[The effect of large-conductance calcium-activated potassium channels on the migration of pericytes in the mice of senile cochlear stria vascularis].

Objective: To investigate whether large conductance calcium-activated potassium channel (BK(Ca)) was involved in the migration of pericytes (PC) in the mice of senile cochlear stria vascularis capillaries PC. Methods: C57BL/6J mice were divided into 3-month (n=10) and 12-month groups (n=10). Auditory brainstem response (ABR) was used to test the hearing threshold of each group. The immunofluorescence was used to detect the expression changes of osteopontin (OPN) and β-BK(Ca) channels on cochlear stria vascularis PC. The morphological changes of perivascular cells in cochlea were observed by transmission electron microscope (TEM). Cell experiment: The PC, which were in the stria vascularis of the cochlea were primary cultured and identified. A cell senile model was made with D-gal. The appropriate intervention concentration of low galactose (D-gal) was determined by CCK8. β-galactosidase (SA-β-gal) staining was used to evaluate the cell decrept level. The change of BK(Ca) channels current on PC were recorded by whole cell patch clamp technique. The expression of BK(Ca) channels on PC was detected by immunofluorescence. The migration and invasion ability of two groups were detected by using Scratch test and Transwell. The levels of OPN and β-BK(Ca) channels were detected by Western blot. SPSS 22.0 software was used to analyze the data. Results: The ABR threshold in the 12-month group was higher than 3-month group (t=12.66, P<0.01). In the 12-month group, the expression of β-BK(Ca) channel was lower and the expression of OPN was increased (t=14.64, P<0.01; t=20.73, P<0.01). In TEM, cochlear stria vascularis PC were tightly connected to endothelial cells in 3-month group, while PC were loosely connected to endothelial cells or PC soma were separated from the capillary in 12-month group. Cell experiment: The positive rate of PC in the primary cultured cochlear stria vascularis is above 95%. Compared with the SA-β-gal stained cells in the control group, the positive rate of 15 mg/ml D-gal intervention PC was 85% (t=36.90, P<0.01). Whole cell patch clamp BK(Ca) channels current decreased in the D-gal group compared with the young group PC (t=12.18, P<0.05). The OPN expression in the senile group was higher than control group (t=16.30, P<0.01), while the β-BK(Ca) channels expression was decreased (t=11.98, P<0.01; t=15.72, P<0.05), and migration ability raised (t=7.91, P<0.01;t=7.59, P<0.01). After intervened of BK(Ca) channels specific blocker IBTX in the D-gal group, the expression of OPN and migration were increased (t=4.26, P<0.05; t=5.88, P<0.01; t=21.97, P<0.01). Conclusion: PC migration capacity were increased during the senile period, and the expression of β-BK(Ca) channel was decreased. The administration of IBTX, a specific blocker of BK(Ca) channel, at the cell level could increase the migration capacity, suggesting that BK(Ca) might be involved in the migration of PC in the stria vascularis of the aging cochlea.目的： 探讨大电导钙激活钾离子通道［large conductance calcium-activated potassium channel，BK（Ca）］是否参与衰老小鼠耳蜗血管纹毛细血管周细胞（pericytes，PC）的迁移。 方法： 将C57BL/6J小鼠分为3月龄（n=10）组和12月龄组（n=10），听性脑干反应（ABR）检测各组听力阈值；免疫荧光检测各组耳蜗血管纹毛细血管PC上β-BK（Ca）通道和骨桥蛋白（osteopontin，OPN）表达变化；透射电镜（transmission electron microscope，TEM）观察各组耳蜗血管纹PC的形态改变。细胞实验：原代培养耳蜗血管纹PC并鉴定；D-半乳糖（D-gal）制备细胞衰老模型，CCK8确定D-gal干预浓度；β-半乳糖苷酶（SA-β-gal）染色评估细胞衰老水平；全细胞膜片钳技术记录PC上BK（Ca）通道激活电流变化；免疫荧光技术检测PC上β-BK（Ca）通道蛋白表达变化；划痕实验和Transwell实验检测2组细胞迁移侵袭能力；Western blot技术检测β-BK（Ca）通道和OPN蛋白表达水平。采用SPSS 22.0软件对数据进行统计学分析。 结果： 动物实验：12月龄组小鼠较3月龄组ABR阈值升高（t=12.66，P<0.01）；12月龄组小鼠耳蜗血管纹PC上β-BK（Ca）通道表达水平较3月龄组降低（t=14.64，P<0.01），OPN表达升高（t=20.73，P<0.01）；TEM中3月龄组PC与内皮细胞紧密相连，12月龄组PC与内皮细胞连接疏松或PC胞体从毛细血管壁分离。细胞实验：耳蜗血管纹原代培养的耳蜗血管纹PC阳性率在95%以上，15 mg/ml D-gal干预的PC较对照组的SA-β-gal染色细胞阳性率高，差异有统计学意义（t=36.90，P<0.01）。与对照组PC相比，D-gal干预组全细胞膜片钳BK（Ca）通道电流减小（t=12.18，P<0.05），β-BK（Ca）通道蛋白和荧光表达水平降低（t=11.98，P<0.01；t=15.72，P<0.05），OPN蛋白表达升高（t=18.53，P<0.01），PC迁移能力增加（t=7.91，P<0.01；t=7.59，P<0.01）。D-gal干预后给予BK（Ca）通道特异性阻断剂Iberiotoxin（IBTX）可使OPN表达明显升高（t=4.26，P<0.05），PC迁移能力增加（t=5.88，P<0.01；t=21.97，P<0.01）。 结论： 衰老小鼠耳蜗血管纹毛细血管PC迁移能力增加，β-BK（Ca）通道表达降低，给予BK（Ca）通道特异性阻断剂IBTX可使迁移能力增加，提示BK（Ca）通道可能参与衰老小鼠耳蜗血管纹毛细血管PC的迁移。.