化学去细胞异体神经复合肝细胞生长因子修复周围神经缺损的研究

Effect of hepatocyte growth factor on peripheral nerve regeneration in aceilular nerve graft

目的 研究化学去细胞异体神经复合人肝细胞生长因子（HGF）修复周围神经缺损的作用。方法 体外试验检测携带人肝细胞生长因子基因的重组腺病毒（Ad-HGF）对小鼠骨骼肌细胞的转染效率以及转染细胞对目的蛋白的表达。化学去细胞异体神经修复大鼠坐骨神经10mm缺损后，近、远端吻合口附近肌肉分别注射腺病毒介导的人肝细胞生长因子（Ad—HGF），与自体神经移植组对照，通过步态分析、肌肉湿重测定、轴突生长速率测定、神经电生理、计算机图像分析等指标评价神经移植后再生效果。结果 流式细胞仪结果表明，随着病毒滴度的增加，Ad—HGF对骨骼肌细胞的转染不断增加。骨骼肌细胞对目的蛋白（HGF）的表达可以持续两周。大鼠动物实验术后16周，去细胞异体神经可以修复周围神经缺损，同自体神经移植对比，肝细胞生长因子明显增强了去细胞异体神经的神经再生能力。结论 复合肝细胞生长因子的化学去细胞异体神经能促进神经轴突生长速度，显著增加移植物内新生血管，满意修复一定长度周围神经缺损，可以成为一种有效的周围神经组织工程修复材料。

Objective To explore the effect of adenoviral transfection with hepatocyte growth factor （HGF） on the functional recovery of transected sciatic nerves repaired by acellular nerve grafting.Methods Primary cultured rat skeletal muscle cells were transfected with replication-deficient recombinant adenoviral vectors carrying the human HGF gene at different multiplicities of infection （MOI） in a range of 0 to 400, The results were observed on fluorescence microscopy after 48 hr and the infect efficiency was determined. The expression of HGF was determined by enzyme-linked immunosorbent assay （ELISA）. Rat sciatic nerves were chemically extracted by Hudson protocol. 30 Rats were divided into three groups （10/group） for autografting and acellular grafting, as well as acellular grafting with adenovirus transfection of HGF （1×109 pfu） injected in muscles around the proximal and distal allograft coapation. 16 weeks after operation the effects of nerve regeneration were evaluated by sciatic nerve function index （SFI）, weight of the gastrocnemius and soleus muscles, histologic and morphometric study and neovascularization in the nerve graf Results Rat skeletal muscle cells could be transfected by Ad-HGF at different MOI （0 to 400 pfu/cell） and transfection effiency increased with increasing MOI. A peak of 131.06 ng/ml Ad-HGF expression was detected on day 2 post-transfection and was maintained for at least 2 weeks. 16 weeks after operation in animal test, autografting gave the best functional recovery, but HGF-treated acellular grafting gave better recovery than acellular grafting alone. Axonal regeneration distance of autografting on the 20th postoperative day was the longest in the three groups,while that of acellular grafting alone was the smallest. Conclusion Acellular nerve grafting may be useful for functional peripheral nerve regeneration, and with human HGF gene transfection may improve on acellular grafting alone in functional recovery.