流体切应力促成骨细胞通过细胞周期G1/S调控点机制的研究

Fluid stress with multiple mechanisms to enhance the cell cycle progression of osteoblastic cells from G1 to S phase

[目的]探讨流体切应力(FSS)促成骨细胞增殖、分化作用与细胞周期从G1期向S期转化机制的关系,为确立最佳生理应力刺激骨再生提供依据。[方法]从KM乳鼠颅盖骨提取原代成骨细胞,在体外流体小室内分别受FSS(12dyn/cm2)作用0、0.25、0.5、1、2、4h,通过MTT法分析细胞增殖能力,并利用碱性磷酸酶(ALP)的表达评价其分化能力;通过流式细胞仪、免疫荧光染色和RT-PCR测定细胞周期G1/S百分比、细胞周期依赖激酶2和4(CDK2、CDK4)的活性改变及E2F1、p27mRNA的表达实现FSS促成骨细胞由G1期转向S期的测定。[结果]FSS促增殖作用在短期(0.25h、0.5h)明显,并且细胞生长曲线前移;但在1、2、4h却有明显抑制增殖作用。FSS同样增加了ALP的活性,尤其在应力作用0.25、0.5h时显著(比对照达128%和158%);而应力作用1、2、4h后减低了ALP的表达。在FSS作用1h内细胞周期S期百分比增高,作用0.5h后与对照组比较明显增高(P<0.05);但随着时间的增加细胞周期S期百分比开始下降,当作用时间持续4h后S期百分比下降明显(P<0.05)。流体切应力增加了活化pRb的CDK2、CDK4的活性,而且与CDK2相关的E2F1表达量也明显增加;而细胞周期依赖激酶抑制剂p27在流体切应力作用下有所下降。其中流体切应力在0.25h、0.5h明显增加了E2FlmRNA的表达水平(P<0.05),此效应在作用1h后减退。[结论]适宜的流体切应力通过上调CDK2、CDK4及E2F1,下调p27,使细胞从G1期转化到S期,在流体切应力促成骨细胞增殖、分化机制中起重要作用;并且这种作用有明显的FSS作用时间限制性。

[ Objective] To investigate into the cellular mechanism of growth promotion due to shear stress by studying G1 - phase events responsible for the suppression of cell transition from the G1 to S phase of the cell cycle, and to establish the most suitable physiological stress to stimulate bone formation. [ Methods] The osteoblasts derived from Kunming murine＇s calvaria were exposed to Fliud shear stress （ FSS： 12 dyn/cm^2） for 0, 0.25, 0. 5, 1, 2, 4 h, respectively. In the flow chamber, its impact on cell proliferation, differentiation and the effection of cell cycle＇ s G1/S checkpoint were recorded. The cell proliferation was studied by MTT assay. The cell differentiation was assessed through alkaline phosphatase （ALP） activity assay. Flow -cytometry, immunofluorescence and RT- PCR techniques were used to evaluate the proportion of S phase in cell cycle, the activity of CDK2, CDK4 and the expression of E2F - 1, p27mRNA, which demonstrate how FSS underlying multiple mecha- nisms to enhance the cell cycle progression from GI to S phase. [ Results] FSS increased proliferation and advanced the time in cell growth curve, but after 1, 2, 4 h, the proliferation was inhibited. The FSS also increased the ALP activity, which were significantly stimulated at 0. 25 and 0. 5 h after shear stress （ 128% and 158 % of control） ; but the FSS decreased ALP activity at 1, 2, and 4 hs. The proportion of S phase in cell cycle raised within the early period. The S phase rate significantly increased at 0. 5 h （ P 〈 0. 05 ） , compared with the controls. The proportion of S phase in cell cycle began to decrease at 0. 5 h timepoint, and the S phase significantly decreased at 4 hs （ P 〈 0. 05） . Shear stress enhanced the activity of cyclin -dependent kinase （cdk） 2 and cdk4, which would phosphorylate pRb, and the amount of E2F1 associated with cdk2 also increased. The level of cdk inhibitor p27, decreased as a result of shear stress. Shear stress significantly elevated the mRNA level of E2F1 at 0. 25 and 0. 5 h （ P 〈 0. 05 ）, this elevation in mRNA faded 1 hour later. [ Conclusion] These results suggest that the suitable FSS can stimulate the transition from the G1 to S phase through the upregulation of CDK2, CDK4 in a way which is similar to the effects of E2F1, the downregnlation of P27. These alterations are involved in key biological processes related to cell proliferation and differentiation, and these regulations showed a time -limit exhibite.