人软骨细胞压力实验模型的建立与评估

目的:人承重关节内受到的多种机械应力(剪切力、张力、静水压力等)在调节关节软骨细胞的生理功能方面起着重要作用。建立对人膝关节软骨细胞施加不同强度周期性静水压的压力模型,观察不同压力强度下软骨细胞的生长形态、增殖和凋亡情况。方法:采用酶消化法分离培养正常成人膝关节软骨细胞,将培养的第3代软骨细胞分为6组:对照组、0.5 MPa组、1.0 MPa组、3.0MPa组、5.0 MPa组、8.0 MPa组,应用高压恒温静水压加载系统分别给予各组不同强度压力作用5 d,每日1 h。甲苯胺蓝染色法和Ⅱ型胶原免疫组织化学染色法鉴定软骨细胞,倒置相差显微镜观察细胞形态和生长状况,流式细胞术检测细胞凋亡,四甲基偶氮唑蓝(MTT)法绘制细胞生长曲线。结果:与对照组相比,0.5 MPa、3.0 MPa组无明显差异(P0.05);1.0 MPa组能促进软骨细胞增殖,抑制凋亡(P0.05);5.0 MPa组出现细胞增殖能力下降,细胞活力降低,凋亡率增加(P0.05);8.0 MPa组则表现出明显的细胞增殖的抑制和细胞凋亡趋势(P0.05),以及细胞形态学的改变。结论:不同强度的周期性压力对人软骨细胞的新陈代谢产生了不同影响,尤其在软骨细胞的增殖和凋亡水平方面。利用本压力实验模型能体外模拟人负重关节软骨细胞的受压情况,初步确定了人软骨细胞压力实验中压力梯度的选择。为软骨细胞的压力损伤研究提供了实验数据,为进一步探寻压力作用与骨关节炎的关系提供了实验平台。

The Establishment and Assessment of the Pressure Experimental Model in Human Chondrocytes*

Objective： Varieties of mechanical stresses inside human weight-bearing joints （shear force, tensile force and hydrosta- tic pressure） play an important role in the regulation of physiological function of articular chondrocytes. To establish a model of different intensities of periodic hydrostatic pressures in human knee joint chondrocytes and observe cell morphology, proliferation and apoptosis under different intensities of pressures. Methods： The normal human knee joint chondrocytes were isolated and cultured fi＇om surgical specimens. The third generation of chondrocytes were treated with different hydrostatic pressures （0, 0.5, 1.0, 3.0, 5.0 and 8.0 MPa） for 1 h daily for 5 days. Toluidine blue staining and immunohistochemical staining of type II collagen were employed to identify the chondrocytes. Cell growth and morphology were observed in all groups by light microscopy. Cell apoptosis and proliferation were examined by flow cytometry and MTT assay respectively. Results： Compared with the control group, the 0.5 MPa and 3.0 MPa groups did not show any significant difference （P〉0.05）; The 1.0 MPa group hydrostatic pressures promoted proliferation of chondrocytes and inhibited apoptosis （P〈0.05）; The proliferation activity of 5.0 MPa group chondrocytes was decreased, and the apoptosis was increased （P〈0.05）; The 8.0 MPa group showed significant trend in inhibition of cell proliferation and promotion of cell apoptosis （P〈0.05）, along with cell morphology changes were observed. Conclusion： Different intensities of periodic pressures had different influence on the metabolism of human chondrocytes, especially in the levels of proliferation and apoptosis. We could simulated the compression on chondrocytes of weight-bearing joints by using this model in vitro, and preliminarily determined the human chondrocytes pressure experiment in the choice of pressure gradient. Our experiment provides experimental data and experimental basis for research of pressure injury to chondrocytes, in order to explore the relationship between pressure and osteoarthfitis for further research.