograft (nerve at equal size) was separately performed in the autogenous nerve graft and nerve allograft groups. In the TGF-β_1 plasmid + nerve allograft group, pAdTrack-CMV-TGF-β_1 plasmid (40 μg) was injected into the local muscle and two sides of transected sciatic nerve of each rat following nerve allograft transplantation. MAIN OUTCOME MEASURES: Motor nerve conduction velocity, pathology and axonal counting were examined 3, 6, 9 weeks after surgery. RESULTS: Motor nerve conduction velocity was higher in the TGF-β_1 plasmid + nerve allograft group than in the nerve allograft group (P < 0.01), which did not show significant difference compared with the autogenous nerve graft group. Axonal counting was greater in the autogenous nerve graft and TGF-β_1 plasmid + nerve allograft groups compared with the nerve allograft group 9 weeks following surgery (P < 0.01). Using optical microscope and electron microscope, nerve fibers were normal and well arranged in the TGF-β_1 plasmid + nerve allograft group. Nerve fibers presented vascular proliferation, good myelin sheath. Abundant regenerated myelin sheath was found in nerve fiber. The number of Schwann cells was obviously increased, and there were prosperous cytoplasm, a large amount of rough endoplasmic reticulum, clear mitochondria. In regenerated axons, microfilament closely arranged, which was similar to the autogenous nerve graft group. In the nerve allograft group, the optical microscope and electron microscope showed a few nerve fibers, disorderly arranged, significant demyelination, axon degeneration and disappearance, without regenerated fibers. CONCLUSION: Local injection of TGF-β_1 plasmid could reduce immune rejection after cold sciatic nerve allograft transplantation.