Phosphorylation of Src protein in rat nucleus pulposus cells under periodic mechanical stress

Abstract

Objective To evaluate the phosphorylation of Src protein and its role in rat nucleus pulposus cells under periodic mechanical stress.Methods Nucleus pulposus cells obtained from 4-week-old rats were cultured in vitro.The second generation cells were planted on glass slides at a concentration of 1 × 105/mL.Firstly,32 slides were randomized into 4 even groups:control group,0.5 h stress group,1.0 b stress group and 2.0 h stress group.The stress groups were cultured under periodic mechanical stress (0 to 200 kPa,0.1 Hz) respectively for 0.5,1.0 and 2.0 hours while the control group was cultured without mechanical stress.Western blot was used to evaluate the expressions of pSrc protein in the 4 groups.Secondly,another 32 slides were randomized into 3 even groups:control group,6 h stress group and PP2 + 6 h stress group in which the inhibitor PP2 was added.Gene expressions of Type Ⅱ collagen and aggrecan were evaluated by Real-time PCR.Results The grey values of pSrc/GAPDH ratio in the control,0.5 h stress,1.0 h stress and 2.0 h stress groups were respectively 0.244 ±0.013,0.477 ±0.044,0.530 ± 0.014 and 0.700 ± 0.063.There were significant differences among the 4 groups (P ＜ 0.05),and between 2 groups except between 0.5 h stress and 1.0 h stress groups (P ＜ 0.05).The gene expressions of type Ⅱ collagen and aggrecan in PP2 +6 h stress group (3.038±0.237 and 1.678 ±0.125,respectively) were significantly higher than those in the control group (1.001 ± 0.039 and 1.004 ± 0.104,respectively) but lower than those in the 6 h stress group (5.404 ±0.219 and 2.127 ±0.028,respectively) (P ＜ 0.05).Conclusions Periodic mechanical stress can promote the metabolism of rat nucleus pulposus cells.The phosphorylation of Src protein may play an important role in the mechanical transductions. Key words: Intervertebral disk; Src protein; Periodic mechanical stress; Nucleus pulposus cell