Study On Effects Of Peripheral Nerve Regeneration By Recombinant Adenovirus-mediated Gene Transfection Of Human Hepatocyte Growth Factor.

Background. Disruption of peripheral nerves due to trauma is a frequentlyoccurring clinical problem,while options for reconstruction of peripheral nerve gaps are currently limited.Gaps in the nerve are bridged by autologous grafts or artificial guides.Among them,acellular nerve was a newly kind of biomaterial graft and has been used to reconstruct the peripheral nerve defect recently. Nerve regeneration can be enhanced by various growth factors which help to sustain the growing nerve. Hepatocyte growth factor is one of the candidates to facilitate the nerve regeneration,by virtue of its function as a powerful neurotrophic factor. Gene therapy can be used as an approach to the treatment of a variety of clinical disorders, increasing levels of recombinant protein within the nervous system. The purpose of this study was to investigate the effects of hepatocyte growth factor (HGF) on the functional recovery of injuried peripheral nerve. Muscles around the injuried peripheral nerve were injected with adenovirus carrying human hepatocyte growth factor gene.Methods: (1) Detection of gene transfection in primary cultured rat skeletal muscle cells. Primary cultured rat skeletal muscle cells (C2C12 Cell Line, Institute of Orthopedics, General Hospital of PLA, Beijing) were transfected with the recombinant Ad-GFP 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 by flow cytometry. The expression of HGF was determined by enzyme-linked immunosorbent assay (ELISA) in C2C12 cells transfected with Ad-HGF (MOI=400pfu/cell). (2) 30 Rats were divided into three groups (10/group) for autografting and acellular grafting, as well as acellular grafting with adenovirus transfection of HGF (1×10~8 pfu) injected in muscles around the proximal and distal allograft coapation. Sciatic functional index, weight of the gastrocnemius and soleus muscles, histologic and morphometric study and neovascularization in the nerve grafts were evaluated at week 16. (3) 20 Rats were divided into two groups (10/group) for crush group and crush group with adenovirus transfection of HGF (1×10~8 pfu) injected in muscles around the injuried nerve. Sciatic functional index, weight of the gastrocnemius and soleus muscles, histologic and morphometric study and neovascularization were evaluated at week 4. (4)Two methods for dog acellular nerve allograft were compared from histology observation,Sondell's protocol and Hudson's protocol. (5) Acellular nerve allograft by Hudson's protocol were transplanted to repair 5.0 cm defect in the dog sciatic nerve. Histologic and morphometric study were evaluated at 6 months.Results: (1) Rat skeletal muscle cells could be transfected by Ad-GFP at different MOI (0 to 400 pfu/cell) and transfection effiency increased with increasing MOI. To determine whether the rat skeletal muscle cells were able to release soluble HGF protein, condition medium was collected from cultured muscle cells transfected with Ad-HGF (400 pfu/cell) or Ad-GFP (400 pfu/cell) and assayed for HGF by ELISA. 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. (2) Autografting gave the best functional recovery, but HGF-treated acellular grafting gave better recovery than acellular grafting alone. Neovascularization was greater with HGF-treated acellular grafting than with autografting and 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. (3) Crush +HGF group gave better recovery than crush group alone. Neovascularization was greater with Crush +HGF group than with crush group alone. (4) Acellular nerve allograft by Hudson's protocol gave better results than that by Sondell's protocol from histology observation. (5) Due to automutilation, 5 dogs were killed early and only 1 dog was left for peripheral nerve regeneration evaluation.Conclusion: (1) Replication-deficient recombinant adenoviral vectors carrying the human HGF gene were constructed successfully. The target muscle cells couldcontinuously release soluble HGF protein. (2) Acellular nerve grafting may be usefulfor functional peripheral nerve regeneration, and with human HGF gene transfectionmay improve on acellular grafting alone in functional recovery.