参附注射液玻璃化保存的大鼠坐骨神经修复异体神经缺损研究

Sciatic nerve preserved by vitrification solution contains Shenfu Injection repaired allogenic nerve defects in a rat model

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 improving sciatic nerve vitrification preservation using Shenfu Injection in rats.Methods At -80℃,SPF male SD rat sciatic nerve segments were preserved in the vitrification solution containing Shenfu Injection （0%,5%,10%,30%,groups A,B,C and D） for 4 weeks.The sciatic nerves were observed by transmission electron microscope （TEM）,the biological activity of the nerve was detected by laser scanning confocal microscope （LSCM） after Calcein-AM and PI double staining,and the expressions of Bcl-2 and Bax of the sciatic nerves were tested by Western blotting.10 mm sciatic nerve defect of the Wistar rats （groups A＇,B＇,C＇ and D＇） was repaired using the corresponding SD rats sciatic nerves preserved for 4 weeks,the gross appearance and sciatic nerve function index （SFI） were detected at different periods after the surgery,the histological and electrophysiological changes of regenerating nerve were detected at 16 weeks post transplantation.Results The results of TEM showed that demyelination of the sciatic nerve in group A was severe,and in groups B,C and D were mild.The results of LSCM showed that there were strong green fluorescent and weak red fluorescent in groups B,C and D,and weak green fluorescent and strong red fluorescent in group A.The expression of Bcl-2 and Bax of the sciatic nerves,there were significant differences between groups B,C,D and group A,and between groups C,D and group B （P〈0.05）,but there was no significant difference between group C and group D （P〉0.05）.The SFI at different periods after the transplantation,the results of electrophysiological detection,the number of myelinated nerve fibers,and the thickness of myelin at 16 weeks after the surgery,there were significant differences between groups C＇,D＇ and groups A＇,B＇（P〈0.05）,but there was no significant differences between group A＇ and group B＇,and between group C＇ and group D＇（P〉0.05）.At 16 weeks after the surgery,the results by TEM observation showed that there were fewer fibers,smaller diameter,sparse distribution,thinner myelin sheath in group A＇ and group B＇,but the number of regenerated fibers was more,the distribution was extensive,and the myelin sheath was thicker in group C＇ and group D＇.Conclusion Shenfu Injection has protective effect on sciatic nerve preserved by vitrification,and can improve nerve regeneration after allograft in rats.