流体切应力下SIVA-1凋亡诱导因子促成骨细胞增殖分化的双向调节作用

Two-way Regulation of Apoptosis-inducing Factor SIVA-1 in the Proliferation and Differentiation of Osteoblasts under Fluid Shear Stress

目的探讨流体切应力(fluid shear stress,FSS)对KM小鼠成骨细胞增殖分化及相关细胞凋亡诱导因子(SIVA-1)的调节作用。方法将传至3代成骨细胞分为应力刺激组(试验组)和非应力刺激组(对照组)。5个试验组分别给予1.2PaFSS作用0.25,0.5,1,2,4h;对照组为0h。MTT法测定细胞增殖,检测碱性磷酸酶(ALP)活性,半定量RT-PCR测定凋亡诱导因子SIVA-1的表达水平。结果FSS促增殖作用在短期(0.25,0.5h)明显,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活性,尤其在应力作用0.5h时显著(ALP含量为2.4320±0.205金氏单位/100ml,相对对照达158%)(P<0.05);而应力作用1,2,4h后减低了ALP的表达。应力初期SIVA-1mRNA表达量明显下降,尤其在FSS作用0.5h后SIVA-1/GAPDH相对表达量为0.099±0.002,相对对照明显下调了71.3%(P<0.01),此效应在作用1h后减退,SIVA-1mRNA表达开始升高,4h后升高明显。结论1.2PaFSS在短时间刺激下可刺激成骨细胞增殖分化,尤其在0.5h后效应明显。早期促细胞增殖和ALP水平升高主要与SIVA-1mRNA表达下调有关,而后期抑制作用可能与SIVA-1mRNA表达升高有关。

Objective To investigate the regulatory effect of fluid shear stress （FSS） on the proliferation and differentiation of osteoblasts, as well as the expression of apoptosis-inducing factor, SIVA-1. Methods The third-passage osteoblasts were divided into five experiment groups and one control group. In the experiment groups, 1.2 Pa FSS were given to the osteoblasts for 0.25, 0.5, 1, 2, and 4 hours respectively, while the control group received no FSS. Afterwards, the cells were harvested to measure MTT value and ALP activity; mRNA level of SIVA-1 were determined by RT-PCR. Results MTT revealed that the cells proliferation markedly increased in the 0.25 h and 0.5 h experiment groups with advanced cell growth curve; whereas significantly inhibited in 1, 2, and 4 h groups. The FSS also increased the ALP activity at 0.25 and 0.5 hour, especially in the 0.5 h group （2. 4320± 0. 205 S unit/100ml, 158% of the control; P 〈 0.05） , while at the 1, 2 and 4 h groups, the expression of ALP deceased significantly. The SIVA-1 mRNA expression decreased as a result of short-time FSS, the relative expression of SIVA-1/GAPDH is 0. 099±0. 002 at 0.5 hour （decreased by 71.3% compared with the control, P 〈 0.01 ）. The expression began to increase at 1 hour, and then increased significantly at 4 hours. Conclusions 1.2 Pa FSS can enhance the proliferation and differentiation of osteoblasts at the early period; the efficacy is especially significant at0. 5 hour. The enhanced proliferation and increased ALP activity at early stage is related to the down-regulation of SIVA-1 expression, and the inhibitory effect in the late state may be associated with the increased expression of SIVA-1.