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前体脑源性神经营养因子抑制大鼠螺旋神经节神经元的存活和神经突再生

童华, 周雷, 刘建平, 高丽, 沈纳, 黄新生^*

复旦大学附属中山医院耳鼻喉科，上海 200032

摘要

本文旨在研究前体脑源性神经营养因子(precursor brain-derived neurotrophic factor, proBDNF)对螺旋神经节神经元(spiral ganglion neurons, SGNs)生长的影响。取新生5天Sprague Dawley (SD)大鼠处死，分离出含有螺旋神经节的蜗轴，将其消化、吹打后用培养液重悬，接种于多聚-D-赖氨酸/层粘连蛋白包被过的8孔腔式载玻片，37 °C培养4 h获得贴壁牢固的原代SGNs。将SGNs随机分为对照组、BDNF组(10 ng/mL BDNF处理)、C10组(10 ng/mL proBDNF处理)、C50组(50 ng/mL proBDNF处理)、C100组(100 ng/mL proBDNF处理)，无血清培养48 h后行βIII tubulin荧光抗体标记染色，荧光显微镜下观察SGNs的存活数目和神经突再生情况。结果显示，C50组和C100组的SGNs存活数目低于对照组(P < 0.001)，且C10组、C50组和C100组的SGNs存活数目均低于BDNF组(P < 0.001)。根据SGNs的形态，可将其分为无突起和有突起两类。C10组、C50组、C100组的无突起SGNs比例均高于对照组，差异有统计意义(P < 0.001)。另外在添加20 μmol/L的特异性c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)抑制剂SP600125后，C50组的SGNs存活数目提升至(91.5 ± 8.2)/孔，高于没有使用SP600125的C50组(P < 0.001)。以上结果说明，proBDNF减少体外培养SGNs的存活数目，并抑制SGNs神经突的出芽生长；使用特异性JNK抑制剂可以部分减轻proBDNF对SGNs存活的抑制作用。

关键词： proBDNF; 螺旋神经节; p75神经营养素受体

分类号：R339.16

[Precursor brain-derived neurotrophic factor reduces survival and axonal sprouting of rat spiral ganglion neurons in vitro.] [Article in Chinese]

TONG Hua, ZHOU Lei, LIU Jian-Ping, GAO Li, SHEN Na, HUANG Xin-Sheng^*

Department of Otolaryngology, Zhongshan Hospital, Fudan University, Shanghai 200032, China

Abstract

The aim of the present study was to investigate the effect of precursor brain-derived neurotrophic factor (proBDNF) on survival and neurite outgrowth of cultured rat spiral ganglion neurons (SGNs). Spiral ganglions (SG) were collected from postnatal day 5 Sprague Dawley (SD) rats, then enzymatically digested and suspended. Dissociated SGNs were plated on poly-D-lysine/laminin coated eight-well chamber plates and maintained at 37 °C for 4 h to promote the attachment of the neurons. Cultured SGNs were randomly divided into five groups: control group, BDNF group (BDNF 10 ng/mL), C10 group (proBDNF 10 ng/mL), C50 group (proBDNF 50 ng/mL), and C100 group (proBDNF 100 ng/mL). All groups were incubated in a serum-free medium. 48 h after incubation, SGNs were fixed and stained for βIII tubulin. Immunostaining of the cultured SGNs showed that, compared with the control group, the cellular survival of C50 group and C100 group were significantly reduced (P < 0.001). Furthermore, surviving numbers of the three proBDNF-treated groups were all lower than the BDNF group. In order to assess the effect of proBDNF on cell morphology, SGNs were divided into two categories: SGNs with or without neurites. The results demonstrated that proBDNF significantly increased the proportions of SGNs without neurites in C10, C50 and C100 groups compared with that in control group (P < 0.001). In addition, c-Jun N-terminal kinase (JNK) inhibitor, SP600125 (20 μmol/L) significantly increased the surviving number of SGNs in C50 group. These results suggest that proBDNF reduces the survival rate of cultured SGNs and inhibits the sprouting of neurites. Furthermore, the inhibition of JNK signaling attenuates the effect of proBDNF on SGNs survival.

Key words: proBDNF; spiral ganglion; p75 neurotrophin receptor

收稿日期：2013-11-13　　录用日期：2014-01-20

通讯作者：黄新生　　E-mail: huangxinsh@hotmail.com

引用本文：

童华, 周雷, 刘建平, 高丽, 沈纳, 黄新生. 前体脑源性神经营养因子抑制大鼠螺旋神经节神经元的存活和神经突再生[J]. 生理学报 2014; 66 (2): 151-157.

TONG Hua, ZHOU Lei, LIU Jian-Ping, GAO Li, SHEN Na, HUANG Xin-Sheng. [Precursor brain-derived neurotrophic factor reduces survival and axonal sprouting of rat spiral ganglion neurons in vitro.] [Article in Chinese]. Acta Physiol Sin 2014; 66 (2): 151-157 (in Chinese with English abstract).