周期性压力下椎间盘细胞中肌动蛋白的变化及其意义

The difference and its significance of actin in inverterbral disc cells under cyclic hydrostatic pressure in vitro

目的探讨在周期性压力下,体外培养的椎间盘细胞中的肌动蛋白的变化及其意义。方法采用10只5～6周龄、体重为25～30kg的猪的椎间盘进行体外细胞的分离和培养,对细胞施加周期性液压,通过对细胞的形态学观察及Western免疫印迹和免疫荧光实验,检测在周期性压力下椎间盘细胞中的肌动蛋白的变化。结果纤维环细胞和髓核细胞经1MPa、1Hz,持续每天3h加压,3d后髓核细胞的存活率大于90%,纤维环细胞的存活率大于85%,加压后的纤维环细胞和髓核细胞体积均缩小,由多角形转变为细长形。Western免疫印迹结果显示,肌动蛋白在纤维环细胞和髓核细胞中的表达均明显减少,以髓核细胞为著。免疫荧光结果显示,肌动蛋白由正常纤维环细胞和髓核细胞内的疏松排列转变为受压细胞内的紧密排列,髓核细胞的体积明显缩小,其中的肌动蛋白在细胞膜周围紧密排列。结论体外培养的单层椎间盘细胞在受力状态下,肌动蛋白的表达减少,细胞的体积缩小,并且肌动蛋白在细胞内重排列。这可能是激发细胞内或细胞表面信号传导的重要机制。

Objective To investigate the difference and its significance of actin in inverterbral disc cells under cyclic hydrostatic pressure in vitro. Methods 10 porcines (range, 5-6 weeks; weight, 25-30 kg) involved in this study. The porcine lumbar intervertebral disc cells were isolated and cultured in vitro, and the cells were underwent cyclic hydrostatic loading. After that, the expression of actin in inverterbral disc cells was studied by means of morphology observing, Western blot and immunofluorescence. Results After loading with cyclic hydrostatic pressure (1 MPa, 1 Hz, 3 h/day) for 3 days, the cell viability was greater than ninety percent in the cells of nucleus pulpous, and eighty-five percent in the cells of anulus fibrous. The morphology of intervertebral disc cells was changed into smaller and flattened shape. A reduction in actin expression was observed by immunoblotting in both nucleus pulpous and anulus fibrous cells, especially in nucleus pulpous cells. Immunofluorescence showed that the actin in both anulus fibrous and nucleus pulpous cells was compact and compressed after loading compared with its loosed form before loading. There was a great reduction of volume of nucleus pulpous cells following loading, and most of the actin in nucleus pulpous cells assembled around its membrane. Conclusion The expression of actin in inverterbral disc cells was decreased under cyclic hydrostatic pressure, which leads to a decrease in cell volume and rearrangement of actin in the disc cells. That may be one of the important mechanisms in activating the signal transduction in and out of the intervertebral disc cells.