摘要
背景:过度力学刺激可引起细胞内质网应激,内质网应激在退行性疾病的发生发展过程中扮演重要角色,半胱氨酸蛋白酶12是内质网应激的特异性分子,内质网应激的强弱程度可由半胱氨酸蛋白酶12表达水平来体现。目的:观察周期性张应力作用下软骨细胞半胱氨酸蛋白酶12的表达规律。方法:以人软骨细胞为实验对象,对实验细胞成功分组后,运用细胞牵张力学加载系统,将加力组及其配对的相应Z-ATAD-FMK抑制剂组分别给予2,12,24,36,48 h的力学刺激条件,空白组(0 h)及其配对的Z-ATAD-FMK抑制剂组除不加力外培养条件和加力组完全相同。规定加力时间终止后,显微镜下观察细胞形态变化及生长状态,随即采用RT-PCR和Western blot检测各组半胱氨酸蛋白酶12基因和蛋白表达变化规律。结果与结论:(1)细胞形态学观察结果显示,从加力2 h开始,细胞出现凋亡,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。说明周期张应力可以使软骨细胞凋亡,内质网应激可能被激活,细胞体外受力模型建立成功;(2)半胱氨酸蛋白酶12基因与蛋白随时间延长表达趋势一致,加力24 h表达量达高峰,与空白和抑制剂组相比,差异均有显著性意义(P<0.05);(3)以此得出,周期性张应力可引起软骨细胞内质网应激,并影响半胱氨酸蛋白酶12表达。
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, cel 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, cel 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 cel s in vitro was established successful y. （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.
出处
《中国组织工程研究》
CAS
北大核心
2016年第29期4334-4340,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金资助项目(81272056)~~
