CAJ | 학술논문

目的探讨周期性应力下大鼠髓核细胞中Src蛋白的磷酸化水平及其意义. 方法体外培养SD大鼠髓核细胞,取第2代细胞按1×105/mL的密度接种于玻片上.将玻片随机分成4组(n=8):对照组、加压0.5h组、加压1.0h组、加压2.0h组.加压0.5h组、加压1.0h组、加压2.0h组细胞以0 ～ 200 kPa的压力、0.1Hz的频率分别加压0.5、1.0、2.0h,对照组在无压力环境下培养.应用Western blot检测各组磷酸化Src(pSrc)蛋白的表达.应用PP2[4-氨基-5-(4-氯苯)-7-(t-丁基)吡唑啉酮(3,4-d)嘧啶]抑制Src蛋白,另取细胞玻片,随机分为3组(n=8):对照组、加压6h组、PP2+加压6h组.使用荧光实时定量多聚酶链式反应(RT-PCR)检测3组细胞中Ⅱ型胶原和蛋白多糖的基因表达.结果对照组、加压0.5h组、加压1.0h组及加压2.0h组pSrc/磷酸甘油醛脱氢酶灰度比值平均分别为0.244 ±0.013、0.477±0.044、0.530±0.014、0.700±0.063,4组之间比较差异有统计学意义(P＜0.05),除加压0.5h组与加压1.0h组之间外,其余组别之间两两比较差异均有统计学意义(P＜0.05).荧光RT-PCR检测3组Ⅱ型胶原和蛋白多糖的基因表达结果显示:对照组表达量最低(1.001±0.039、1.004 ±0.104),加压6h组最高(5.404 ±0.219、2.127 ±0.028),PP2+加压6h组居中(3.038 ±0.237、1.678±0.125),3组之间两两比较差异均有统计学意义(P＜0.05).结论周期性压力能促进髓核细胞Ⅱ型胶原和蛋白多糖的分泌,Src蛋白磷酸化在压力传导中起信号传递作用.
목적 탐토주기성응력하대서수핵세포중Src단백적린산화수평급기의의. 방법 체외배양SD대서수핵세포,취제2대세포안1×105/mL적밀도접충우파편상.장파편수궤분성4조(n=8):대조조、가압0.5h조、가압1.0h조、가압2.0h조.가압0.5h조、가압1.0h조、가압2.0h조세포이0 ～ 200 kPa적압력、0.1Hz적빈솔분별가압0.5、1.0、2.0h,대조조재무압력배경하배양.응용Western blot검측각조린산화Src(pSrc)단백적표체.응용PP2[4-안기-5-(4-록분)-7-(t-정기)필서람동(3,4-d)밀정]억제Src단백,령취세포파편,수궤분위3조(n=8):대조조、가압6h조、PP2+가압6h조.사용형광실시정량다취매련식반응(RT-PCR)검측3조세포중Ⅱ형효원화단백다당적기인표체.결과 대조조、가압0.5h조、가압1.0h조급가압2.0h조pSrc/린산감유철탈경매회도비치평균분별위0.244 ±0.013、0.477±0.044、0.530±0.014、0.700±0.063,4조지간비교차이유통계학의의(P＜0.05),제가압0.5h조여가압1.0h조지간외,기여조별지간량량비교차이균유통계학의의(P＜0.05).형광RT-PCR검측3조Ⅱ형효원화단백다당적기인표체결과현시:대조조표체량최저(1.001±0.039、1.004 ±0.104),가압6h조최고(5.404 ±0.219、2.127 ±0.028),PP2+가압6h조거중(3.038 ±0.237、1.678±0.125),3조지간량량비교차이균유통계학의의(P＜0.05).결론 주기성압력능촉진수핵세포Ⅱ형효원화단백다당적분비,Src단백린산화재압력전도중기신호전체작용.
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.