论文部分内容阅读
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 purpose of this study was to investigate whether ex vivo olfactory ensheathing cells (OECs) can promote the survival of cochlear auditory afferent neurons, spiral ganglion cells (SGCs), and their possible mechanisms. The OECs and SGCs were isolated from the olfactory bulb of adult rat and the tissue of the neonatal rat. The OECs were purified by differential adherence. Experimental OECs and SGCs co-culture group and SGCs alone group. OECs and SGCs were observed under inverted phase contrast microscope. OECs and neuron specific marker βIII-tubulin labeled SGCs were identified by immunohistochemical staining of neurotrophic factor receptor p75. In order to study the possible mechanism of the former survival in the co-culture system of OECs and SGCs, BDNF (500pg / mL) and BDNF antibody (IgY, 50μg / mL) The control group was untreated co-culture group, and then examined the survival and survival time of SGCs in each culture group. The results showed that OECs formed a cell monolayer after adherent culture for 7 days. In the co-culture system of OECs and SGCs, SGCs grew on the surface of cell monolayer formed by OECs and extended into long protuberances, showing typical bipolar neuron morphology ; In the first 6 days of culture, SGCs in both groups decreased with the increase of culture time, but the number of SGCs in co-culture group was significantly higher than that in SGCs alone group (P <0.01); in cultured group The number of SGCs decreased significantly on the 6th day of culture, and there was almost no growth on the 9th day of culture. There was no significant change in the number of SGCs in the co-culture group (P> 0.05). The addition of BDNF to co-culture promoted the survival of SGCs No significant effect, while the addition of BDNF antibody (IgY) survival SGCs decreased (P <0.01). Our results suggest that co-culture of OECs and SGCs can promote survival and protuberance of SGCs in neonatal rats and prolong their survival time. Secretion of BDNF by OECs may be one of the mechanisms that promote the survival of SGCs.