化学去细胞异体神经添加不同组织来源雪旺细胞对周围神经损伤修复的功能评价

FUNCTIONAL EVALUATION OF CHEMICALLY EXTRACTED ACELLULAR NERVE ALLOGRAFT SUPPLEMENT WITH DIFFERENT TISSUES OF SCHWANN CELLS FOR PERIPHERAL NERVE REGENERATION

目的构建不同组织来源雪旺细胞(Schwann cells,SCs)及化学去细胞异体神经(chemically extracted acellular nerve allograft,CEANA)移植物,比较其修复周围神经缺损的效果。方法 4周龄SD大鼠3只,体重80～120g,体外培养、扩增和鉴定BMSCs及脂肪来源干细胞(adipose-derived stem cells,ADSCs)。体外诱导BMSCs和ADSCs分化为类雪旺细胞(dMSC,dADSC),并采用胶质细胞标记物p75和抗胶原纤维酸性蛋白(glial fi brillary acidic protein,GFAP)进行鉴定。取出生3d SD大鼠10只,体重6～8g,分离培养SCs。20只成年Wistar大鼠,体重200～250g,取双侧约20mm坐骨神经制备CEANA。40只成年雄性SD大鼠,制备左侧15mm坐骨神经缺损模型,根据神经缺损修复方法不同随机分为5组(n=8):A组,取自体坐骨神经翻转后吻合;B组,取15mm CEANA吻合后补充5×105个SCs;C组,取15mm CEANA吻合后补充5×105个dMSC;D组,取15mm CEANA吻合后补充5×105个dADSC;E组,取15mm CEANA吻合。术后12周行感觉和运动功能恢复评价及组织学评价。结果 BMSCs和ADSCs表面抗原标志为CD34-、CD45-、CD90+。经诱导后BMSCs和ADSCs形态变化与SCs相似,呈双极、星形结构,SCs标记物p75和GFAP均表达阳性。术后12周,A、B、C、D、E组肢体50%回缩阈值分别为(13.8±2.3)、(15.4±6.5)、(16.9±5.3)、(16.3±3.5)和(20.0±5.3)g,A组与E组比较差异有统计学意义(P<0.01),B、C、D组间比较差异无统计学意义(P>0.05)。A、B、C、D、E组小腿三头肌收缩力恢复率分别为87.0%±9.7%、70.0%±6.6%、69.0%±6.7%、65.0%±9.8%和45.0%±12.1%,A、B、C、D组与E组比较差异有统计学意义(P<0.05)。各组远端吻合口神经坚牢蓝染色显示神经纤维排列整齐,无炎性反应。甲苯胺蓝染色和透射电镜观察显示,B、C、D组有髓神经纤维计数及有髓神经纤维髓鞘厚度均大于E组,差异均有统计学意义(P<0.01);B组轴突直径大于C、D组,差异有统计学意义(P<0.05)。结论 CEANA补充dADSC修复周围神经缺损与补充dMSC和SCs具有类似的修复效果。dADSC来源广泛,可作为组织工程神经理想的种子细胞,在复合CEANA修复周围神经缺损发挥重要作用。

Objective To construct chemically extracted acellular nerve allograft（CEANA） with Schwann cells（SCs） from different tissues and to compare the effect of repairing peripheral nerve defect. Methods Bone marrow mesenchymal stem cells（BMSCs） and adipose-derived stem cells（ADSCs） were isolated and cultured from 3 4-week-old SD mice with weighing 80-120 g.BMSCs and ADSCs were induced to differentiated MSC（dMSC） and differentiated ADSC（dADSC） in vitro.dMSC and dADSC were identified by p75 protein and glial fibrillary acidic protein（GFAP）.SCs were isolated and cultured from 10 3-day-old SD mice with weighing 6-8 g.CEANA were made from bilateral sciatic nerves of 20 adult Wistar mice with weighing 200-250 g.Forty adult SD mice were made the model of left sciatic nerve defect（15 mm） and divided into 5 groups（n=8 per group） according to CEANA with different sources of SCs：autografting（group A）,acellular grafting with SCs（5 × 105）（group B）,acellular grafting with dMSCs（5 × 105）（group C）,acellular grafting with dADSCs（5 × 105）（group D）,and acellular grafting alone（group E）.Motor and sensory nerve recovery was assessed by Von Frey and tension of the triceps surae muscle testing 12 weeks after operation.Then wet weight recovery ratio of triceps surae muscles was measured and histomorphometric assessment of nerve grafts was evaluated. Results BMSCs and ADSCs did not express antigens CD34 and CD45,and expressed antigen CD90.BMSCs and ADSC were differentiated into similar morphous of SCs and confirmed by the detection of SCs-specific cell-surface markers.The mean 50% withdrawal threshold in groups A,B,C,D,and E was（13.8 ± 2.3）,（15.4 ± 6.5）,（16.9 ± 5.3）,（16.3 ± 3.5）,and（20.0 ± 5.3） g,showing significant difference between group A and group E（P 0.01）.The recovery of tension of the triceps surae muscle in groups A,B,C,D,and E was 87.0% ± 9.7%,70.0% ± 6.6%,69.0% ± 6.7%,65.0% ± 9.8%,and 45.0%± 12.1%,showing significant differences between groups A,B,C,D,and group E（P 0.05）.No inflammatory reaction existed around nerve graft.The histological observation indicated that the number of myelinated nerve fiber and the myelin sheath thickness in group E were significantly smaller than that in groups B,C,and D（P 0.01）.The fiber diameter of group B was significantly bigger than that of groups C and D（P 0.05） Conclusion CEANA supplementing with dADSC has similar repair effect in peripheral nerve defect to supplementing with dMSC or SCs.dADSC,as an ideal seeding cell in nerve tissue engineering,can be benefit for treatment of peripheral nerve injuries.