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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 explore a method that can provide more quantity and longer time to retain the activity of guinea pig isolated type Ⅰ vestibular hair cells. Methods Forty-eight variegated guinea pigs with normal auricle reflex were randomly divided into four groups: collagenase Ⅳ + common separation group (group A), trypsin + common separation group (group B), Collagenase Ⅳ + modified isolation group (C Group) and trypsin + modified separation group (group D). Twelve rabbits in each group were treated with 0.25% collagenase Ⅳ and 0.05% trypsin respectively, and the common liquid and modified liquid plus mechanical separation were used to separate the guinea pig vestibular type Ⅰ hairs The cells were observed, identified and evaluated by light microscopy, patch clamp and intracellular calcium imaging. Results In group A, 25 surviving monovalent vestibular hair cells isolated per ear were obtained. About 14 cells survived after 3 hours, 8 survived cells after 6 hours, and resting membrane potential averaged -55 ± 10 mV after 3 hours. Intracellular calcium The average ion concentration was 105 ± 15nmol / L in group B. The average number of surviving mononuclear vestibular hair cells in group B was 40 per well. The number of viable cells was about 19 at 3h, 6 at 6h, Was -60 ± 10mV, the average concentration of intracellular calcium was 95 ± 10nmol / L. In group C, 24 surviving monovalent vestibular hair cells isolated per ear were isolated. After 3 hours, the number of viable cells was about 17, and survived after 6 hours About 11, 3h resting membrane potential average of -55 ± 10mV, intracellular calcium concentration average of 95 ± 15nmol / L; D group average per ear isolated type Ⅰ vestibular hair cells 42, 3h surviving cells after about 30 There were about 23 surviving cells after 6 hours. The average resting membrane potential was -60 ± 10 mV after 3 hours, the average intracellular calcium concentration was 90 ± 10 nmol / L, and the intracellular calcium concentration was more stable. Conclusion Compared with the traditional collagenase Ⅳ enzymatic hydrolysis plus mechanical separation method, trypsin digestion combined with modified liquid plus mechanical separation technology is simple and easy to provide enough guinea pigs to survive for a long time separated from the type Ⅰ vestibule Hair cells, suitable for cell activity demanding experiments such as patch-clamp electrophysiological studies.