组织工程神经联合川芎嗪移植修复大鼠坐骨神经缺损

Tissue-engineered nerve graft and tetramethylpyrazine for repair of rat ischiadic nerve defect

目的观察组织工程神经联合川芎嗪移植修复大鼠坐骨神经缺损的效果。方法55只成年SD大鼠按随机数字表法分为4组，A、B、C组各15只，D组10只，实验侧均为右后肢坐骨神经。10g/L戊巴比妥钠腹腔注射麻醉后，显露坐骨神经，从梨状肌下缘0．5cm处切除神经1．5cm，分别用4种移植物桥接神经缺损：去细胞异体神经种植神经干细胞（NSCs）在含川芎嗪培养液中培养1周构成组织工程神经（A组）、去细胞异体神经种植NSCs在普通培养液中培养1周构成组织工程神经（B组）、去细胞异体神经（C组）、自体神经（D组）。术后每天A组术侧注射川芎嗪液，B、C、D组注射等体积9g／L等渗盐水，连续注射12周至实验结束。通过术后2周行坐骨神经功能指数（SFI）测定、神经电生理检测、免疫荧光、免疫组化检测；术后12周行SFI测定、神经电生理检测、免疫荧光、免疫组化、辣根过氧化物酶（HRP）逆行示踪、再生神经纤维检测、透射电镜、腓肠肌湿重等指标观察神经修复情况。结果术后2周各组SFI测定和电生理检测结果差异无统计学意义；免疫荧光A、B组均可见大量强荧光，C组未见荧光细胞；免疫组化A、B组以巢蛋白阳性细胞为主。术后12周，A组SFI为-17．52±2．41，B组为-25．74±2．85，C组为-36．12±3．41，D组为-15．87±2．26；A组较B、C组差异有统计学意义（P〈0．05），与D组差异无统计学意义（P〉0．05）。A组神经传导速度为（9．43±0．40）m／s，B组为（7．76±0．31）m／s，C组为（5．87±0．67）m／s，D组为（10．16±0．39）m／s；A组较B、C组差异有统计学意义（P〈0．05），与D组差异无统计学意义（P〉0．05）。NSCs能在体内存活、迁移并分化为神经元和胶质细胞。HRP标记的细胞数A组为（885．40±19．91）个，B组为（684．57±38．37）个，C组为（390．33±43．41）个，D组为（941．67±32．54）个；A、D组较B、C组差异有统计学意义（P〈0．05）。甲苯胺蓝染色示A组移植段再生神经纤维较多，以有髓神经纤维为主。结论去细胞异体神经种植NSCs在含川芎嗪培养液中培养构成组织工程神经，能有效修复大鼠坐骨神经缺损，促进周围神经再生及下肢运动功能的恢复。

Objective To evaluate the effect of tissue-engineered nerve graft combined with tetramethylpyrazine in the repair of ischiadic nerve defect in rats. Methods Fifty-five adult SD rats were allocated into groupsA （n=15）, B （n=15）, C （n=15） and D （n=10）, according to the random number table. All experimental sciatic nerves were at the right hind side. After the rats were anesthetized with 10 g/L pentobarbital through abdominal injection, a 1.5 cm nerve sciatic nerve was excised at the point 0.5 cm away from the lower margin of the piriformis. Four grafts were used to bridge the nerve defect, including tissue-engineered nerve transplanted neural stem cells （ NSCs ） that were cultured in medium containing tetramethylpyrazine for 1 week （group A） , tissue-engineered nerve trans- planted NSCs that were cultured in tetramethylpyrazine-free medium for 1 week （group B ） , acellularnerve allograft （ group C） , and nerve autograft （ group D ）. After operation, group A was administered tetramethylpyrazine for 12 weeks in the operative site. For other groups, the same volume of normal saline was used instead of tetramethylpyrazine. Repair results were detected with the sciatic function index （SFI）, nerve electrophysiological evaluation, fluorescent microscopy, horseradish peroxidas （HRP） retrograde tracer and nerve fiber regeneration study. Results At postoperative 2 weeks, no differences were found in SFI and electrophysiological evaluation among the groups, there was an intensive fluorescence in groups A and B and no fluorescence in group C, and large nestin - positive cells were observed in groups A and B. At postoperative 12 weeks, SFI was -17.52 ±2.41 in group A, -25.74 ± 2.85 in group B, -36. 12 ± 3. 41 in group C and -15. 87 ± 2. 26 in group D; electrophysiological evaluation showed nerve conduction velocity of （9.43 ±0. 40） m/s in group A, （7.76 ± 0.31 ） m/s in group B, （5.87 ±0.67）m/s in group C and （10.16 ±0.39）m/s in group D; fluorescence microscope and histological staining showed NSCs could survive in vivo, migrate and differentiate into neurons and gliocytes ; HRP-positive neurons were 885.40 ±19.91 in group A, 684.57 ± 38.37 in group B, 390.33 ±43.41 in group C and 941.67±32.54 in group D; regenerated nerve fibers, often myelinated were large in group A. All the measures in group A were better compared to groups B and C （ P 〈 0.05 ） , but didn＇t differ from those in group D （P 〉0.05）. Conclusion Tissue-engineered nerve transplanted NSCs cultured in medium including tetramethylpyrazine can effectively repair rat sciatic nerve defect, and promote peripheral nerve regeneration and lower limb motor function recovery.