Résumé
Objective:To investigate the effect of endogenous neurotrophic factor (ENTFs) on nerve regeneration after cryopreserved sciatic nerve allograft in rats. Methods:The 15-mm sciatic nerves from female Sprague-Dawley rats were placed in DMEM solution and pretreated in vitro for 1 day, 3 days, 7 days, 14 days, and 21 days at 37 ℃ with 5% CO2 (groups A, B, C, D and E) respectively. Fresh nerve group (group F) was set up. The protein expression of glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), Bcl-2, Bax, Caspase-3, major histocompatibility complex (MHC)-I and MHC-II was detected by Western blotting. The above six groups were cryopreserved in liquid nitrogen for four weeks. The living cells and dead cells of the nerves after cryopreservation were observed by Calcein-AM/propidium iodide staining. In addition, the above six cryopreserved groups and another fresh nerve group (group G) were transplanted to the Wistar rats by allografting (groups A', B', C', D', E', F' and G'). Isograft group (group H') was set up. One week after transplantation, the expression of CD8+ T cells and macrophages of the graft were observed by immunofluorescence staining, and the plasma levels of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were detected by ELISA. Twenty weeks after transplantation, the compound muscle action potential (CMAP) and nerve conduction velocity (NCV) were examined by electrophysiology. The wet weight ratio of gastrocnemius muscle was calculated by the operational side compared with the contralateral side. The expression of neurofilament protein (NF) 200 of the transplanted nerves was observed by immunofluorescence staining. The number of myelinated nerve fibers was analyzed by toluidine blue staining. The thickness of myelinated was analyzed by electron microscopy. Results:Compared with group F, the protein expression of GDNF and NGF increased in groups C, D and E (P < 0.05); the protein expression of Bcl-2 reduced and the protein expression of Bax and Caspase-3 increased in groups B, C, D, and E (P < 0.05); the protein expression of MHC-I and MHC-II decreased in all the pretreated groups (P < 0.05). Four weeks after cryopreservation, compared with groups F and G, the number of living cells decreased in groups C, D and E. One week after transplantation, compared with groups F' and G', the expression of CD8+ T cells and macrophages decreased, and the plasma concentration of IL-2 and TNF-α decreased in groups C', D' and E' (P<0.05). Twenty weeks after transplantation, CMAP, NCV, the wet weight ratio of gastrocnemius muscle, the number of axon and thickness of myelin sheath were better in groups C', D' and E' than in groups F' and G' (P<0.05), as well as the expression of NF200. Conclusion:ENTFs can be induced by pretreating the sciatic nerve in vitro. Cryopreserved sciatic nerve with high expression of ENTFs could promote nerve regeneration and functional recovery after allograft.
Résumé
The latest developments of techniques that prolong the half-life of protein drugs were reviewed.The therapeutic potential of several proteins is limited by their rapid clearance from the body,resulting in the need for frequent treatment of individuals to maintain drug levels.Several techniques including chemical modification,protein fusion,microsphere,glycosylation,and protease-resistant variants,and so on,prevent rapid degradation of such drugs and prolong their half-lives in vivo.Several successful protein drugs with prolonged half-life have already been on the market or on clinical trials.The principles,efficiencies and scopes of application of these techniques were described,compared and discussed.