周期性牵张椎间盘纤维环细胞肌动蛋白骨架的重排

Effects of cyclic tensile strain on actin cytoskeleton rearrangement in annulus fibrosus cells

背景:当椎间盘承受应力时,髓核内部产生的液压使纤维环向外扩张、膨胀,纤维环胶原纤维被拉伸,使纤维环细胞外基质也受到压力的作用。蛋白聚糖是椎间盘中主要的蛋白多糖成分,基质金属蛋白酶2是椎间盘降解细胞外基质的主要酶,金属蛋白酶组织抑制剂是特异性抑制基质金属蛋白酶活性的多功能因子,与基质金属蛋白酶相互调节,保持平衡。目的:探讨周期性牵张在椎间盘纤维环基质代谢中的作用机制。方法:采用Flexcell4000牵张系统对体外培养的大鼠椎间盘纤维环细胞施加牵张应变为2%和10%、频率为1.0 Hz,时间为2 h和12 h的周期性牵张,分别于2 h和12 h卸载并收集细胞和条件培养基分别进行基因和蛋白检测。采用荧光定量PCR检测aggrecan、基质金属蛋白酶2以及组织基质金属蛋白酶抑制剂2 mR NA的表达;采用明胶酶谱法检测基质金属蛋白酶2蛋白活性。结果与结论:(1)2%牵拉对肌动蛋白骨架形成的应力纤维影响不大,10%牵拉则使肌动蛋白骨架发生明显解聚。(2)2%牵拉12 h Aggrecan上调,牵拉2 h基质金属蛋白酶2、组织基质金属蛋白酶抑制剂2上调,二者保持动态平衡,基质金属蛋白酶2活性无显著变化。(3)10%牵拉对Aggrecan无影响。无论牵拉2 h或12 h,均使基质金属蛋白酶2上调,组织基质金属蛋白酶抑制剂2下调,二者失衡,基质金属蛋白酶2活性无显著变化。(4)结果提示周期性牵张可以在基因水平上对纤维环细胞Aggrecan、基质金属蛋白酶2和组织基质金属蛋白酶抑制剂2基因进行调节,通过调节肌动蛋白骨架从而对力学刺激产生响应。

BACKGROUND： When the intervertebral disc is under stress, the hydraulic pressure generated inside the nucleus pulposus makes the annulus fibrosus extend outward and expand, and the annulus collagen fibers are stretched so that the extracellular matrix of annulus fibrosus cells is also under the pressure. In the intervertebral disc, aggrecan is the main component of proteoglycans, matrix metalloproteinase-2 is a major enzyme for extracellular matrix degradation, and tissue inhibitor of metalloproteinase is a multifunctional specific inhibition factor for matrix metalloproteinase activity. There is a mutual regulation between the latter two to keep the homeostasis between them. OBJECTIVE： To investigate the mechanism of cyclic tensile strain in the metabolism of intervertebral disc annulus matrix. METHODS： Rat anulus fibrosus cells were subjected to 2% or 10% cyclic tensile strain at 1.0 Hz for 2 and 12 hours using Flexcell4000 tension system. Then cells were collected and cultured in conditioned medium for gene and protein detection. Real-time quantitative PCR was used to detect m RNA expression of aggrecan, matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2. Gelatin zymography was used to detect matrix metalloproteinases-2 activity. RESULTS AND CONCLUSION： The use of 2% cyclic tensile strain had no obvious effect on the stress fiber of actin cytoskeleton, whereas actin cytoskeleton was depolymerized in response to 10% cyclic tensile strain. The 2% cyclic tensile strain raised the expression of Aggrecan at 12 hours; whereas raised the matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2 at 2 hours, both of which were in homeostasis; matrix metalloproteinases-2 activity had no significant changes. 10% cyclic tensile strain had no effect on the mR NA expression of Aggrecan. No matter stretching 2 or 12 hours, the matrix metalloproteinases-2 was up-regulated, and the tissue inhibitor of metalloproteinase-2 was down-regulated, both of which were not in balance. Moreover, the matrix metalloproteinases-2 activity was not significantly changed. These findings indicate that the mR NA expressions of Aggrecan, matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2 alter in response to cyclic tensile strain in rat anulus fibrosus cells, and the tensile strain induces different mechano-responses in the actin cytoskeleton.