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离体培养嗅鞘细胞促进新生大鼠螺旋神经节细胞的存活

刘全, 余洪猛, 戴春富, 李雯, 朱雅颖, 顾瑜蓉, 李华伟

复旦大学附属眼耳鼻喉科医院 耳鼻喉科； 中心实验室，上海 200031；复旦大学脑科学研究院，上海 200032

摘要

本文旨在探索离体实验中的嗅鞘细胞(olfactory ensheathing cells, OECs)有无促进耳蜗听觉传入神经元——螺旋神经节细胞(spiral ganglion cells, SGCs)存活作用及其可能机制。取成年大鼠嗅球和新生大鼠蜗轴组织块进行OECs 与SGCs 的培养，采用差速贴壁法纯化培养OECs。实验分OECs 与SGCs 共培养组和SGCs 单独培养组。倒置相差显微镜下观察OECs和SGCs 生长状态，神经营养因子受体p75 免疫组织化学法鉴定OECs，神经元特异性标志物βIII-tubulin 标记SGCs。为了研究OECs 与SGCs 共培养体系中，前者促进后者存活的可能机制，共培养组中分别加入脑源性神经生长因子(BDNF, 500pg/mL)和BDNF 抗体(IgY 型，50 μg/mL)，对照组为未加任何处理的共培养组，然后检查各培养组中SGCs 存活数量和存活时间。结果显示，OECs 贴壁培养7 d 后形成一细胞单层，在OECs 与SGCs 共培养体系中，SGCs 在OECs 形成的细胞单层的表面生长，并伸出长突起，呈现典型的双极神经元形态；在培养的前6 天内，随着培养时间的增加，两组中的SGCs 都较接种前减少，但共培养组中SGCs 存活数量明显高于SGCs 单独培养组(P<0.01)；单独培养组的 SGCs 数量在培养的第6 天出现大幅度减少，在培养的第9 天几乎没有生长；共培养组的SGCs 数量未见明显变化(P>0.05)；共培养中加入BDNF 对OECs 促进SGCs 存活无明显影响，而加入BDNF 抗体(IgY)后存活的SGCs 减少(P<0.01)。本研究结果提示，OECs 与SGCs 共培养能够促进新生大鼠SGCs 存活和突起生长，延长存活时间，OECs 分泌BDNF 可能是促进SGCs 存活的机制之一。

关键词： 嗅鞘细胞; 螺旋神经节细胞; 培养; 脑源性神经营养因子; 存活

分类号：R339.12

[Olfactory ensheathing cells promote the survival of newborn rat spiral ganglion cells in vitro.] [Ariticle in Chinese]

LIU Quan, YU Hong-Meng, DAI Chun-Fu, LI Wen, ZHU Ya-Ying, GU Yu-Rong, LI Hua-Wei

Department of Otolaryngology, and Central Laboratory, Eye Ear Nose and Throat Hospital, Fudan University, Shanghai 200031, China； The Institutes of Brain Science, Fudan University, Shanghai 200032, China

Abstract

The objective of this study is to explore whether olfactory ensheathing cells (OECs) can promote the survival of newborn ratspiral ganglion cells (SGCs) and the underlying possible mechanisms. Co-culture of OECs from adult rats with SGCs from newborn ratcochlea was established and single culture of SGCs acted as control. OECs were obtained and purified based on their special rate ofattachment which was different from the other harvested cell types during culture. OECs and SGCs were immunocytochemicallycharacterized and confirmed by expression of low-affinity nerve growth factor receptor p75 or positive label of neuron-specific βIIItubulin.To investigate the mechanisms of the role of OECs in survival of SGCs, brain derived neurotrophic factor (BDNF) and anti-BDNF antibody (IgY) were added into the media of the co-cultures respectively, and the surviving SGCs were examined aftertreatment. Single layer of OECs (92% pure) was seen seven days after plating. Surviving SGCs, which extended their primary neurites,were found on the surface of the layer in the co-cultures. When OECs and SGCs were co-cultured, the number of surviving SGCs wassignificantly greater than that in the single culture (P<0.01). Nine days after culture, there was even no change in the number of survivingSGCs in the co-culture while the number reduced to almost zero in the single culture. In comparison with co-culture without treatment,addition of BDNF (500 pg/mL) into the media had no obvious promoting effect on the survival of SGCs. The number of surviving SGCsreduced significantly when anti-BDNF antibody was applied into the media of co-cultures (P<0.01). These results suggest that OECs can promote the survival of SGCs when they are co-cultured in vitro. BDNF released from OECs, as one of the survival factors, playsan important role in the survival of SGCs.

Key words: olfactory ensheathing cell; spiral ganglion cell; culture; brain derived neurotrophic factor; survival

收稿日期：2009-12-21　　录用日期：2010-02-09

通讯作者：余洪猛　　E-mail: hongmengyush@yahoo.com.cn

引用本文：

刘全, 余洪猛, 戴春富, 李雯, 朱雅颖, 顾瑜蓉, 李华伟. 离体培养嗅鞘细胞促进新生大鼠螺旋神经节细胞的存活[J]. 生理学报 2010; 62 (2): 115-121.

LIU Quan, YU Hong-Meng, DAI Chun-Fu, LI Wen, ZHU Ya-Ying, GU Yu-Rong, LI Hua-Wei. [Olfactory ensheathing cells promote the survival of newborn rat spiral ganglion cells in vitro.] [Ariticle in Chinese]. Acta Physiol Sin 2010; 62 (2): 115-121 (in Chinese with English abstract).