Preparation And Function Study Of The Blocker Of An Important Signal Transduction Pathway In Rheumatoid Arthritis

Discoidin domain receptors (DDRs) belong to a kind of receptor protein tyrosine famlily. Protein tyrosine kinases (PTKs) play a significant role in cell signal transduction in cell proliferation and apoptosis.DDRs not only correlate with cell proliferation,but also with MMPs over expression.They participate in many cell physiology process,such as cell proliferation, differentiation, migration and celluar metabolism etc.especially play important roles in controlling cartilage cell proliferation and bones development.DDR2 is one of the most significant molecule in rheumatoid arthritis(RA) pathologic process. DDR2 is highly expressed in RA synovial cell and in high phosphorylation level.It can be activated by collagenⅡthen regulate the expression of matrix metalloproteinases (MMPs). MMPs gradually eroded joint cartilage,result in inreversible destruction of joint cartilage.So, we prepared and studied the blocker of collagenⅡ-DDR2-MMPs pathway,making sure it can function in interrupting constant expressing of MMPs mediated by DDR2.This method will provide a new drug in RA clinical treatment in future.Our study has obtained following results:1. We successfully constructed DDRs/IgG fusion express vector and expressed them in HEK293 cell and CHO cell.We expressed the fusion protein of DDR2 DS domain and IgG Fc fraction,DS domain is binding domain of DDR2 to collagenⅡ,we combine it with Fc because DDR2 DS domain will form a dimmer using the disulfide linkage of Fc, and it will competitively bind to collagenⅡand suppress the binding of natural receptor, thus block collagenⅡ-DDR2-MMPs pathway.2. We got a strong positive clone expressing fusion protein by stably transfecting and G418 slecting using CHO express system.3. Culturing the positive cell clone,we collected the cell cultural supernatant fluid and purificated them using Protein A. We successfully purificated the protein with concentrate 1.14μg/mL(7.98μg/107cell).4. ELISA and cell experiment indicated that the blocker we expressed has effective function.5. We constructed DDR2/Fc recombinant adenovirus vector and primaryily packaged the recombinant adenovirus, aiming for acquiring adenovirus with high titre and then transfecting RA animal model.