骨骼肌来源细胞移植早期修复小鼠周围神经缺损的实验研究

Experimental study on early repair of peripheral nerve defect in mice by transplantation of muscle-derived cells

目的通过观察损伤周围神经早期生长情况,探讨骨骼肌来源细胞(muscle-derived cells,MDCs)修复小鼠坐骨神经缺损的作用机制。方法收集携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)小鼠的后肢骨骼肌,提取EGFP-MDCs并培养至P1代备用。构建C57BL/6小鼠右侧坐骨神经5 mm长缺损模型,并用7 mm长的聚氨酯(polyurethane,PUR)导管桥接两神经断端,将EGFP-MDCs注入PUR导管内(MDC组),与空白组(PUR组)比较。于术后1、2周取术侧坐骨神经近侧断端及远侧断端神经组织,大体观察神经早期生长情况;对神经组织的纵行冰冻切片行雪旺细胞标志物S100β免疫荧光染色,计算小鼠再生神经内雪旺细胞迁移的最远距离总和,并观察表达S100β的雪旺细胞来源;对神经组织横行冰冻切片行磷酸化酪氨酸激酶受体(phosphorylated erb-b2 receptor tyrosine kinase 2,p-ErbB2)及磷酸化黏着斑激酶(phosphorylated focal adhesion kinase,p-FAK)免疫荧光染色,计算二者蛋白阳性率。结果大体观察示术后1、2周PUR组术侧神经的近侧和远侧断端均未相连,而MDC组术后2周两侧神经断端已连接。免疫荧光染色示,术后1周,MDC组及PUR组术侧神经的近侧和远侧断端内表达S100β的雪旺细胞并未相连;术后2周,MDC组两侧神经断端内表达S100β的雪旺细胞已连接,而PUR组仍未连接。术后2周,两组再生神经内雪旺细胞迁移的最远距离总和分别较各组术后1周显著增加,术后1、2周MDC组显著高于PUR组,差异均有统计学意义(P<0.05)。术后1周,MDC组近侧断端p-ErbB2及p-FAK蛋白阳性率均显著高于PUR组(P<0.05);术后2周两组近侧断端p-ErbB2蛋白阳性率差异无统计学意义(t=0.327,P=0.747),MDC组p-FAK蛋白阳性率显著高于PUR组(t=4.470,P=0.000)。术后1、2周,MDC组远侧断端p-ErbB2及p-FAK蛋白阳性率均显著高于PUR组(P<0.05)。术后1、2周,MDC组再生神经内均可见部分表达S100β的雪旺细胞来源于EGFP-MDCs。结论 MDCs可促进小鼠坐骨神经断端内ErbB2及FAK蛋白磷酸化,促进雪旺细胞迁移。MDCs可分化为表达雪旺细胞标志物S100β的细胞,或作为其他细胞成分参与周围神经损伤的早期修复。

Objective To investigate the mechanism of muscle-derived cells(MDCs)in repairing sciatic nerve defects in mice by observing the early growth of damaged peripheral nerves.Methods The hind limb skeletal muscles of mice carrying enhanced green fluorescent protein(EGFP)was collected to extract and culture EGFP-MDCs to P1 generation for later experiments.Five-mm-long nerve defects were created in the right sciatic nerves of C57 BL/6 mice to establish a peripheral nerve defect model.The two stumps of sciatic nerve were bridged with 7-mm-long polyurethane(PUR)conduit.For the MDC group,EGFP-MDCs were injected into the PUR conduit.The PUR group without EGFPMDCs was used as the negative control group.At 1 and 2 weeks after operation,the proximal and distal nerve stumps of the surgical side were collected to generally observe the early growth of nerve.Immunofluorescence staining of S100β,the marker of Schwann cells,was performed on longitudinal frozen sections of nerve tissues to calculate the maximum migration distance of Schwann cells,and observe the source of the Schwann cells expressing S100β.Immunofluorescence staining of phosphorylated erb-b2 receptor tyrosine kinase 2(p-ErbB2)and phosphorylated focal adhesion kinase(p-FAK)in transverse frozen sections of nerve tissue was performed to calculate the positive rates of both proteins.Results The general observation showed that the proximal and distal stumps of the surgical side in PUR group were not connected at 1 and 2 weeks after operation,while the bilateral nerve stumps in the MDC group were connected at 2 weeks after operation.Immunofluorescence staining showed that the Schwann cells expressing S100βin proximal and distal nerve stumps of PUR group and MDC group was not connected at 1 week after operation.At 2 weeks after operation,the Schwann cells expressing S100βin the two nerve stumps of the MDC group were connected,but not in the PUR group.At2 weeks after operation,the sum of the maximum migration distance of Schwann cells in the regenerated nerve in both two groups was significantly increased when compared with that in each group at 1 week after operation,and that of MDC group was significantly higher than that in the PUR group at both 1 and 2 weeks after operation,the differences were all significant(P<0.05).At 1 week after operation,the positive rates of p-ErbB2 and p-FAK in the proximal nerve stump of MDC group were significantly higher than those in PUR group(P<0.05).There was no significant difference in the positive rate of p-ErbB2 of proximal stump between the two groups at 2 weeks after operation(t=0.327,P=0.747),while the positive rate of p-FAK of MDC group was significantly higher than that of PUR group(t=4.470,P=0.000).At 1 and2 weeks after operation,the positive rates of p-ErbB2 and p-FAK in the distal stump of MDC group were significantly higher than those in PUR group(P<0.05).At 1 and 2 weeks after operation,part of Schwann cells expressing S100β,which were derived from EGFP-MDCs,could be observed in the regenerated nerves of MDC group.Conclusion MDCs can promote the phosphorylation of ErbB2 and FAK in the nerve stumps of mice,and promote the migration of Schwann cells.MDCs can be differentiated into cells expressing the Schwann cell marker S100β,or as other cellular components,to involve in the early repair of peripheral nerves.