肌动蛋白结合蛋白细丝蛋白A参与肿瘤坏死因子α诱导的支持细胞屏障功能降低的研究

Study on participation of filamin A in tumor necrosis factor α-induced Sertoli cell barrier function decrease

目的:探讨肌动蛋白结合蛋白细丝蛋白A(filamin A,FLNa)在肿瘤坏死因子α(tumor necrosis factorα,TNFα)调节支持(Sertoli)细胞屏障功能过程中发挥的作用。方法:体外分离培养20日龄大鼠睾丸支持细胞,用10μg/L TNFα处理后测定跨上皮电阻(trans-epithelial electrical resistance,TER),反映屏障功能改变;Western blotting分析TNFα处理后FLNa表达水平变化;细胞免疫荧光或鬼笔环肽染色分别检测FLNa及微丝(F-actin)的定位改变;FLNa si RNA干扰后检测支持细胞屏障功能。结果:TNFα处理后TER值较对照组显著降低(P<0.01);Western blotting结果显示TNFα处理组FLNa水平明显下降(P<0.01);细胞形态学检测亦表明TNFα处理后FLNa及F-actin在支持细胞的分布有显著改变;TER测定结果表明FLNa经si RNA沉默后可降低血睾屏障(BTB)功能(P<0.01)。结论:FLNa可通过调节F-actin在支持细胞中的排布参与TNFα引起的BTB功能降低。

Objective： To investigate the role of actin-binding protein filamin A （FLNa） in tumor necrosis factor α（TNFα）-regulated Sertoli cell barrier function change. Methods： Sertoli cells were isolated from 20-day- old rat testis and cultured in vitro. Trans-epithelial electrical resistance （TER） was measured after TNFα treatment to reflect barrier function change. Western blotting was applied to analyze FLNa level after TNFα treatment. Immunofluorescence and phalloidin staining were used to detect the localization of FLNa and F-actin, respectively. siRNAs targeting FLNa was transfected into cultured Sertoli cells to silence FLNa, followed by TER measurement to detect the effect of FLNa knockdown on blood-testis barrier （BTB） function in vitro. Results： The TER value of Sertoli cell barrier decreased significantly after TNFα treatment （P〈0.01）, accompanied by an obvious decline in FLNa level revealed by Western blotting. Immunofluorescence and phalloidin staining also showed discriminate distribution of FLNa and F-actin between TNFα group and control group. TER measurement also showed a compromised Sertoli cell barrier function after FLNa was silenced as compared with the control. Conclusion： FLNa particapated in TNFα-induced BTB function decrease by regulating F-actin arrangement in Sertoli cells.