T16Ainh-A01对耳蜗血管纹毛细血管内皮细胞凋亡及衰老的影响*

目的: 原代培养豚鼠耳蜗血管纹毛细血管内皮细胞(ECs),探讨跨膜蛋白16A(TMEM16A)在耳蜗血管纹毛细血管ECs衰老过程中的变化及对耳蜗血管纹毛细血管ECs凋亡及衰老的影响。方法: 原代培养耳蜗血管纹毛细血管ECs,细胞传代构建衰老模型并根据CCK-8及β-半乳糖苷酶(SA-β-gal)染色评估细胞衰老程度,衰老细胞被随机分为衰老组(P12)、溶剂组(P12+DMSO)、T16Ainh-A01组(P12+T16Ainh-A01),免疫荧光及Western blot检测TMEM16A在ECs上的表达及分布,流式细胞术检测各组细胞凋亡率,Western blot检测各组Bax、Bcl-2、cleaved casepase-3蛋白表达水平。结果: 原代培养的耳蜗血管纹毛细血管ECs阳性率在95%以上,并确定第12代耳蜗血管纹毛细血管ECs为衰老组,与年轻组ECs相比,衰老组ECs上TMEM16A荧光及蛋白表达显著增强(P＜0.05),细胞凋亡率升高,衰老组给予T16Ainh-A01干预24 h后,Bax、cleaved casepase-3的蛋白表达下调(P＜0.01),Bcl-2的蛋白表达上调(P＜0.05),凋亡率下降且SA-β-gal阳性细胞率明显下降(P＜0.01)。结论: 衰老耳蜗血管纹毛细血管ECs凋亡增多且TMEM16A表达增加,TMEM16A特异性阻断剂T16Ainh-A01可以降低耳蜗血管纹毛细血管ECs的凋亡和衰老程度,提示TMEM16A可能参与耳蜗血管纹毛细血管ECs的凋亡和衰老过程。

Effects of T16Ainh-A01 on the apoptosis and senescence of endothelial cells in the cochlea stria vascularis

Objective: Primary cultured cochlear stria vascularis endothelial cells (ECs) of guinea pig were used to investigate the expression changes of TMEM16A and its effect on apoptosis and senescence of ECs in the cochlear stria vascularis. Methods: Primary cultured ECs in the cochlear stria vascularis were used to establish aging models according to CCK-8 and SA-β-galactosidase. Senescent cells were randomly divided into senescent group (P12), DMSO group (P12+DMSO), T16Ainh-A01 group (P12+T16Ainh-A01). Immunofluorescence and Western blot were used to detect the expression of TMEM16A in ECs. Flow cytometry was used to detect the apoptotic rate. Western blot was used to detect the protein expressions of Bax, Bcl-2 and cleaved casepase-3 in each group. Results: The positive rate of primary cultured cochlear stria vascularis ECs was above 95%, and the 12th generation cochlear stria vascularis ECs were determined as the senescence group, and the expression of TMEM16A in protein and fluorescence was increased (P＜0.05). After intervention with T16Ainh-A01 for 24 h, the protein expressions of Bax and cleaved casepase-3 were down-regulated (P＜0.01), the protein expression of Bcl-2 was increased (P＜0.05), the apoptotic rate and the positive rate of SA-β-gal were down-regulated (P＜0.01). Conclusion: It was found that apoptosis and TMEM16A expression were increased in cochlear stria vascularis senescent ECs, TMEM16A specific blocker T16Ainh-A01 could reduce the apoptosis and senescence in ECs of the cochlear stria vascularis. These results suggest that TMEM16A may participate in apoptosis and senescence of ECs in the cochlear stria vascularis.