TMEM16A对衰老耳蜗血管纹周细胞凋亡的作用

The Effect of TMEM16A on Apoptosis of Senescent Pericytes in the Cochlea Stria Vascularis

目的原代培养豚鼠耳蜗血管纹毛细血管周细胞(PCs),探讨TMEM16A在耳蜗血管纹PCs上的表达变化及其对衰老耳蜗血管纹PCs凋亡的影响。方法原代培养耳蜗血管纹PCs并鉴定;运用β-半乳糖苷酶染色确定细胞衰老模型;全细胞膜片钳技术记录PCs上CaCCs的电流变化;免疫荧光检测耳蜗血管纹PCs上TMEM16A的表达变化;Western Blot技术检测TMEM16A和凋亡相关蛋白Caspase-3、Bax、Bcl-2蛋白水平;流式细胞术检测各组细胞凋亡率。结果原代培养的耳蜗血管纹PCs阳性率在95%以上,确定第13代耳蜗血管纹PCs为衰老组。全细胞膜片钳记录到衰老组较年轻组PCs上CaCCs电流密度增大。与年轻组PCs相比,衰老组周细胞上TMEM16A、Caspase-3、Bax表达升高,Bcl-2表达下降,细胞凋亡率明显升高。衰老组给予30μmol/L T16Ainh-A01干预24 h后,Caspase-3、Bax表达下降,Bcl-2表达升高,细胞凋亡率明显下降。结论衰老的耳蜗血管纹PCs凋亡增多且TMEM16A表达增加,TMEM16A特异性阻断剂T16Ainh-A01可以减少衰老耳蜗血管纹PCs的凋亡。提示TMEM16A可能参与衰老耳蜗血管纹PCs的凋亡。

Objectives Primary cultured cochlear stria vascularis periytes(PCs)of guinea pig were used to investigate the expression changes of TMEM16A and its effect on apoptosis of senescent PCs in the cochlear stria vascularis.Methods Primary culture and identification of PCs in the cochlear stria vascularis;The cell aging model was determined byβ-Galactosidase Staining Kit.Whole-cell patch clamp technique recorded the changes of CaCCs current in PCs.Immunofluorescence detected the expression changes of TMEM16A on cochlear stria vascularis PCs.Western Blot technique detected the levels of TMEM16A and apoptosis-related proteins Caspase-3,Bax and Bcl-2.Flow cytometry detected the apoptosis rate of each group.Results The positive rate of primary cultured cochlear stria vascularis PCs is above 95%,and the 13th generation cochlear stria vascularis PCs is determined as the senescence group.Whole-cell patch clamp records an increasing in CaCCs current density on PCs in senescence group than young group.Compared with young PCs group,the expression of TMEM16A,Caspase-3 and Bax in senescence group is increased,the expression of Bcl-2 is decreased,and the apoptosis rate is significantly increased.After intervene for 24 h with 30μmol/L T16Ainh-A01,the expression of Caspase-3 and Bax are decreased,the expression of Bcl-2 is increased,and the apoptosis rate is significantly decreased in the senescence group.Conclusion It was found apoptosis and TMEM16A expression were increased in cochlear stria vascularis senescent PCs,TMEM16A specific blocker T16Ainh-A01 can reduce the apoptosis of senescent PCs in the cochlear stria vascularis.These results suggest that TMEM16A may participate in apoptosis of senescent PCs in the cochlear stria vascularis.