豚鼠Ⅰ型前庭毛细胞改良分离法  

Modified Dissociation Methods for Type I Vestibular Hair Cells from Guinea Pigs

作　　者：朱云[1] 孔维佳[1] 夏交[1] 姚琦 郭长凯[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院耳鼻咽喉科,武汉430022

出　　处：《听力学及言语疾病杂志》2008年第3期241-245,共5页Journal of Audiology and Speech Pathology

基　　金：国家杰出青年科学基金(39925035);国家自然科学基金(30371525);“十一五”国家科技支撑计划(2007BA118B13)资助项目

摘　　要：目的探索一种能提供更多数量并更长时间保留豚鼠单离Ⅰ型前庭毛细胞细胞活性的分离方法。方法将耳廓反射正常的杂色豚鼠48只随机分为胶原酶Ⅳ+常用分离法组(A组)、胰蛋白酶+常用分离法组(B组)、胶原酶Ⅳ+改良分离法组(C组)和胰蛋白酶+改良分离法组(D组),每组12只,分别用0.25%胶原酶Ⅳ和0.05%胰蛋白酶结合文献报道常用液体和改良液体外加机械分离法分离豚鼠前庭Ⅰ型毛细胞,通过光镜、膜片钳、胞内钙成像等方法观察、鉴定和评价Ⅰ的活性。结果A组平均每耳分离出存活单离Ⅰ型前庭毛细胞25个,3h后存活细胞约14个,6h后存活细胞约8个,3h静息膜电位平均为-55±10mV,胞内钙离子浓度平均为105±15nmol/L;B组平均每耳分离开存活单离Ⅰ型前庭毛细胞40个,3h后存活细胞约19个,6h后存活细胞约6个,3h静息膜电位平均为-60±10mV,胞内钙离子浓度平均为95±10nmol/L;C组平均每耳分离出存活单离Ⅰ型前庭毛细胞24个,3h后存活细胞约为17个,6h后存活细胞约11个,3h静息膜电位平均为-55±10mV,胞内钙离子浓度平均为95±15nmol/L;D组平均每耳分离出Ⅰ型前庭毛细胞42个,3h后存活细胞约30个,6h后存活细胞约23个,3h静息膜电位平均为-60±10mV,胞内钙离子浓度平均为90±10nmol/L,且胞内钙离子浓度更稳定。结论与传统的胶原酶Ⅳ酶解加机械分离法相比,以胰蛋白酶酶解结合改良式液体外加机械分离技术分离方法简便易行,能提供足够量的存活时间较长的豚鼠单离Ⅰ型前庭毛细胞,适用于对细胞活性要求较高的实验如膜片钳电生理研究等。Objective To evaluate a modified method of dissociating vestibular type I hair cells （VHC I ） from guinea pigs, which could provide more cells with longer survival time and better cell activity. Methods VHCs I were dissociated from guinea pigs separately by 0.25 % collagenase Ⅳ,according to the general dissociation meth- ods,and 0. 05% trypsin with two different dissociation methods and were investigated, identified and evaluated with microscopy, patch clamping and calcium imaging techniques. Results General dissociation methods with 0. 25% collagenase Ⅳ ： on average 25 VHCs I were isolated every otocyst, about 14 and only 8 cells survived at the end of 3 h and 6 h, the RP was -55±10 mV and the intracellular calcium concentration was 105±15 nmol/ L at the end of 3 h, conglomerate cell aggregates could be easily found by light microscope. General dissociation methods with 0. 05% trypsin showed that on average 40 VHCs I were isolated every otocyst, 19 and 6 cells survived at the end of 3 h and 6 h, the RP was -60±10 mV and the intracellular calcium concentration was 95±10 nmol/L at the end of 3 h. Modified dissociation methods with 0.25 % collagenase Ⅳ indicated that on average 24 VHCs I were isolated every otocyst, about 17 and 11 cells survived at the end of 3 h and 6 h, the RP was --554-10 mV and intracellular calcium concentration was 954-15 nmol/L at the end of 3 h. Modified dissociation methods with 0. 05 % trypsin showed that on average 42 VHCs I were isolated every otocyst, about 32 and 23 cells survived at the end of 3 h and 6 h, the RP was --60±10 mV and intracellular calcium concentration was more stable average to 90±10 nmol/ L at the end of 3 h. Conglomerate cell aggregates could be rarely found by light microscope. Conclusion Modified dissociation methods with trypsin digestion followed by trituation can supply sufficient amounts of VHCs I with satisfactory sur- vival time compared with general methods. This is more adequate for the experiments that need better cell activity as patch

关 键 词：豚鼠 毛细胞 前庭 膜片钳术 钙成像 

分 类 号：R764.3[医药卫生—耳鼻咽喉科]