离体培养嗅鞘细胞促进新生大鼠螺旋神经节细胞的存活

Olfactory ensheathing cells promote the survival of newborn rat spiral ganglion cells in vitro

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,50μg/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7d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。

The objective of this study is to explore whether olfactory ensheathing cells （OECs） can promote the survival of newborn rat spiral ganglion cells （SGCs） and the underlying possible mechanisms.Co-culture of OECs from adult rats with SGCs from newborn rat cochlea was established and single culture of SGCs acted as control.OECs were obtained and purified based on their special rate of attachment which was different from the other harvested cell types during culture.OECs and SGCs were immunocytochemically characterized and confirmed by expression of low-affinity nerve growth factor receptor p75 or positive label of neuron-specific βIII- tubulin.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 after treatment.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 was significantly greater than that in the single culture （P〈0.01）.Nine days after culture,there was even no change in the number of surviving SGCs 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 SGCs reduced 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,plays an important role in the survival of SGCs.