Shi Chen-Hui, Wang Wei-Shan, Zhang Zhen-Dong, Li Chang-Jun, Guo Feng-Jing, Li Feng, Chen An-Ming
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Department of Orthopedics, The First Affiliated Hospital of Medical School, Shihezi University, Shihezi, 832008, China.
J Huazhong Univ Sci Technolog Med Sci. 2015 Feb;35(1):111-116. doi: 10.1007/s11596-015-1398-1. Epub 2015 Feb 12.
The lentivirus-mediated uPA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin (IL)-1β (10 ng/mL) for 24 h and then divided into three groups: uPA-siRNA group (cells transfected with uPA-siRNA lentiviruses), blank control group (untreated cells), and negative control group (cells transfected with empty vectors). Western blotting and real-time quantitative reverse transcription-PCR (RT-QPCR) were performed to detect the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after uPA-siRNA transfection, the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased (P<0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P<0.01 for uPA, MMP-3 and MMP-13). Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in uPA-siRNA group than in control groups (P<0.01). The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the uPA-siRNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase (P<0.01). It was suggested that the down-regulation of uPA gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.
本研究检测了慢病毒介导的uPA干扰对骨关节炎软骨细胞增殖、凋亡及分泌的影响。细胞取自新西兰白兔的软骨组织。用白细胞介素（IL）-1β（10 ng/mL）培养24小时，然后分为三组：uPA-siRNA组（用uPA-siRNA慢病毒转染的细胞）、空白对照组（未处理的细胞）和阴性对照组（用空载体转染的细胞）。采用蛋白质免疫印迹法和实时定量逆转录聚合酶链反应（RT-QPCR）检测骨关节炎软骨细胞中uPA、基质金属蛋白酶（MMP）-1、MMP-3、MMP-9、MMP-10、MMP-13和MMP-14的蛋白质和mRNA表达水平。使用细胞计数试剂盒-8、流式细胞术和集落形成试验检测软骨细胞的增殖和凋亡。结果显示，uPA-siRNA转染后，uPA、MMP-1、MMP-3、MMP-9、MMP-10、MMP-13和MMP-14的蛋白质和mRNA表达水平显著降低（MMP-1、MMP-9、MMP-10和MMP-14，P<0.05；uPA、MMP-3和MMP-13，P<0.01）。uPA-siRNA组的细胞增殖和集落形成率显著高于对照组，细胞凋亡率显著低于对照组（P<0.01）。对照组G0/G1期细胞比例明显增加，S期细胞比例降低，细胞周期阻滞在G1/S期。在uPA-siRNA组中，S期细胞比例显著增加，导致细胞周期各期细胞比例不同（P<0.01）。提示下调uPA基因可抑制MMPs蛋白表达和细胞凋亡，增加骨关节炎软骨细胞的增殖和集落形成。
