| BackgroundOsteOArthritis (OA) is a progressive degradation of articular cartilage badly threathen the health of human beings, with a arthromeningitis and subchondral bone reconstruction as the main feature,which is considered as the most common chronic joint disease. With the acceleration of the process of ageing population and obesity, the incidence of OA among the elderly increased year. Cartilage belongs to Hyaline cartilage with no blood vessel, Lymphatic drainage and nerves, consisted of chonodrocyte and ECM. Chonodrocyte, the only cell in cartilage, involves in the synthesis and maintains the composition of ECM, playing a crucial role in the repair process of OA. The investigation of the mechanisms responsible for the stimulation of chondrocyte proliferation and the suppression of chondrocyte apoptosis in vitro is of clinical significance. It has been previously demonstrated that a variety of cytokines, hormones, modifiers and drugs affect the biosynthesis of chondrocytes in in vitro experiments. ECM is a core factor to regulate the chonodrocyte connection, growth and distribution and repair, involving the aggrecan and collagen. Collagen can increase the tensile strength of cartilage tissue. The main function of aggrecan is to maintain a large number of water molecules in ECM, which help cartilage tissues reduce lOAd and withstand the pressure.Integrin is one of the members of the family of cell adhesion molecules, which has a important effect in cell adhesion and signal transduction involving in the regulation of cell polarity,shape, adhesion, motility, proliferation growth and differentiation. It has been found many kinds of integrin existing in the chondrocyte, regulating the gene expression and function through signal transduction and Conducting mechanical stimulation. The interaction between integrin of chondrocyte and their surrounding extracellular matrix is considered to be having significant effects on the metabolic homeostasis of the articular cartilage. The degradation and synthesis of cartilage keeps a balance under normal condition, whereas OA will happen following the broken of balance.GIT1 exerts its effects on cell proliferatin and migration through interacting with the cell surface protein and a variety of cytokines. Until now, the researches about GIT1 in bone cells focus on osteoblast and osteoclast. The relationship between integrin β1 and GIT1 is still unclear, some researchers suggest that GIT1 may be act as a crucial protein dowmstream of the integrin-mediated pathway.ObejectiveThe OA model was established to exploring the influence induced by integrinβ1 and GIT1, and exploring the relationship between them.Methods1. Took 201-week-old Sprague-Dawley (SD) nemnatal rats. The OA model was established according Hulth method in order to obtain limb joints under sterile conditions. After the separation and harvest for articular chondrocyte. which was cultured and passaged in vitro, type Ⅱ collagen immuneofluorescene assay were employed to identify the articular chondrocyte. The Trizol method was used to extract the total RNA of chondrocyte, and amplified the integrin-β1 and GIT1 coding regions. They were then cloned into the pcDNA3.1 vector through Kpn 1 and EcoR I (TaKaRa) restriction enzymes. Plasmid and siRNA transfection was carried out using Lipofectamine 2000. Quantitative PCR and wester blot analysis were used to detect difference of mRNA and protein relative expression of integrinβ1 and GIT1 among control groug、pcDNA3.1 group、pcDNA3.1-GIT1 group、pcDNA3.1-integrin-β1 group、integrin-β1 siRNA group and NC siRNA group.2. First generation OA chondrocyte were used to cultured throuth the same method as the first part. Quantitative PCR and wester blot analysis were used to detect difference of mRNA and protein relative expression of aggrecan and type Ⅱ collagen among control groug、pcDNA3.1 group、pcDNA3.1-GIT1 group、 pcDNA3.1-integrin-β1 group、integrin-β1 siRNA group and NC siRNA group. The cell proliferation ability and cell apoptosis was dectected by BrdU assay and TUNEL-DAPI co-staining assay.Results1. Following transfection with a vector expressing integrin-β1, the quality PCR analysis show that after the knockdown of integrin-β1 reduced GIT1 mRNA expression, whereas the overexpression of integrin-β1 enchanced GIT1 mRNA expression. The difference was significant in statistic compared to the control group (P<0.05). The western blot analysis found that the expression of protein was the same with mRNA. after the knockdown of integrin-β1 reduced GIT1 protein expression, whereas the overexpression of integrin-β1 enchanced GIT1 protein expression. The difference was significant in statistic compared to the control group (P<0.05). On the contrary, after enchacing the GIT1 expression, the expression of integrin-β1 had no change.2. Transfect of chondrocyte with integrin-β1 siRNA and NC siRNA, incubated 48 hours. The proliferation ability of OA chondrocyte was reduced by integrin-β1 siRNA and increased by integrin and GIT1 detected by BrdU assay (P<0.05). The mRNA and protein expression was down-regulated in the infected with integrin-β1 siRNA groups, whereas which was up-regulated by integrin-β1 and GIT1, thus promoting the chondrocyte proliferation. The apoptosis was inhibited by the overexpression of integrin-β1 through TUNEL-DAPI analysis.ConclusionsIntegrin-β1 has a significant effect on the proliferation, apoptosis and differentiation of chondrocytes. What’s more, integrin-β1 stimulates the chondrocyte proliferation and differentiation, and suppresses chondrocyte apoptosis through increasing the relative expression of GIT1. GITφ has similar effects on the proliferation, apoptosis and differentiation of chondrocytes as a downstream effector of integrin-β1. However, the role of integrin-β1 and its underlying mechanisms of how to regulate the proliferation,differentiation and apoptosis of cartilage cells remain unclear. Therefore, further research is necessary to provide more convinced evidence of the effects of integrin-β1 on chondrocytes. |
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