低氧对原代支持细胞增殖能力和occludin蛋白表达的影响

Hypoxia reduces the proliferation and occludin expression of primary Sertoli cells

目的:观察低氧对原代大鼠睾丸支持细胞生长活性、occludin蛋白表达的影响。方法:18～22日龄Wistar大鼠,通过两步酶消化法,建立原代支持细胞培养体系,经油红O、免疫荧光法鉴定。传代后随机分为以下氧浓度组进行培养:20%、15%、10%、5%和1%。分别培养至6、12、24、48、72 h,CCK-8测定细胞增殖活性,West-ern印迹测定occludin蛋白表达量,并进行差异性分析。结果:油红O染色见胞质内脂滴染成红色,免疫荧光见FasL蛋白表达阳性,细胞纯度>95%。与20%氧浓度相比,细胞在15%和10%氧浓度下增殖率逐渐降低,5%和1%氧浓度下,细胞增殖率明显下降(P<0.01);从12 h开始,随着氧浓度降低和时间延长,occludin表达量明显下降(P<0.01)。结论:低氧会抑制支持细胞生长,并减少occludin蛋白表达。推测低氧环境损伤支持细胞参与的血睾屏障完整性,影响睾丸的生精过程。

[ Abstract] Objective： To investigate the effect of hypoxia on the proliferation and occludin expression of primary rat Sertoli cells. Methods： We constructed a primary Sertoli cell system by two-step enzymatic digestion in 18 -22 days old Wistar rats and identified it by oil red 0 and immunofluorescence methods. We randomly divided the Sertoli cells into five groups to be cultured in oxy- gen at the concentrations of 20%, 15%, 10%, 5% and 1%, respectively, for 6, 12, 24, 48 and 72 hours. We detected the proliferation of the Sertoli ceils by CCK-8 assay, determined the expression of occludin by Western blot, and analyzed the differences among the five groups. Results ： Oil red 0 staining revealed red lipid droplets in the cytoplasm of the Sertoli cells, and immunofluo- rescence showed the positive expression of the FasL protein, with the purity of Sertoli cells over 95% in vitro. Compared with the 20% normoxic group, the proliferation of the Sertoli cells was gradually reduced in the 15% and 10% hypoxia groups, and significantly de- clined in the 5% and 1% groups （P 〈0.01 ）. At 12 hours, the expression of occludin began to decrease with the prolonging of time and reduction of oxygen concentration （P 〈 0.01 ）. Conclusion ： Hypoxia suppresses the proliferation of Sertoli ceils and reduces the expression of occludin. It could be inferred that hypoxia could damage the integrity of blood-testis barrier and spermatogenesis of the testis.