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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。
Objective To investigate the effects of sustained stretch stress on proliferation and differentiation of bone marrow stromal cells (BMSCs) in vitro by means of an external force device. Methods 3-month-old healthy SD female rats were used to isolate and culture BMSCs by whole-blood adherent culture. Well-growing 3rd to 5th passage cells were seeded on a Flexercell stress loading system (10%, 1 Hz) and were divided into 1, 6, 12, 24 h and 48 h groups according to different stress time. Observe and analyze the effect of sustained stretch on the morphous, proliferation activity and osteogenic capacity of BMSCs in rats. Results (1) Compared with the control group, the morphology of the cells in the experimental group showed a certain regularity with the extension of the energization time, and the long axis of the cells was perpendicular to the radial direction of the force. (2) 10% continuous tensile stress can inhibit the proliferation of BMSCs. (3) Continuous tensile stress can increase the expression of alkaline phosphatase (ALP), collagenⅠ (COLⅠ) and core binding factor Cbfa1 (aka Runx2) mRNA, Dependency. The expression of ALP in the experimental group was significantly higher than that of the corresponding control group at 24 h, the expression of COL Ⅰ was significantly higher at 24 and 48 h, and the expression of Runx2 at 6 h was significantly higher than that of the control group (P <0.05) . The content of osteocalcin (OC) was significantly higher than that of the control group at the initial stage of afterburner, and gradually decreased with time, significantly lower than that of the control group at 48 h (P <0.05). (4) Continuous tension can promote Runx2 protein levels, and in the 6 h experimental group was significantly higher than the control group (P <0.05). Then decreased slowly, which was significantly lower than that of the control group at 24 h (P <0.05). CONCLUSIONS: The morphology of BM-SCs cells is regularly arranged under sustained tension, and its proliferation activity is inhibited. However, the differentiation ability of BM-SCs in early osteoblast is significantly increased.