pH对衰老相关β-半乳糖苷酶染色的影响

Effect of pH on senescence associated β-galactosidase staining

目的观察正常老化及应激诱导老化（stress—inducedprematuresenescence，SIPS）的小鼠成纤维细胞在不同pH值条件下，衰老相关β-半乳糖苷酶（senescence—associatedp—galactosidase，SA—β-Gal）染色效果的变化规律。方法取新生1～3dC57BL／6小鼠背部皮肤成纤维细胞进行传代培养，UVB照射应激诱导老化细胞，以不同代次的细胞和应激诱导老化细胞为实验对象，进行衰老相关蛋白检测，并在不同pH条件下进行衰老相关β-半乳糖苷酶染色，对其着色情况进行观察和分析。结果在P1代细胞、P6代细胞、应激诱导老化细胞和P12代细胞中，p53／p21蛋白表达依次增多；正常P1代细胞衰老相关β-半乳糖苷酶染色阴性，其余细胞组随着pH值的由低到高（6．0～8．0），着色情况均呈现由强到弱的变化趋势，P6细胞、SIPS细胞、P12细胞分别在pH7．0、7．5和8．0时阳性染色消失。结论随着细胞衰老程度的增加，衰老相关p一半乳糖苷酶染色将在较高pH时才会呈现假阴性，提示染色时安全pH范围应根据细胞代次而定。

Objective To investigate the alteration of the level of senescence-associated β-galactosidase （SA-β-Gal） in the mouse dermal fibroblasts （MDF） of replicative senescence and stressinduced premature senescence （SIPS） in different pH staining solutions. Methods MDF were harvested from dorsal skin of newborn 1 -3 d C57BL/6 mice for serial subcultivation. The SIPS cells were induced by repeated UVB irradiation. The aging process was evaluated by western blotting of p53/p21 protein and senescence-associated and β-galactosidase histochemical staining was also performed in different pH solutions. The staining results were observed and analyzed. Results The expression of p53 and p21at the P1 ,P6,SIPS and P12 cells was increased in turn; SA-β-Gal staining was negative at PI cells. With the increasing of pH （6.0 - 8.0）, the positive percentage of SA-β-Gal staining in the other cells decreased, the positive expression in the P6,SIPS and P12 disappeared at pH of 7.0, 7.5, and 8.0, respectively. Conclusion With the development of cell senescence, SA-β-Gal staining would show the a false negative when the pH value increased, suggesting that the safe pH range for SA-β-Gal staining should be determined with corresponding passages taken into account.