目的 探讨雷公藤甲素(T10)对冷保存大鼠坐骨神经生物活性和异体移植后神经再生的影响。方法 取对数生长期施万细胞(SCs),CCK-8检测1×10-6 mol/L、1×10-7 mol/L、1×10-8 mol/L、1×10-9 mol/L T10溶液对SCs增殖的影响。取Sprague-Dawley大鼠双侧坐骨神经15 mm,分别在含0 mol/L、1×10-6 mol/L、1×10-7 mol/L、1×10-8 mol/L、1×10-9 mol/L T10的DMEM液中,4 ℃或37 ℃ 放置24 h (n = 6),Western blotting检测神经生长因子(NGF)、神经胶质细胞源性神经营养因子(GDNF)、脑源性神经营养因子(BDNF)表达。另取大鼠坐骨神经15 mm,随机分成新鲜神经组(A组, n = 30)、DMEM保存组(B组, n = 30)、T10保存组(C组, n = 30)、T10预处理后DMEM保存组(D组, n = 30)、T10预处理后T10保存组(E组, n = 30),4 ℃保存4周,Calcein-AM/PI双染激光共聚焦显微镜观察、流式细胞术检测神经活细胞、死细胞数,Western blotting检测主要组织相容性复合体-Ⅰ(MHC-Ⅰ)、MHC-Ⅱ、细胞间黏附分子-1 (ICAM-1)表达。用上述坐骨神经修复Wistar大鼠坐骨神经10 mm缺损(A′组、B′组、C′组、D′组、E′组, n = 10),设立同系移植新鲜组(F′组, n = 10)。术后16周,电生理检测肌肉复合动作电位(CMAP)和运动神经传导速度(MNCV);取移植段神经,观察神经纤维数和神经超微结构。结果 SCs在浓度为1×10-9~1×10-7 mol/L T10溶液下,细胞增殖与对照组无显著性差异(P > 0.05)。各浓度T10溶液下,大鼠坐骨神经各神经营养因子表达均37 ℃明显高于4 ℃;相同温度时,1×10-8 mol/L组各神经营养因子表达均高于其他浓度组(P < 0.05)。大鼠坐骨神经冷保存4周后,与B、C、D组相比,E组神经活细胞数量多,MHC-I、MHC-II、ICAM-1表达降低(P < 0.05)。移植术后16周,E′组CMAP、MNCV、再生有髓神经纤维数量均优于A′、B′、C′、D′组(P < 0.05),E′、F′组有髓神经纤维数量多,粗细均匀,分布广泛,髓鞘厚。结论 一定浓度T10体外能诱导大鼠坐骨神经神经营养因子表达,提高冷保存神经生物活性,降低免疫原性,促进异体移植后受体神经再生;冷保存前用一定浓度T10预处理效果更好。
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.
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