嗅黏膜嗅鞘细胞与肌基膜管联合移植修复脊髓损伤

目的：观察嗅黏膜来源的嗅鞘细胞与肌基膜管联合移植后对脊髓损伤的修复效果。 方法：1只SD大鼠行背正中切口，顺椎旁肌纤维切除约1.5 cm×0.8 cm×0.6 cm的肌条，复温、漂洗并挤压肌条以排出肌浆，制成肌基膜管。4只SD大鼠麻醉后取出嗅黏膜，胶原酶消化法分离培养嗅鞘细胞，调整浓度至1011 L-1。取SD大鼠50只，随机分成5组：嗅鞘细胞+肌基膜管联合组、嗅鞘细胞组、肌基膜管组、模型组、正常组，10只/组。除正常组外，其余组均建立脊髓损伤模型，于T10横断脊髓并切除约2 mm，将培养7 d的嗅鞘细胞与肌基膜管按组别分别植入脊髓断端，模型组用浸有DMEM的凝胶海绵桥接横断的脊髓。 结果：移植后第8周，嗅鞘细胞+肌基膜管联合组、嗅鞘细胞组大鼠运动功能明显恢复，出现大关节大幅度运动，且前者运动功能BBB评分升高尤为显著（P < 0.01）；肌基膜管组大鼠仅见小关节轻微活动；模型组大鼠后肢挛缩重，无明显功能恢复。嗅鞘细胞+肌基膜管联合组后肢体感诱发电位及运动诱发电位的潜伏期显著低于其他各组（P < 0.01）。苏木精-伊红染色和核转录因子免疫组化染色结果显示，嗅鞘细胞+肌基膜管联合组、嗅鞘细胞组的移植物与损伤脊髓整合较好，未见明显空洞，有大量染色呈阳性的纤维，纤维较长，由近侧端长入远侧端；肌基膜管组有空洞形成，染色阳性的纤维数量少，纤维细小且排列紊乱；模型组端断间充满瘢痕组织，未见明显染色阳性纤维。 结论：嗅鞘细胞移植可促进脊髓损伤后的轴突再生，肌基膜管作为一种生物管道，两者联合应用可明显促进脊髓损伤后的轴突再生及功能恢复。

Combination of olfactory ensheathing cells from the olfactory mucosa and muscle basal lamina for the treatment of spinal cord injury

OBJECTIVE: To study the effect of olfactory ensheathing cells from olfactory mucosa and muscle basal lamina on spinal cord injury following combined transplantation of muscle basal lamina. METHODS: One Sprague Dawley rats received a median incision on the back. About 1.5 cm×0.8 cm×0.6 cm muscle was removed along the muscle fiber, re-warmed, washed and crushed to discard sarcoplasm so as to make muscle basal lamina. Olfactory mucosa was harvested from 4 Sprague Dawley rats following anesthesia. Olfactory ensheathing cells were collected and culture by collagenase digestion at 1011/L. A total of 50 Sprague Dawley rats were randomly assigned into 5 groups, olfactory ensheathing cells + muscle basal lamina group, olfactory ensheathing cells group, muscle basal lamina group, model group and normal group, 10 rats for each group. Except the normal group, spinal cord injury models were created in other groups. 2-mm spinal cord was excised following transaction at T10. Olfactory ensheathing cells and muscle basal lamina were implanted into the broken ends of the spinal cord at 7 days. Bridge grafting was performed using gel sponge dipped with DMEM in the model group. RESULTS: At 8 weeks following transplantation, motor function of rats in the olfactory ensheathing cells + muscle basal lamina group, olfactory ensheathing cells group was significantly improved, with the presence of gross movement of big joints. Basso Beattie and Bresnahan score was greater in the former group (P < 0.01). Slight activity of small joints were shown in the muscle basal lamina group. Contracture of the posterior limb was severe, no significant improvement in the model group. Latencies of somatosensory evoked potential and motion evoked potential were significantly lower in the olfactory ensheathing cells + muscle basal lamina group compared with other groups (P < 0.01). Hematoxylin-eosin staining and nuclear factor immunohistochemistry showed a good integration of grafts and the injured spinal cord, with the presence of positive long fibers in the olfactory ensheathing cell + muscle basal lamina group and olfactory ensheathing cell group; Porosis was displayed in the muscle basal lamina group, with a few irregular positive fibers. Scar tissues were filled in the broken region in the model group. CONCLUSION: Olfactory ensheathing cell transplantation can promote axon regeneration after spinal cord injury. As a biological pipe, the combination of muscle basal lamina and olfactory ensheathing cells can significantly improve the axonal regeneration and function recovery after spinal cord injury.