CAJ | 학술논문

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0～3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10 mM、20 mM、40 mM 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48 h 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（cal-pain）、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20 mM 和40 mM 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。
목적：관찰불동농도곡안산（Glu）손상라선신경원중조망유도인자（apoptosis inducing factor，AIF）적표체여분포변화，탐토이용곡안산손상체외배양라선신경원시부여 AIF 조망도경상관。방법취40지출생후0～3천 SD 자서라선신경원행체외배양，소득배양세포평균분위정상조화10 mM、20 mM、40 mM Glu 조，정상조부급여정상 SGNs 배양액배양，기여삼조분별가입상응농도곡안산화 SGNs 배양액，배양48 h 후，응용광학현미경、면역형광염색급실시형광정량 PCR 방법관찰사조중 SGN 세포형태、SGN 중 AIF 적분포급 AIF、개단백매（cal-pain）、반광안산천동안산3（caspase 3）적표체변화；용 TUNEL 법관찰세포조망。결과20 mM 화40 mM Glu 조SGNs 세포형태개변화세포조망명현，병반유 AIF 핵전위；불동농도 Glu 조 AIF 화 calpain 기인표체명현고우정상조（P＜0．05），단 caspase 3표체무명현승고（P＞0．05）。결론곡안산손상체외배양적라선신경원과정중，AIF 조망도경삼여료세포조망，calpain 적고표체야촉진료흥강성신경체질대 SGNs 적손상，단 caspase3도경무명현작용。
Objective The study aimed to explore the relationship between AIF related pathway and the inju-ring of cultured SGNs (spiral ganglion neurons)by glutamate toxicity,and to find AIF expression and distribution changes in SGNs.Methods SGNs of 40 newborn rats within 3 day were obtained and cultured in vitro.Cultured cells were divided into four groups:the normal control group,10 mM,20 mM and 40 mM glutamate injured group, separately.After 48 h hours culturing,optical microscopy,immune fluorescence staining and real-time fluores-cence quantitative PCR were used to observe the morphology,AIF distribution,and AIF,calpain,Caspase3 expres-sion changes in SGNs in vitro.TUNEL was used to verify the cell apoptosis.ResuIts Noticeable morphological chan-ges and cell apoptosis were occurred in 20 mM glutamate group,with AIF nuclear translocation.AIF gene expression was significantly higher than normal after glutamate administration (P<0.05);moreover,calpain gene expression increased(P<0.05);but caspase3 expression was not statistically significantly increased in all glutamate treated groups (P>0.05). ConcIusion In the process of cultured SGNs injured by glutamate,AIF participated in the cell apoptosis.Noticeable cell apoptosis were occurred in 20 mM glutamate group with AIF nuclear translocation.Calpain up-expression also contributed to excitatory neurotransmitter injury on SGNs,but Caspase 3 had no obvious effects.