Effects of cyclic tensile stress on caspase-12 expression in chondrocytes

doi:10.3969/j.issn.2095-4344.2016.29.011

Abstract

Abstract:

BACKGROUND: Excessive mechanical stimulation can lead to endoplasmic reticulum stress. Endoplasmic reticulum stress plays an important role in the occurrence and development of degenerative diseases. Caspase-12 is a specific molecule of endoplasmic reticulum stress, and the intensity of endoplasmic reticulum stress can be reflected by caspase-12 expression.
OBJECTIVE: To observe the expression rule of Caspase-12 in chondrocytes under the cyclic tensile stress.
METHODS: Human chondrocytes were used as test subjects. After group assignment, mechanical loading system was used. The loading group and corresponding Z-ATAD-FMK inhibitor group received 2, 12, 24, 36, and 48 hours of mechanical stimulation. The blank group (0 hour) and the paired Z-ATAD-FMK inhibitor group received the same process as the loading group except the loading. After loading, cell morphology and growth were observed under the microscope. RT-PCR and western blot assay were used to detect the expression changes of caspase-12 gene and protein in each group.
RESULTS AND CONCLUSION: (1) Morphological observation results demonstrated that apoptosis appeared at 2 hours after loading, peaked at 24 hours. With prolonged time, cell growth showed the trend along stress, but apoptosis weakened. It is indicated that cyclic tensile stress could make chondrocyte apoptosis. Endoplasmic reticulum stress might be activated. The model of cells in vitro was established successfully. (2) Caspase-12 gene and protein expression showed consistent trend, and peaked at 24 hours, which showed significant differences as compared with the blank and inhibitor groups (P < 0.05). (3) Cyclic tensile stress can induce chondrocyte endoplasmic reticulum stress, and affect caspase-12 expression.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Chondrocytes, Endoplasmic Reticulum, Cysteine Proteases, Tissue Engineering

CLC Number:

R318

Chen Zhu-ke, Li Xiao-fei, Zhang Hai-ning. Effects of cyclic tensile stress on caspase-12 expression in chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(29): 4334-4340.

Figures/Tables 1

2.1 加力细胞光镜下形态正常细胞呈多角形，形态不规则，空白组(0 h)多此特征。从加力2 h开始，细胞开始不同程度脱落，边缘模糊变圆钝，体积缩小，形态结构不清，细胞出现凋亡(细胞体积变小，染色质浓缩，染色质边集)，到24 h此现象最明显，随着加力时间延长，细胞出现顺应力生长趋势，但细胞凋亡现象减弱(图1)，提示周期张应力可以使软骨细胞凋亡，内质网应激可能被激活，实验模型建立成功。 2.2 周期性张应力作用下半胱氨酸蛋白酶12基因表达空白组(0 h)中并非没有半胱氨酸蛋白酶12表达，从2 h表达量开始升高，到24 h表达量达到峰值，加力时间24 h后，表达量递减，加力组与空白组(0 h)对比差异均有显著性意义。加力组中的抑制剂对半胱氨酸蛋白酶12的表达有明显抑制，但没有完全被抑制，空白组(0 h)与抑制剂组表达量上相当(图2)，说明周期性张应力可促使细胞表达半胱氨酸蛋白酶12相关基因。不可否认的是，细胞受力同时也发生不同程度的内质网应激，值得一提的是Z-ATAD-FMK对半胱氨酸蛋白酶12具有特异性抑制作用，但不同时间段内其抑制程度差别原因有待进一步研究。 2.3 周期性张应力对半胱氨酸蛋白酶12蛋白表达的影响在张应力作用下，随着加力时间的延长，加力24 h蛋白表达量最高，越过峰值后，表达量再次趋减，和半胱氨酸蛋白酶12基因表达规律相似。在蛋白表达方面，加力组与空白组差异有显著性意义，同样，Z-ATAD-FMK对其蛋白表达也有不同程度的抑制(图3，4)，表明蛋白是基因表达的结果，力学刺激对半胱氨酸蛋白酶12的表达有着重要影响，内质网应激同样也是半胱氨酸蛋白酶12蛋白产生的原因。综上结果，细胞在周期性力学因素影响下，软骨细胞可发生内质网应激反应，在促使半胱氨酸蛋白酶12的表达发生变化的同时，也同样导致了细胞不同程度的凋亡。"

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