持续张应力对骨髓基质干细胞增殖及骨向分化的影响

目的通过体外加力装置研究持续牵张应力对大鼠骨髓基质干细胞(bone marrow stromal cells,BMSCs)增殖及骨向分化能力的影响。方法选用3月龄健康SD雌性大鼠,采用全血贴壁培养法分离及培养BMSCs。取生长良好的第3～5代细胞接种于Flexercell应力加载系统(10%、1 Hz),根据应力作用时间不同分为1、6、12、24 h组和48 h组。观察并分析持续牵张力对于大鼠BMSCs形态、增殖活性以及成骨能力变化的影响。结果 (1)随着加力时间的延长,与对照组相比,实验组细胞形态呈现一定规律性,细胞长轴多垂直于受力径向。(2)10%持续张应力作用可抑制BMSCs增殖活性。(3)持续张应力可增高碱性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型胶原(collagenI,COLⅠ)、核心结合因子Cbfa1(core binding factor a1,又名Runx2)mRNA的表达量,且呈现时间依赖性。其中实验组ALP表达量在24 h明显高于相应对照组,COLⅠ表达量在24 h及48 h均明显高于对照组,Runx2表达量在6 h与对照组相比显著增高(P<0.05)。骨钙素(osteocalcin,OC)含量在加力起始阶段显著高于对照组,随时间推移逐渐下降,48 h时明显低于对照组(P<0.05)。(4)持续张力可以促使Runx2蛋白水平增高,且在6 h实验组明显高于对照组(P<0.05)。之后缓慢下降,在24 h时显著低于对照组水平(P<0.05)。结论持续牵张力作用下BM-SCs细胞形态呈现一定规律性排列,其增殖活性受到抑制,但早期成骨向分化能力却显著提高。

Effect of continuous strain on proliferation and osteogenic differentiation of bone marrow stromal cells

Objective To study the effect of continuous strain on the proliferation and osteogenic differentiation of rat bone marrow stromal cells(BMSCs) in vitro.Methods Rat BMSCs were obtained from adult female Sprague-Dawley rats(3-month old),and purified by full-blood attachment culture.BMSCs between passage 3—5 were seeded on Flexercell mechanical loading system(10%,1 Hz),and divided into 1 h group,6 h group,12 h group,24 h group,48 h group,respectively,according to the time subjected to strain.Effects of continuous strain on the morphology,proliferation and osteogenic differentiation of BMSCs were observed and analyzed.Results(1) Compared with the control group,cells subjected to 10% strain showed the particular orientation.Their alignment elongated mostly in the direction perpendicular to the strain axis in a time-dependent manner.(2)10% continuous strain could significantly decrease the proliferation of BMSCs.(3) Continuous strain could increase mRNA expression of ALP,COLⅠand Runx2 in a time-dependent manner.Compared with the control group,mRNA expression of ALP was increased significantly at 24 h,COLⅠat 24 h and 48 h,and Runx2 at 6 h.mRNA expression of osteocalcin(OC) ascended greatly in the beginning,but went down gradually and was significantly lower than that of control at 48 h(P<0.05).(4) Continuous strain could induce an increase in Runx2 protein level.A sharp increase in Runx2 protein was observed at 6 h(P<0.05),then Runx2 protein level decreased slowly with its mRNA expression being significantly lower than that of control at 24 h(P<0.05) Conclusions Continuous strain could induce rat BMSCs to orient in an orderly manner,suppress its proliferation activity,but stimulate the osteoblastic differentiation at the early stage.