Effect Of Sirna Targeting Connexin43 On Expression Of MMP-1,13 In Cultured Osteoarthirits Chondrocytes

Objective: Progressive degeneration of articular cartilage is the main cause in osteoarthritis,and there is the upregulation of Cx43 expression in articular cartilage.Studies showed that Cx43 affects the secretion level of MMPs in synovial cells.We transfected the siRNA targeting Cx43 to inhibit it’s expression in Osteoarthritis Chondrocytes,to investigate the effect on the secretion of MMP-1,13 in cultured human Osteoarthritis Chondrocytes in vitro.Methods: The articular cartilage were required from patients with osteoarthritis who were taken the total knee replacement surgery.Chondrocytes required by enzyme digestion method was used for Osteoarthritis Chondrocytes Cultured in vitro.The cells were seeded in 6-well plates when growed to cover 90% of culture plate.Type II collagenimmunocytochemistry method were used to identificate the Chondrocytes.Cx43 siRNA by commercial synthesis was transfected in cells to inhibit the expression of Cx43,and the RT-PCR was used to screen the highest inhibition efficiency of siRNA.The total RNA and total protein extracted form the highest inhibition efficiency group were uesd for RT-PCR and Western-Blotto test the mRNA and protein expression levels of MMP-1 and MMP-13.All the data was measured with independent t-test.Results: Compared with the control group,the immunocytochemistry experiment showed human Osteoarthritis Chondrocytes was weakly positive.The screening results of siCx43 sequence showed that GAJ3 had the highest inhibition efficiency.3.RT-PCR result showed that,compared with the negative control group,the expression of mRNA of MMP-1,13 decreased in the experimental group,with statistical significance(P< 0.05).4.Western-Blot results showed that the protein level of MMP-1,13 in the experimental group decreased with statistical significance(P< 0.05)compared with the negative control group.Conclusion: The experimental results showed that the inhibition of Cx43 by siRNA transfection,can reduce the MMP-1,13 expression in cultured human osteoarthritis in vitro.These results suggest that Cx43 and gap junction channel may affect the initiation and progression of OA by affecting the proteolytic enzyme such as MMPs.