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目的:在体外条件下,探讨周期张应力作用对人牙周膜成纤维细胞凋亡的影响及PI3k/Akt信号通路在细胞凋亡中的作用。方法:应用多通道细胞牵张应力加载系统,以HPDLFs(人牙周膜成纤维细胞)为对象构建细胞体外培养-力学刺激模型,对照组为0h,0h+LY294002,加力组1 h,6 h,12 h,12 h+LY294002,24 h,力值定为15%,频率为1/6HZ,即10循环/分钟。采用Hoechst33258染色检测细胞形态和凋亡情况,应用RT-PCR技术检测Bcl-2、Bax的表达情况。结果:Hoechst 33258细胞染色结果显示,对照组的细胞核为弥散均匀的圆形或椭圆形荧光,实验组的细胞核或细胞质内出现可见致密浓染的颗粒、新月体或环状荧,RT-PCR结果显示Bcl-2与Bax基因表达均呈现时间依赖性。12 h HPDLFs的细胞凋亡数达最高峰值(P<0.01),24 h细胞凋亡峰值开始下降,但仍高于未加力组(P<0.05)。与对照组相比加入LY294002后,Bcl-2/Bax比值较加载相同时间的加力组小(P<0.05)。结论:一定的时间范围内,周期性张应力能促进HPDLFs凋亡;随着时间的延长(24h),细胞凋亡受到抑制;PI3K/Akt信号传导通路可能参与在周期性张应力介导的HPDLFs的凋亡。
OBJECTIVE: To investigate the effect of cyclic tensile stress on apoptosis of human periodontal ligament fibroblasts and the role of PI3k / Akt signaling pathway in apoptosis in vitro. Methods: Multi-channel cell stretch stress loading system was used to establish a model of HPDLFs (human periodontal ligament fibroblasts) culture in vitro. The control group was 0h, 0h + LY294002, h, 12 h, 12 h + LY294002, 24 h, the force value is set at 15%, the frequency is 1 / 6HZ, that is, 10 cycles / min. Cell morphology and apoptosis were detected by Hoechst33258 staining. The expression of Bcl-2 and Bax was detected by RT-PCR. Results: Hoechst 33258 staining results showed that the nuclei of the control group were round or oval fluorescence with uniform dispersion. In the nuclei or cytoplasm of the experimental group, densely-stained particles, crescent or ring-like fluorescence appeared, and RT-PCR The results showed that Bcl-2 and Bax gene expression in a time-dependent manner. The number of apoptotic cells in HPDLFs reached the peak value at 12 h (P <0.01), and peaked at 24 h, but still higher than those in non-stressed cells (P <0.05). After adding LY294002 to the control group, the Bcl-2 / Bax ratio was lower than that in the Jiali group loaded with the same time (P <0.05). CONCLUSION: Cyclic tensile stress can promote the apoptosis of HPDLFs in a certain time frame. The apoptosis of HPDLFs is inhibited with time prolonging (24h). PI3K / Akt signaling pathway may be involved in the cyclic tension-mediated HPDLFs Apoptosis.