H_2O_2氧化应激诱导兔椎间盘髓核细胞衰老的研究

Reseach of oxidative stress induces aging in rabbit intervertebral disc nucleus pulposus cells injured by H_2O_2

目的:研究H2O2不同浓度对兔椎间盘髓核细胞形态、活力、增值、周期等的影响。方法:新西兰大白兔(2～3kg,雌)10只,无菌条件下酶消化法分离髓核细胞。含15%FBS的DMEM/F12(1:1)培养液培养,细胞90%融合后传第1代。按照H2O2不同浓度(0μmol/L、130μmol/L、216μmol/L、360μmol/L、600μmol/L、1000μmol/L)分组,0μmol/LH2O2为空白对照组。在细胞对数生长期,不同浓度H2O2处理1h后原培养液继续培养48h,通过检测,分析比较各组髓核细胞与空白对照组间形态、活力、增值、周期的差异性。结果:与空白对照组比较,当H2O2浓度为130μmol/L、216μmol/L时,髓核细胞生物学特性未见显著性改变(P>0.05);当H2O2浓度为360μmol/L、600μmol/L、1000μmol/L时,髓核细胞出现衰老改变:细胞质减少并出现空泡、增值减慢、衰老相关-β-半乳糖苷酶蓝染率增加、细胞周期阻滞于G1期而进入S期减少(P<0.05)。随着H2O2浓度的增高,衰老程度不断升高。结论:一定浓度的H2O2能够导致髓核细胞提前衰老,引起髓核细胞生物学特性的改变。

Objective： To study the effect of H202 on the morphological pattern, vitality, proliferation, cycle period of rabbit intervertebral disc nucleus pulposus cells. Methods：Ten New Zealand white rabbits （2 to 3 kg, female） were used for isolating nucleus pulposus cells under sterilized condition. The culture solution with 15% FBS and DMEM/F12 （ 1：1 ） was applied for cell cultivation. After 90% cell fusion ,the first generation was obtain and stimulated by H202 with different concentrations of 0μmol/L （control group）, 130 I.Lmol/L, 216 μmol/L, 360 μmol/L, 600 μmol/L, and 1000 μmol/L. Results：Compared with the control group ,there was little difference of the biological property （P〉0.05） in 130 μmol/L and 216 μmol/L H202-treated groups. When the concentration of H202 attained 360μmol/L, 600 μmol/L, and 1 000 bLmol/L, the cells suffered aging, with increased cell vacuoles, decreased proliferation, and aging：related increase of β-galactosidase dyeing. The cell cycle of many nucleus pulposus cells was blocked in G1 stage other than entering S stage. With increasing H202 concentrations ,the aging degree was increased. Conclusion： A certain concentration of H202 could induce early aging of nucleus pulposus cells, resulting in biological abnormalities of these cells.