谷氨酸损伤体外培养大鼠螺旋神经元中凋亡诱导因子的表达与分布

AIF Distribution and Expression in Cultured Spiral Ganglion Neuron Injured by Glutamate

丁忠家;唐晓旭;陈鑫;宋勇莉;米文娟;王剑;陈福权;邱建华;

1:第四军医大学西京医院耳鼻咽喉头颈外科

摘要
目的观察不同浓度谷氨酸(Glu)损伤螺旋神经元中凋亡诱导因子(apoptosis inducing factor,AIF)的表达与分布变化,探讨利用谷氨酸损伤体外培养螺旋神经元是否与AIF凋亡途径相关。方法取40只出生后03天SD仔鼠螺旋神经元行体外培养,所得培养细胞平均分为正常组和10mM、20mM、40mM Glu组,正常组仅给予正常SGNs培养液培养,其余三组分别加入相应浓度谷氨酸和SGNs培养液,培养48h后,应用光学显微镜、免疫荧光染色及实时荧光定量PCR方法观察四组中SGN细胞形态、SGN中AIF的分布及AIF、钙蛋白酶(calpain)、半胱氨酸天冬氨酸3(caspase 3)的表达变化;用TUNEL法观察细胞凋亡。结果 20mM和40mM Glu组SGNs细胞形态改变和细胞凋亡明显,并伴有AIF核转位;不同浓度Glu组AIF和calpain基因表达明显高于正常组(P<0.05),但caspase 3表达无明显升高(P>0.05)。结论谷氨酸损伤体外培养的螺旋神经元过程中,AIF凋亡途径参与了细胞凋亡,calpain的高表达也促进了兴奋性神经递质对SGNs的损伤,但caspase3途径无明显作用。
关键词
螺旋神经元;谷氨酸;凋亡诱导因子;钙蛋白酶;半胱氨酸天冬氨酸3
基金项目(Foundation):
国家自然科学基金(81120108008,30930098,81271069);; 国家“973”项目课题(2011CB504505)联合资助
作者
丁忠家;唐晓旭;陈鑫;宋勇莉;米文娟;王剑;陈福权;邱建华;
参考文献

1 Delettre C,Yuste VJ,Moubarak RS,et al.Identification and characterization of AIFsh2,a mitochondrial apoptosis-inducing factor(AIF)isoform with NADH oxidase activity[J].J Biol Chem,2006,281:18507.

2 Cande C,Cohen I,Daugas E,et al.Apoptosis-inducing factor(AIF):a novel caspase-independent death effector released from mitochondria[J].Biochimie,2002,84:215.

3 Zhu C,Wang X,Deinum J,et al.Cyclophilin A participates in the nuclear translocation of apoptosis-inducing factor in neurons after cerebral hypoxia-ischemia[J].J Exp Med,2007,204:1741.

4 Vahsen N,Cande C,Briere JJ,et al.AIF deficiency compromises oxidative phosphorylation[J].EMBO J,2004,23:4679.

5 Berlin CI,Hood LJ,Cecola RP,et al.Does type I afferent neuron dysfunction reveal itself through lack of efferent suppression[J]?Hear Res,1993,65:40.

6 张琰敏,马蓓,高元元,等.谷氨酸受体在噪声致豚鼠螺旋神经节细胞损伤中的作用[J].生理学报,2007,59:103.

7 Cardon G,Campbell J,Sharma A.Plasticity in the developing auditory cortex:evidence from children with sensorineural hearing loss and auditory neuropathy spectrum disorder[J].J Am Acad Audiol,2012,23:396.

8 Wang H,Yu SW,Koh DW,et al.Apoptosis-inducing factor substitutes for caspase executioners in NMDA-triggered excitotoxic neuronal death[J].J Neurosci,2004,24:10963.

9 Steinbach S,Lutz J.Glutamate induces apoptosis in cultured spiral ganglion explants[J].Biochem Biophys Res Commun,2007,357:14.

10 Schmutzhard J,Glueckert R,Pritz C,et al.Sepsis otopathy:experimental sepsis leads to significant hearing impairment due to apoptosis and glutamate excitotoxicity in murine cochlea[J].Dis Model Mech,2013,6:745.

11 Abaamrane L,Raffin F,Schmerber S,et al.Intracochlear perfusion of leupeptin and z-VAD-FMK:influence of antiapoptotic agents on gunshot-induced hearing loss[J].Eur Arch Otorhinolaryngol,2011,268:987.

12 Otera H,Ohsakaya S,Nagaura Z,et al.Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space[J].EMBO J,2005,24:1375.

13 Norberg E,Gogvadze V,Ott M,et al.An increase in intracellular Ca2+is required for the activation of mitochondrial calpain to release AIF during cell death[J].Cell Death Differ,2008,15:1857.

14 Yamashita D,Miller JM,Jiang HY,et al.AIF and EndoG in noise-induced hearing loss[J].Neuroreport,2004,15:2719.

15 Han W,Shi X,Nuttall AL.AIF and endoG translocation in noise exposure induced hair cell death[J].Hear Res,2006,211:85.

16 Parameswaran S,Sharma RK.Altered expression of calcineurin,calpain,calpastatin and HMWCaMBP in cardiac cells following ischemia and reperfusion[J].Biochem Biophys Res Commun,2014,443:604.