中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2009年
6期
494-498
,共5页
何景春%于栋祯%丁大连%殷善开%Richard J Salvi
何景春%于棟禎%丁大連%慇善開%Richard J Salvi
하경춘%우동정%정대련%은선개%Richard J Salvi
毛细胞,听觉%器官培养技术%链霉素%细胞凋亡%半胱氨酸天冬氨酸蛋白酶
毛細胞,聽覺%器官培養技術%鏈黴素%細胞凋亡%半胱氨痠天鼕氨痠蛋白酶
모세포,은각%기관배양기술%련매소%세포조망%반광안산천동안산단백매
Hair cells,auditory%Organ culture techniques%Streptomycin%Apoptosis%Caspases
目的 探讨在离体培养条件下硫酸链霉素是否通过半胱氨酸天冬氨酸蛋白酶(caspase)的激活而导致大鼠耳蜗毛细胞凋亡.方法 将出生后3～4 d的大鼠耳蜗基底膜取出进行离体培养,培养过夜后用0.1 mmol/L硫酸链霉素培养液继续培养24 h.终止培养前1 h应用碳氧荧光素标记的肽荧光甲基酮(carboxyfluorescein labeled peptide fluoromethyl ketone,FAM-peptide-FMK)标记的caspase-6,caspase-8,或caspase-9指示剂作用1 h,然后应用异硫氰酸四甲基罗丹明(tetramethyl rhodamine iso-thiocyanate,TRITC)标记的鬼笔环肽(phalloidin)标记毛细胞的纤毛和表皮板,再应用Topro-3 DNA探针对细胞核进行染色,最后在共聚焦显微镜下观察.结果 1 mmol/L硫酸链霉素引起的耳蜗毛细胞缺失在底回约为80%,在顶回约为20%.经链霉素作用后,耳蜗毛细胞的细胞核发生浓缩和碎裂,同时可见cmpase-6,caspase-8和caspase-9阳性表达.结论 硫酸链霉素可以引起离体培养的大鼠耳蜗毛细胞凋亡,上游和下游的caspase激活可能是其主要途径.
目的 探討在離體培養條件下硫痠鏈黴素是否通過半胱氨痠天鼕氨痠蛋白酶(caspase)的激活而導緻大鼠耳蝸毛細胞凋亡.方法 將齣生後3～4 d的大鼠耳蝸基底膜取齣進行離體培養,培養過夜後用0.1 mmol/L硫痠鏈黴素培養液繼續培養24 h.終止培養前1 h應用碳氧熒光素標記的肽熒光甲基酮(carboxyfluorescein labeled peptide fluoromethyl ketone,FAM-peptide-FMK)標記的caspase-6,caspase-8,或caspase-9指示劑作用1 h,然後應用異硫氰痠四甲基囉丹明(tetramethyl rhodamine iso-thiocyanate,TRITC)標記的鬼筆環肽(phalloidin)標記毛細胞的纖毛和錶皮闆,再應用Topro-3 DNA探針對細胞覈進行染色,最後在共聚焦顯微鏡下觀察.結果 1 mmol/L硫痠鏈黴素引起的耳蝸毛細胞缺失在底迴約為80%,在頂迴約為20%.經鏈黴素作用後,耳蝸毛細胞的細胞覈髮生濃縮和碎裂,同時可見cmpase-6,caspase-8和caspase-9暘性錶達.結論 硫痠鏈黴素可以引起離體培養的大鼠耳蝸毛細胞凋亡,上遊和下遊的caspase激活可能是其主要途徑.
목적 탐토재리체배양조건하류산련매소시부통과반광안산천동안산단백매(caspase)적격활이도치대서이와모세포조망.방법 장출생후3～4 d적대서이와기저막취출진행리체배양,배양과야후용0.1 mmol/L류산련매소배양액계속배양24 h.종지배양전1 h응용탄양형광소표기적태형광갑기동(carboxyfluorescein labeled peptide fluoromethyl ketone,FAM-peptide-FMK)표기적caspase-6,caspase-8,혹caspase-9지시제작용1 h,연후응용이류청산사갑기라단명(tetramethyl rhodamine iso-thiocyanate,TRITC)표기적귀필배태(phalloidin)표기모세포적섬모화표피판,재응용Topro-3 DNA탐침대세포핵진행염색,최후재공취초현미경하관찰.결과 1 mmol/L류산련매소인기적이와모세포결실재저회약위80%,재정회약위20%.경련매소작용후,이와모세포적세포핵발생농축화쇄렬,동시가견cmpase-6,caspase-8화caspase-9양성표체.결론 류산련매소가이인기리체배양적대서이와모세포조망,상유화하유적caspase격활가능시기주요도경.
Objective To evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells. Methods F344 rats at postnatal day 3 or 4 were used for the study in cochlear organotypic cultures. The cochlear basilar membrane was micro-dissected out and cultured overnight, and then treated with 1 mmol/L streptomycin for 24 hours. Before the termination, the activity of caspase-8, 9 or 6 were detected with FAM-peptide-FMK labeled caspase-8, 9 or 6, respectively. The stereocilia and cuticular plate of hair cells were stained with TRITC conjugated phalloidin, and the nuclei were stained with Topro-3 DNA probe. The specimens were observed and photographed under confocal fluorescent microscope. Results Streptomycin with 1 nunol/L causes about 80% cochlear hair cells missing in the basal turn and 10% hair cell loss in the apex. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in most cochlear hair cells, and the caspase-8, caspase-9 and caspase-6 were greatly activated. Conclusions Apoptosis is involved in the cochlear hair cells death induced by Streptomycin in vitro. The caspase activities in upstream and downstream are maybe the major apoptotic pathway.
