机械负荷调节猪腰椎间盘细胞表达α5β1整合素的体外研究

The effect of mechanical load on integrin subunits α5β1 in the cells of inverterbral disc cultured in vitro

目的研究α5β1整合素在体外培养的猪腰椎间盘细胞中的表达和机械负荷对其的影响。方法取10只年龄5～6周、体重25～30kg的猪,处死后12h内,无菌条件下切取完整的腰椎,剔除腰椎周围的韧带和软组织,尤其是椎间盘周围的韧带。从腹侧一次性切开椎间盘取出髓核(nucleuspulpous,NP),仔细分离纤维环(anulusfibrous,AF),将两者立即置于Hanks平衡盐溶液中,制成细胞悬液。分别对腰椎间盘的AF和NP细胞施加1MPa、1Hz,3h/d,共3d的周期性液压,通过对细胞的形态学观察、Western免疫印迹和免疫组织化学染色,检测α5β1整合素在正常腰椎间盘AF细胞和NP细胞中的表达及周期性压力对其的影响。结果经周期性液压后,NP细胞的存活率大于90%,AF细胞的存活率大于85%,加压后的AF细胞和NP细胞均可见体积缩小。α5β1整合素在正常腰椎间盘AF细胞和NP细胞中的表达呈强阳性。Western免疫印迹结果显示:加压后α5β1整合素在AF细胞中的表达均明显减少,P值分别为0.000、0.003,与对照组比较差异有统计学意义;α5β1整合素在NP细胞中的表达也明显减少,P值分别为0.001、0.015,与对照组比较差异有统计学意义。结论机械负荷可引起腰椎间盘细胞中α5β1整合素的变化,提示α5β1整合素在腰椎间盘细胞中可能发挥力学传感器的作用。

Objective To investigate the expression of integrin α5β1 in the cells of porcine intervertebral disc cultured in vitro and the effect of mechanical load on them. Methods The porcine lumbar cells were got from 10 pigs （age for 5-6 weeks and weight for 25-30 kg）. Within killed 12 hours, the lumbar intervertebral disc was got asepticly, and the peripheral ligament and soft tissue were rejected. The nucleus pulpous （NP） cells and anulus fibrous （AF） cells in lumbar intervertebral disc were isolated and cultured in vitro respectively, and then both cells underwent cyclic hydrostatic loading at 1 MPa, 1 Hz for 3 days（3 h/d）. After that, the expressions of integrin α5β1 in inverterbral disc NP and AF cells and the effects of cyclic loading were studied by means of morphological observing, Western blot and immunohistochemistry staining. Results Cell viability of greater than ninety percent was observed in the NP cells, while eighty-five percent in the AF cells after 3 days cyclic hydrostatic loading. The morphology of intervertebral disc cells were changed into smaller and flatten shape, and the expression of integrin α5β1 was strong positive both in AF and NP cells. After cyclic loading, there were significant decreases in the expressions of integrin α5β1 in both AF and NP cells with the Western blot compared to the control groups （P=0.000, 0.003 and 0.001, 0.015）. Conclusion The effect of mechanical load on integrin α5β1 in the cells of inverterbral disc cultured in vitro indicates that integrin α5β1 may act as a mechanical signal transducer.