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目的探讨参附注射液提高大鼠坐骨神经深低温玻璃化保存效果的可行性。方法 SPF级SD雄性大鼠坐骨神经分别在含有不同体积分数参附注射液(0、5%、10%、30%)的玻璃化液(A、B、C、D组)中深低温(-80℃)保存4周,透射电镜观察神经超微结构,Calcein-AM/PI双染色激光扫描共聚焦显微镜(LSCM)观察神经生物活性,Western blotting检测Bcl-2、Bax蛋白表达。用保存后的大鼠坐骨神经修复对应Wistar雄性大鼠(A′、B′、C′、D′组)坐骨神经10 mm缺损,术后分期观察大鼠一般情况、坐骨神经功能指数(SFI),第16周行电生理检测,再生神经组织学观察。结果透射电镜观察,A、B、C、D组均存在不同程度的脱髓鞘改变,但B、C、D组轻于A组;LSCM观察,B、C、D组绿色荧光较强,红色荧光较弱,A组绿色荧光较弱,红色荧光最广;Bcl-2和Bax蛋白表达,B、C、D组与A组间,C、D组与B组间差异显著(P<0.05),C、D组间差异不显著(P>0.05)。移植术后各期SFI,术后16周后电生理、再生神经有髓纤维数及髓鞘厚度,均显示C′、D′组与A′、B′组间差异显著(P<0.05),但A′、B′组间及C′、D′组间差异不显著(P>0.05)。术后16周再生神经超微结构,A′、B′组有髓神经纤维数较少、直径较细,分布稀疏,髓鞘较薄;C′、D′组再生有髓神经纤维数量较多,分布广泛、髓鞘较厚。结论参附注射液能提高大鼠坐骨神经玻璃化保存效果,促进异体移植后受体神经再生。
Objective To investigate the feasibility of Shenfu injection in enhancing the vitrification of deep sciatic nerve in rats. Methods The sciatic nerve of SPF SD male rats were exposed to deep hypothermia (-80) in vitreous humor (groups A, B, C and D) with different volume fractions of Shenfu injection (0, 5%, 10% and 30% ℃) for 4 weeks. The ultrastructures of neurons were observed under transmission electron microscope. The neurobiological activity was observed by Calcein-AM / PI double staining laser scanning confocal microscope (LSCM). The protein expressions of Bcl-2 and Bax were detected by Western blotting. The 10-mm sciatic nerve defect in Wistar male rats (A ’, B’, C ’and D’ groups) was repaired with the preserved sciatic nerve in rats. The general condition, sciatic nerve function index (SFI) Weekly electrophysiological examination, regeneration of nerve histology. Results The results of TEM showed that there were some demyelination changes in groups A, B, C and D, but the levels in groups B, C and D were lighter than those in group A. LSCM showed that the green fluorescence was stronger in groups B, C and D The fluorescence of A group was weak, the red fluorescence was the most extensive, the expression of Bcl-2 and Bax was the highest in group A, but there was significant difference between group B, C, D and group A, C and D (P <0.05) There was no significant difference between groups C and D (P> 0.05). SFI at each stage of transplantation, electrophysiological and medullary nerve fiber thickness and myelin sheath thickness at 16 weeks postoperatively showed significant differences (P <0.05) between C ’and D’ groups and A ’and B’ However, there was no significant difference between A ’, B’ groups and C ’, D’ groups (P> 0.05). At 16 weeks after operation, the ultrastructures of the regenerated nerve were observed. The number of myelinated nerve fibers in A ’and B’ groups was smaller, the diameter was thinner, the distribution was sparse and the myelin sheath was thinner. The number of regenerated myelinated nerve fibers in C ’and D’ groups was more , Widely distributed, thicker myelin. Conclusion Shenfu injection can enhance the vitrification of rat sciatic nerve and promote the regeneration of the recipient after allotransplantation.