关键词: 异体神经移植, 周围神经保存, 冷保存损伤, 雷公藤甲素, 神经再生, 坐骨神经, 大鼠

Abstract: Objective To investigate the effects of triptolide (T10) on biological activity of sciatic nerve in cold preservation and nerve regeneration after allogeneic transplantation. Methods Cell Counting Kit-8 (CCK-8) was used to test the proliferation of SCs in logarithmic phase in 1×10^-6 mol/L, 1×10^-7 mol/L, 1×10^-8 mol/L and 1×10^-9 mol/L of T10 solution. The sciatic nerves from Sprague-Dawley rats were pretreated in 0 mol/L, 1×10^-6 mol/L, 1×10^-7 mol/L, 1×10^-8 mol/L and 1×10^-9 mol/L of T10 solution at 4 ℃ or 37 ℃ for 24 hours (n = 6). The expression of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) was detected with Western blotting. Other sciatic nerve fragments were randomly divided into fresh nerve group (group A, n = 30), DMEM preservation group (group B, n = 30), T10 preservation group (group C, n = 30), T10 pretreatment DMEM preservation group (group D, n = 30) and T10 pretreatment T10 preservation (group E, n = 30), and were stored under 4 ℃ for four weeks. Calcein-AM/PI double staining laser confocal microscope and flow cytometry were used to detect the living cells and dead cells. The expression of the major histocompatibility complex (MHC)-I, MHC-II and intercellular cell adhesion molecule-1 (ICAM-1) was detected with Western blotting. The corresponding sciatic nerves were used to repaire 10 mm defects in Wistar rats (named groups A', B', C', D' and E'), and fresh sciatic nerve from Wistar rats were also used to do it (group F'). Compound muscle action potential (CMAP) and motor nerve conduction velocity (MNCV) were tested 16 weeks after transplantation, and then the grafts were observed for the nerve regeneration. Results SCs proliferated as the controls in the T10 solution with a concentration of 1×10^-9 to 1×10^-7 mol/L (P > 0.05). The expression of all the neurotrophic factors was more under 37 ℃ than under 4 ℃ in all the concentrations of T10 solution, and it was the most in the concentration of 1×10^-8 mol/L whenever under 37 ℃ or 4 ℃ (P < 0.05). After four weeks of cold preservation, compared with groups B, C and D, the living nerve cells were the most in group E, and the expression of MHC-I, MHC-II and ICAM-1 was the least (P < 0.05). CMAP, MNCV and the never regeneration were better in group E' than in groups A', B', C' and D' (P < 0.05). A large number of myelinated nerve fibers were observed in groups E' and F', uniformity in size, wide distribution, and with myelin sheath, compared with those in groups A', B', C' and D'. Conclusion A certain concentration of T10 can induce the sciatic nerve of rats to express neurotrophic factor in vitro, which can improve the biological activity of cold preservation nerves, reduce the immunogenicity, and promote the regeneration of recipient nerve after allogeneic transplantation. It is even better to be pretreated with T10 before cold preservation.

Key words: allogeneic nerve transplantation, peripheral nerve preservation, cold preservation damage, triptolide, nerve regeneration, sciatic nerve, rats