大鼠耳蜗大上皮嵴的分离和鉴定  

Isolation and identification of greater epithelial ridge in rat cochlea

作　　者：宋巍[1] 胡吟燕[1] 郭维[1] 郑贵亮[1] 翟所强[1] 高维强 

机构地区：[1]解放军总医院耳鼻咽喉研究所,北京1008531 [2]美国旧金山基因技术公司,美国圣佛朗西斯科ca94080

出　　处：《临床耳鼻咽喉科杂志》2005年第13期611-613,共3页Journal of Clinical Otorhinolaryngology

基　　金：海外青年学者合作研究基金资助项目(No:30228029)

摘　　要：目的:探讨利用酶消化和机械解剖相结合分离大鼠耳蜗大上皮嵴(GER)的方法。方法:取出生后0～3d的SD大鼠耳蜗基底膜后,将其放入含有嗜热菌蛋白酶和少许DNA酶的DHank溶液中,37℃孵箱孵育25min进行酶消化,然后在高倍显微镜下进行显微机械分离;对分离的GER细胞用免疫组织化学和RTPCR扩增GER细胞特异性表达基因Hes1的方法进行鉴定,并进行GER的原代培养以验证其活性。结果:免疫组织化学染色表明分离的GER细胞是单一的上皮组织;对Hes1基因进行RTPCR分析表明,分离的GER细胞表达该基因;分离的GER具有活性并可以进行原代培养。结论:利用酶消化和机械解剖相结合的方法可以分离出纯粹的有活性的GER,为进一步建立GER细胞系,进行各种基因转染试验以及耳蜗内的各种基础实验建立细胞模型提供有效的实验手段。Objective:To investigate the method for isolating greater epithelial ridge by use of thermolysin digestion combined with dissection under microscope.Method:The basement membrane prepared from postnatal day 0 to postnatal day 3 rat was placed into D-Hank's solution with thermolysin and DNase, then incubated at 37℃ for 25minute and dissected under microscope. After GER had been isolated, morphological comparison, immunohistochemistry, RT-PCR and GER culture were performed for the purpose of identification of GER.Result:Immunohistochemistry indicated that the isolated GER was purely epithelial tissue. RT-PCR analysis certified that the isolated GER expressed Hes1 which was selectively expressed in GER at early postnatal in cochlea.The isolated GER could be successfully cultured in the presence of 5% serum and could survive at least 12 day in the serum-free medium.Conclusion:GER isolation can be successfully performed by use of thermolysin combined with microdissection. This method may provide a good technique to establish the GER cell lines or perform series of gene transfection experiments.

关 键 词：大鼠 耳蜗 大上皮嵴 嗜热菌蛋白酶 

分 类 号：Q959.837[生物学—动物学] R322[医药卫生—人体解剖和组织胚胎学]