| Objective:This project intended to study the regulation role of Osteopontin (OPN) in Osteoarthritis (OA) and explored its possible pathways, preliminary observation on function effects of OPN on OA cartilage cells.Methods:1Take the13non-OA patients (8men and5women,53.1±12.8years of age) and13patients with OA (6men and7women,68.8±7.9years of age) of the hip and knee cartilage, the mRNA expression of OPN and MMP-13in the two group specimens was detected by real-time QPCR, the correlation between expression of OPN and MMP-13also was analysed by SPSS19.0. OA cartilage cell was separated from digesting OA cartilage tissue, and taken first generation for training, treated by lug/ml concentration of OPN, then respectively detected by real-time QPCR and Western Blotting at different time points (Oh,24h,48h,72h). On the other hand, OA cartilage cell was treated by different concentration of OPN (0,0.5,1,2,4μg/ml). The difference of the mRNA and protein expression of MMP-13among the cells was detected to identify the most optimal concentration and time of OPN intervention.2First generation OA cartilage cells were divided into two groups, the blank control group and PDTC (Pyrrol idinedithiocarbamic acid, inhibitor of NF-κd) treated group, each group subdivided into Con-siRNA subgroup, OPN-siRNA and OPN. The expression of internal and external NF-Kb(nuclear factor-kappaB) was detected by Immunofluorescence. The protein expression of NF-Kb p65, MMP-13and COL2A1was detected by Western Blotting.3The artilage cells were infected with Con-shRNA and OPN-shRNA lentivirus particle, trasfected with TGFA ORF clone. Then the cells were divided into control, Con-shRNA, OPN-shRNA, OPN four groups for detection of cell proliferation ability by MTT and Brdu assays.Result:1The expression of OPN and MMP-13(OPN±0.068VS0.848±0.085, P=0.0010; MMP-13±0.079, P=0.00910.971±VS1.269) from OA cartilage tissues compared with non-OA tissues. The expression of MMP-13in OPN were positively correlated (R2=0.761,p<0.001);1μ g/ml OPN for24h, the mRNA and protein expression of MMP-13was up-regulated significantly (P=0.0039; P=0.0041), for48h was the peak (P=0.0001; P=0.0004), the expression level was still at a high level after72h (P=0.0001; P=0.0004); The OA cartilage cells treated with different concentration of OPN protein for48h, the mRNA and protein expression of MMP-13was up-regulated significantly (P=0.007; P=0.003) when the concentration of OPN was1ug/mL2The OA cartilage cells were treated with OPN, the expression of NF-Kb p65in the nuclear increased and decreased when tranfected with OPN-siRNA. The expression of NF-Kb p65(P=0.0027), p-NF-Kb p65(P=0.0021) and MMP-13(P=0.0079) was up-regulated and the expression of COL2A1was down-regulated (P=0.0188) when OPN was over-expressed. On the contrary, the expression of NF-Kb p65(P=0.0026), p-NF-Kb p65(P=0.0168) and MMP-13(P=0.0023) was down-regulated and the expression of COL2A1(P=0.0226) was up-regulated when OPN was knocked down by siRNA. NF-Kb inhibitor (PDTC) could reverse the effects of OPN (p65P=0.00320; p-p65P=0.0346; MMP-13P=0.0213; P=0.0422of COL2Al).3The mRNA and protein expression was down-regulated in the infected with OPN-shRNA lentivirus particles. The proliferation ability of OA cartilage cells was reduced by OPN-shRNA and increased by OPN detected by MTT (P<0.05) and BrdU (P<0.05) assays.Conclusions:1The expression of MMP-13in OA cartilage tissues and cells was up-regulated by OPN;2OPN could accelerate NF-κb p65moving into the nucleus of OA cartilage cell, and raise NF-κb p65, and p-NF-κb p65, and MMP-13relative protein expression level, and reduce the expression of COL2A1;3The proliferation ability OA cartilage cell could be induced by OPN;4OPN activated NF-κb pathway, raised MMP-13protein expression, hydrolysis of COL2A1may be an important component of the pathogenesis of OA,... |
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