壳聚糖支架复合嗅鞘细胞修复坐骨神经损伤

doi:10.3969/j.issn.2095-4344.2014.21.015

摘要/Abstract

摘要：

背景：嗅鞘细胞可促进中枢神经损伤修复。将壳聚糖与胶原结合制备复合工程材料，已经广泛应用于组织工程化神经导管的构建。
目的：探讨嗅鞘细胞复合壳聚糖用于治疗大鼠坐骨神经损伤的修复作用。
方法：建立坐骨神经缺损大鼠模型，用联合培养的原代培养大鼠嗅鞘细胞和壳聚糖支架连接于缺损处，设为嗅鞘细胞结合壳聚糖组；单纯壳聚糖支架组用单纯壳聚糖支架连接缺损；空白单纯壳聚糖支架组不作任何处理。
结果与结论：建模后1-4周分别检测大鼠坐骨神经功能指数，运动诱发电位潜伏期以及组织学检查发现，与嗅鞘细胞结合壳聚糖组大鼠坐骨神经功能指数显著升高(P < 0.05)，运动诱发电位潜伏期显著低于单纯壳聚糖支架组和空白对照组(P < 0.01)，且嗅鞘细胞结合壳聚糖组有新生的神经达到远侧端，周围少有炎症反应。说明嗅鞘细胞结合壳聚糖支架修复坐骨神经损伤效果较好。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

关键词: 组织工程, 材料相容性, 嗅鞘细胞, 壳聚糖, 支架, 坐骨神经, 周围神经损伤, 炎症

Abstract:

BACKGROUND: Olfactory ensheathing cells can promote the repair of the central nervous system. Composite engineering materials prepared by the combination of chitosan and collagen have been widely used in the construction of tissue-engineered nerve conduits.
OBJECTIVE: To explore the repair effect of olfactory ensheathing cells and chitosan in the treatment of sciatic nerve injury in rats.
METHODS: Rat models of sciatic nerve injury were prepared. Olfactory ensheathing cells combined with chitosan scaffold were used to connect the injured sciatic nerve. In the chitosan scaffold group, only the chitosan scaffold was utilized. In the control group, no treatment was done.
RESULTS AND CONCLUSION: At 1–4 weeks following surgery, sciatic functional index and motion evoked potential were monitored and histological examination was performed. Sciatic functional index was significantly improved in the olfactory ensheathing cells+chitosan scaffold group (P < 0.05). Motion evoked potential was significantly lower in the olfactory ensheathing cells+chitosan scaffold group compared with other groups (P < 0.001). Histological examinations showed new nerve fibers and rare inflammatory reaction in the olfactory ensheathing cells+chitosan scaffold group. These findings indicate that autologous olfactory ensheathing cells combined with chitosan scaffold exerts good repair effects on treatment of sciatic nerve injury, and can be considered as an ideal tissue engineering material.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: peripheral nerves, chitosan, sciatic nerve, inflammation

中图分类号:

R318

常瑞，阴小龙，尚保生，何鹏. 壳聚糖支架复合嗅鞘细胞修复坐骨神经损伤[J]. 中国组织工程研究, 2014, 18(21): 3361-3366.

Chang Rui, Yin Xiao-long, Shang Bao-sheng, He Peng . Combination of olfactory ensheathing cells and chitosan in treatment of peripheral nerve injury[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(21): 3361-3366.

图/表（结果） 4

2.1 实验动物数量分析 纳入大鼠30只，无死亡和感染，均进入结果分析。

2.2 大鼠嗅鞘细胞的分离、纯化和鉴定结果 嗅鞘细胞原代培养24 h内大部分细胞已贴壁，呈球形，透亮，培养第7天后有细胞分化，发出纤细的突起，细胞成束排列，培养第10天，双极细胞与三极细胞连成网络状，突起更细更长。免疫细胞化学染色结果显示，培养10天的细胞染色深，以三角形和条索状为主，多极性非常明显，多为阳性免疫反应。见图2。

2.3 嗅鞘细胞复合壳聚糖支架修复坐骨神经损伤大鼠一般形态变化 各组大鼠建模后大体观察所有大鼠均正常存活，各组均出现足肿胀，但无足底溃疡、化脓或感染。表明细胞移植不会引起排斥反应，安全良好。建模后1 d，各组SD大鼠患肢足趾弯曲，无法伸展，行走时拖拽，针刺无收缩反应。建模后2周，除空白单纯壳聚糖支架组外，其他2组SD大鼠肌肉萎缩有恢复，行走时拖曳程度降低，针刺能引起微弱的收缩。修复后4周，除空白单纯壳聚糖支架组外，其他2组大鼠肌肉萎缩恢复，僵直消失，其中嗅鞘细胞复合壳聚糖支架组的大鼠足趾有较大握力，存在明显针刺反应。

2.4 嗅鞘细胞复合壳聚糖支架修复后大鼠坐骨神经功能变化 表1显示建模后1-4周各组大鼠坐骨神经指数变化。建模后1周，各组坐骨神经功能指数大致相同，约为-83.6，1周后各组大鼠的坐骨神经功能指数均随时间的延长而提高。修复后4周，嗅鞘细胞复合壳聚糖支架组修复速率最快，3组间坐骨神经功能指数比较均差异有显著性意义(P < 0.05)。

2.5 嗅鞘细胞复合壳聚糖支架修复后大鼠坐骨神经运动诱发电位变化 建模后2，4周，各组运动诱发电位潜伏期检测结果见表2。结果显示，修复后4周时，3组大鼠均可引出运动电位，但嗅鞘细胞复合壳聚糖支架组运动电位潜伏期短于其他两组(P < 0.01)。

2.6 嗅鞘细胞复合壳聚糖支架修复后大鼠坐骨神经组织学形态 苏木精-伊红染色显示，修复后4周，坐骨神经组织纵切面光学显微镜可见，嗅鞘细胞复合壳聚糖支架组有新生的神经达到远侧端，且再生神经周围没有炎症反应，证明壳聚糖支架具有良好的组织相容性，见图3A。空白单纯壳聚糖支架组神经束内大部分空间被纤维组织填充，仅见少量神经纤维，见图3B。单纯壳聚糖支架组新生神经干数目明显多于空白单纯壳聚糖支架组，但是神经束排列没有规律，密度较小，见图3C。扫描电镜下显示，嗅鞘细胞复合壳聚糖支架组嗅鞘细胞与支架复合情况显示，材料的内部结构疏松排列，多孔结构规则有序，说明嗅鞘细胞与支架复合贴合良好，见图3D。

2.7 移植后生物相容性及不良反应评价 嗅鞘细胞复合壳聚糖支架修复坐骨神经损伤大鼠生物相容性良好，无不良反应发生。

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引言

材料方法

设计：随机对照动物实验。

时间及地点：于2012年1月至2013年2月在西安交通大学医学院实验室完成。

材料：

动物：成年健康SD大鼠，30只，雌雄各半，体质量(220.0±10.8) g，由西安交通大学医学院实验动物中心提供。实验对动物的处理方法符合中华人民共和国科学技术部颁发的《关于善待实验动物的指导性意见》^[5]。

方法：

嗅鞘细胞的分离、纯化和鉴定：无菌条件下取SD大鼠嗅球，剪成小组织块，用体积分数0.25%胰酶消化，后使用添加胎牛血清的DMEM-F12培养基中和，吹打后接种。采用改良的NASH差速贴壁法纯化3次^[6]，培养第10天取部分嗅鞘细胞用多克隆兔抗人p75，NGFR抗体进行免疫细胞化学染色鉴定，镜下表现为嗅鞘细胞可染色为深棕黄色的多级性细胞。

壳聚糖与明胶复合支架的制备：壳聚糖溶于体积分数2%的醋酸水溶液，过滤。将明胶溶于60 ℃蒸馏水中，过滤得体积分数6%的明胶水溶液。将明胶溶液和壳聚糖凝胶按1∶1比例混匀一定比例混合于50 ℃热水浴中充分搅拌混合，50 ℃水浴48 h，注入玻璃培养皿，置于-20 ℃和-80 ℃冷冻12 h后，再抽干48 h，制成壳聚糖明胶多孔复合支架备用。

嗅鞘细胞与壳聚糖支架材料的复合：将纯化的嗅鞘细胞稀释为1×10⁸ L^-¹，与壳聚糖明胶支架材料混合培养。培养至1周时，吸去培养液，常规电镜切片，JEN-100SX扫描电镜观察。

坐骨神经损伤大鼠模型的建立及分组：选用成年健康SD大鼠30只，切除约1 cm坐骨神经制作动物模型。随机分为3组，嗅鞘细胞复合壳聚糖支架组，用联合培养的原代培养大鼠嗅鞘细胞和壳聚糖支架连接于缺损处，单纯壳聚糖支架组用单纯壳聚糖支架连接缺损，空白单纯壳聚糖支架组不作任何处理，每组10只。

壳聚糖支架桥接损伤神经：解剖显微镜下将神经断端分别套入壳聚糖支架内，8-0无损伤线缝合1针固定神经外膜和支架，使神经两断端在导管内相距1 cm，见图1。

大鼠坐骨神经功能测定：将大鼠四肢涂抹墨水后让其在白纸上行走进行观察，对比正常足与受损足的足迹差异。建模后1-4周测定坐骨神经功能指数，正常大鼠坐骨神经功能指数为0，-100表示坐骨神经功能完全丧失。

大鼠坐骨神经运动诱发电位检测：建模后0，2，4周对各组动物坐骨神经运动诱发电位测定，将双极电极置于距离神经损伤修复区大约1 cm的近端，记录电极刺入腓肠肌肌腹，记录坐骨神经运动电位的潜伏期及传导速度，评价其功能修复效果。

大鼠坐骨神经组织学观察：修复后4周，用40 g/L多聚甲醛磷酸盐缓冲液对大鼠行心室灌注并取材，修剪后石蜡包埋切片，进行苏木精-伊红染色，光学显微镜下观察。

主要观察指标：大鼠建模后坐骨神经功能指数、运动诱发电位和组织学形态。

统计学分析：计量资料以x(_)±s表示，采用SPSS 17.0统计软件进行数据分析，组间均数差异的比较采用两样本t检验，P < 0.05为差异有显著性意义。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

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讨论

周围神经损伤的临床治疗目前尚无有效治疗方案，且临床上周围神经损伤远较中枢神经损伤常见，容易造成神经支配区域的功能障碍，如感觉丧失，运动障碍等症状，加之损伤后再生过程复杂，因此治疗效果不满意。现有治疗周围神经损伤的方法主要为药物治疗和自体神经移植等外科手术方法。随着组织工程研究的兴起，构建人工神经修复周围神经缺损已经取得了一定的研究进展^[7-8] 。
壳聚糖做为组织工程三要素之一的支架材料，目前研究较多^[4] ，其优点为生物相容性好，内部多孔结构排列规则，具有较高的生物相似性，有利于损伤局部种子细胞的迁移和聚集。因此，壳聚糖复合神经支架是一种较为理想的神经导管材料，可用于周围神经组织的修复^[10-11] 。实验采用壳聚糖明胶支架，扫描电镜下观察证实嗅鞘细胞与壳聚糖支架有良好的整合性，内部结构规则，细胞复合后生长良好。
壳聚糖复合支架用于修复大鼠坐骨神经缺损，毒性和免疫原性较小，在体内具有刺激肌肉细胞生长且抑制纤维细胞生长的作用，而且能够被溶菌酶降解为葡萄糖而被机体吸收利用，避免周围纤维瘢痕的形成。实验中镜下再生神经生长良好，各组动物均未出现明显的全身排斥反应和局部明显的炎症反应。嗅鞘细胞结合壳聚糖组均与两端的原位神经连接良好，空白单纯壳聚糖支架组神经缺损间隙由结缔组织相连，是一种良好的组织工程用生物材料。其作用可能为：①支架改善再生轴突的微环境，避免非神经组织侵入。②支架可以避免坐骨神经局部张力过大，避免再次断裂等并发症。③支架有利于同时植入种子细胞、导入外源营养因子以促进神经再生。
嗅鞘细胞作为种子细胞，在神经损伤中的作用已经得到共识。单纯嗅鞘细胞移植修复脊髓损伤的报道众多，研究表明：嗅鞘细胞可为中枢神经系统的轴突再生提供适宜的环境^[12] 。嗅鞘细胞有一定的迁徙能力，可跨越胶质瘢痕，促进轴突发芽。嗅鞘细胞覆盖于再生轴突的表面可能使其避免暴露于神经再生抑制分子的环境中^[13] 。轴突再生还与嗅鞘细胞所产生的黏附分子和生长因子相关^[14-57] 。实验中嗅鞘细胞壳聚糖嗅鞘细胞复合壳聚糖支架组可使大鼠恢复足趾握力，出现明显针刺反应，足趾观察拖曳程度明显降低，坐骨神经功能指数明显增高，与其他2组相比均差异有显著性意义。实验证实了嗅鞘细胞移植对周围神经损伤确有良好的修复作用。
实验结果证实了联合修复周围神经损伤与单独应用一种方法修复相比具有较显著的促进功能恢复作用，实验应用嗅鞘细胞与壳聚糖复合支架修复坐骨神经损伤，发现坐骨神经功能指数、运动诱发电位和组织学观察等结果均高于单独应用壳聚糖支架修复，两者比较提示嗅鞘细胞修复效果优于单纯生物支架。因此，嗅鞘细胞与壳聚糖支架复合，是一种较为理想的组织工程学材料，能够为进一步研究神经修复支架提供研究的基础和方向。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
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