Construction Of The DDR2 Recomboinant Adenovirus And The Preliminary Study Of The Secretion Of MMP-13 Induced By DDR2

The hyper-proliferation and invasiveness of synovial tissue is the main reason for the destruction of joint cartilage in rheumatoid arthritis (RA). Our previous studies have indicated that discordin domain receptor 2 (DDR2) was highly expressed in the RA fibroblast-like synoviocytes and increased the expression levels of MMPs upon collagen stimulation, and especially the MMP-13 was considered as the rate-limited enzyme in the destruction of joint cartilage in RA patients, we raised the following hypothesis:"type II collage---DDR2---MMP-13"such a positive feedback loop may be the critical process in the cartilage destruction in RA. To further explore the potential roles of DDR2---MMP-13 signaling pathway in the cartilage destruction of RA, we constructed the DDR2-related recombinant adenoviruses and established a cell model containing an assay system of MMP-13 expression.1. Construction of DDR2-related recombinant adenoviruses The AdEasy system was used to complete the construction of recombinant adenovirous. Firstly, sequence of enchanced GFP, wild type coding sequence of DDR2 tagged with flag, Fc/DDR2 chimeric sequence and DDR2 RNA interference (RNAi) sequence was respectively cloned into the shuttle vectors, which were then cotransfected with rescue plasmids pAdEasy-1 into BJ5183. After recombination in E coli, the recombinants were linearized by Pac I and then transfected HEK 293 cells to acquire the recombinant adenovirous. When most of the cells show typical cytopathic effect (CPE), the cells were collected, frozen, molten and vortexed repeatedly, then the cell lysates were centrifugated and the supernatants were collected and stored at -70℃. The results of the RT-PCR and Westen blotting indicates that these DDR2-related adenovirs were successfully constructed.2. Establishment of the stable assay system of the expression of MMP-13 induced by DDR2 The sequence of MMP-13 promoter and luciferase were amplified by PCR , subcloned into pcDNA4Cmyc/his plasmid to replace the original CMV promoter. The new recombined plasmid was then cotransfected with the constructively activated DDR2 plasmids into 293T cells. The MMP-13 promoter activity was subsequently analyzed by measuring luciferase activity after 24 hours'serum free culture. The expression of constructively activated DDR2 led to an increase in MMP-13 promoter activity compared with the vector control. Stable transfections of the new plasmid into Tet-on293 cells were done via the LipofectAMINE2000 method. Zeocin-resistant colonies were selected by adding Zeocin(50μg/ml) to the medium for 2 weeks. Viable colonies were further screened by measuring luciferase activity.