丹参酮Ⅱ_A磺酸钠促进低温保存的大鼠坐骨神经同种异体移植后神经再生

Effects of tanshinone Ⅱ_A sulfonate on nerve regeneration after cryopreserved sciatic nerve allograft in rats

探讨丹参酮ⅡA磺酸钠（tanshinoneⅡAsulfonate）对深低温保存大鼠坐骨神经活性以及同种异体移植后神经再生、功能恢复的保护作用及其可能的作用机制。将SPF级标准雄性SD大鼠坐骨神经片段15 mm,分别在含有不同浓度丹参酮ⅡA磺酸钠（A 0 mg·L-1,B 80 mg·L-1,C 160 mg·L-1,D 480 mg·L-1）中低温（-80℃）保存24周,另设有新鲜对照组,通过电镜观察保存后神经超微结构,calcein-AM/PI荧光染色激光共聚焦显微镜观察活细胞、死细胞数量,Western blot法检测Bax,Bcl-2的蛋白表达变化;冻存后神经体外培养7 d,PCR,Western blot法检测NGF,GDNF的mRNA以及蛋白表达水平。用保存24周的SD大鼠坐骨神经,修复对应的Wistar雄性大鼠坐骨神经10 mm缺损（A’,B’,C’,D’组）,术后16周,电生理检测肌肉复合动作电位潜伏期和神经传导速度,甲苯胺蓝染色分析再生有髓神经纤维数目和髓鞘厚度,电镜观察再生神经超微结构。结果显示,大鼠坐骨神经保存24周,与A,B组相比,C,D组脱髓鞘、空泡化程度较弱,活细胞数量较多,Bax表达降低,Bcl-2表达升高;与此同时,保存24周后的坐骨神经NGF,GDNF mRNA及蛋白表达,C,D组均显著高于A,B组。同种异体移植16周后,与A’,B’组相比,C’,D’组再生有髓神经纤维数量较多,分布广泛、髓鞘较厚,肌肉复合动作电位潜伏期缩短,神经传导速度升高。这表明丹参酮ⅡA磺酸钠对-80℃长期保存的大鼠坐骨神经具有保护作用,能维持保存后神经的生物活性,促进同种异体移植后受体神经再生,其中以中、高浓度（C,D组）效果更好。

In order to investigate the protective effect of tanshinone ⅡAsulfonate on the sciatic nerve activty in rats after cryopreservation as well as the nerve regeneration and functional recovery after allograft and its possible mechanism,Sprague-Dawley（ SD） rats were divded into four groups at different doses of tanshinone ⅡAsulfonate（ A 0 mg·L-1,B 80 mg·L-1,C 160 mg·L-1,D 480 mg·L-1） cryopreserved at-80 ℃ for 24 weeks. Fresh control group nerve segments were harvested without cryopreservation. The ultrastructure and the viable cells of the nerve segments after cryopreservation were observed by electron microscopy,calcein-AM/propidium iodide staining,respectively. The expression of Bax and Bcl-2 was detected by Western blot. After cryopreservation,the nerve segments were cultured in vitro for one week,the mRNA and protein level of NGF and GDNF were detected by PCR and Western blot respectively. In addition,the above four cryopreserved groups transplanted to the Wistar rats by allografting（ A’,B’,C’,D’）. At 16-week postoperation,muscle compound action potential latency and nerve conduction velocity were examined by electrophysiological.The number and the thickness of myelinated nerve fibers were analyzed by toluidine blue staining. The ultrastructure of the sciatic nerve by electron microscopy was observed. According to the results,after the cryopreserved for 24 weeks,compared with groups A and B,the nerve demyelination and vacuolation were weak,and the more viable cells,the decreased Bax and increased Bcl-2,the increased NGF and GDNF in group C and D. At 16-week poseoperation,the results demonstrated that the more larger and thickly regenerated myelinated axons,the shorter latency of muscle compound action potentials and higher nerve conduction velocity in groups C’ and D’compared with groups A’ and B’. According to these results,tanshinone ⅡAsulfonate exerted a significant protective effect on the viability of the nerves during cryopreservation at-80 ℃ and promoted nerve regeneration and functional recovery after transplantation especially in middle-and high-dose of tanshinone ⅡAsulfonate.