자외선에 의한 각막 섬유아세포노화와 안지오제닌의 항노화 역할
Ultraviolet B-induced Senescence Model Using Corneal Fibroblasts and the Anti-aging Effect of Angiogenin
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목적: 각막 섬유아세포의 ultraviolet (UV) B에 의한 노화모델을 확립하고 angiogenin (ANG)의 항노화 역할에 대해 알아보고자 하였다. 대상과 방법: 섬유아세포를 UVB (1 mJ/cm2)에 노출 후 ANG 포함 배지로 24시간 배양했으며, 자외선 조사 후 배양하는 과정을 3일간 반복하였다. 세포생존능력은 3-[4, 5–dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay로 평가하였으며, 세포노화 정도는 전체 세포 수 중 senescence-associated (SA)-β-gal 염색된 세포 수의 비를 구하여 확인하였다. 노화관련 인자의 발현 및 DNA 손상 정도는 Western blot을 통해 확인하였다. 실험은 non-UVB군, UVB 조사 후 ANG 처리를 하지 않은 UVB군, ANG 처리한 UVB + ANG군으로 나누어 비교하였다. 결과: UVB + ANG군은 UVB군에 비해 세포생존능력이 11% 유의하게 높았으며, 세포노화 정도는 10% 유의하게 낮았다(p<0.05, p<0.05). 노화관련 인자의 발현은 UVB + ANG군이 UVB군에 비해 p53, p21, p16, RB의 발현이 감소하였다. 인산화된 히스톤 단백질(γ-H2AX)과 p38의 발현은 UVB + ANG군이 UVB군에 비해 적게 나타났다. 결론: UVB는 각막 섬유아세포에서 세포노화를 유발하며, ANG은 p53, p21, p16, RB를 통해 세포주기를 조절하고 DNA 손상을 감소시켜 항노화 효과를 가지는 것으로 생각된다.
Abstract
Purpose: To establish a corneal-fibroblast senescence model induced by ultraviolet B (UVB) exposure and to investigate the anti- senescence effect of angiogenin (ANG). Methods: Fibroblasts were exposed to UVB (1 mJ/cm2) and then cultured with ANG-containing solution for 24 hours. The 24-hour culture procedure was repeated for three days after UVB irradiation. Cell viability was evaluated using the 3-[4, 5–dimethylthiazol- 2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. The degree of senescence was assessed using the ratio of senescence- associated (SA)-β-gal-stained cells to total cells. The expression of age-related factors and degree of DNA damage were assessed via Western blot. Samples were divided into a non-UVB group, a UVB group without ANG treatment, and a UVB group with ANG treatment after irradiation (UVB + ANG). Results: Cell viability in the UVB + ANG group was 11% higher than that in the UVB group (p < 0.05). The UVB + ANG group exhibited a 10% lower degree of SA-β-gal staining compared with the UVB group (p < 0.05). Western blot analysis showed that there was reduced expression of p53, p21, p16, and RB in the UVB + ANG group compared with the UVB group. The expression of phosphorylated histone (γ-H2AX) and p38 in the UVB + ANG group was less than that in the UVB group. Conclusions: Senescence in corneal fibroblasts is induced by UVB, and ANG may exert an anti-aging effect by regulating the cell cycle through p53, p21, p16, and RB and reducing DNA damage.
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