周期性张应力对成纤维细胞凋亡的影响及其调控机制

背景：在体内条件下，细胞力学的功能研究因其所处生理环境的复杂性、实验条件的不易控制而很难得到满意结果。 目的：探讨周期性张应力对成纤维细胞凋亡的作用及影响机制。 方法：采用多通道细胞牵张应力加载系统，以成纤维细胞为对象构建细胞体外培养-力学刺激模型。加力组成纤维细胞分别给予周期性张应力刺激6，12，24 h，力值定为12% surface elongation，频率为6 cycles/min，每一循环包括 3 s stretch/3 s relaxation，并设立正常对照组。采用Hoechst33258染色检测细胞凋亡情况，以流式细胞仪分析细胞周期，应用酶标仪测定半胱氨酸天冬氨酸蛋白酶3的活性。 结果与结论：正常对照组细胞核为弥散均匀的圆形或椭圆形荧光，加力组细胞核或细胞质内可见致密浓染的颗粒、新月体或环状荧光。与正常对照组比较，加力组细胞周期显著延长(P < 0.01)；与加力6 h组比较，加力12，24 h组细胞周期均显著缩短(P < 0.01)，而加力24 h组细胞周期略长于加力12 h组。与正常对照组比较，加力组细胞半胱氨酸天冬氨酸蛋白酶3活性均显著升高(P < 0.01)；加力12 h组细胞半胱氨酸天冬氨酸蛋白酶3活性显著高于加力6 h组(P < 0.01)。表明周期性张应力可以诱导成纤维细胞凋亡，在12 h内加力时间越长，早期凋亡越明显，且能够改变其细胞周期，半胱氨酸天冬氨酸蛋白酶3参与了此过程。

Effect of cyclic tensile stress on fibroblast apoptosis and its regulatory mechanism

BACKGROUND: In vivo study of cellular mechanics obtains poor results due to complexity of physiological environment and uncontrollable experiment conditions. OBJECTIVE: To study the cyclic tensile stress on fibroblasts apoptosis and its mechanism. METHODS: In vitro culture-tensile stimulate models of fibroblast were established by using a multi-passage load adding system. Cyclic tensile stress was applied on the fibroblasts for 6, 12, and 24 hours, respectively, the load was set for 12% surface elongation, with frequency of 6 cycles per minute. A cycle of stress comprised 3 s stretch/3 s relaxation. At the same time, the normal control group was established. The cell apoptosis was determined by Hoechst 33258 staining, cell cycle was analyzed by flow cytometry, and the caspase-3 activity was detected by ELISA. RESULTS AND CONCLUSION: The nucleus in the normal control group presented with dispersed uniformly round or oval-shaped fluorescent, while dense stain particles, crescent or ring fluorescence could be seen within the nucleus or cytoplasm in the load groups. Compared with the normal control group, load adding significantly prolonged cell cycles (P < 0.01). The cell cycle appeared to be notably shortened in the 12 and 24 hours groups than that of 6 hours group (P < 0.01); however, the 24 hours group appeared to be extended the cell cycle than that of the 12 hours group. The caspase-3 activity was obviously increased in of the load adding group that that of the normal control group (P < 0.01), especially in the 12 hours group, which was greater than that of the 6 hours group (P < 0.01). Cyclical tensile stress not only can induce fibroblast apoptosis in a time-dependent manner within 12 hours, but also can change the cell cycle. Caspase3 involves in this process.