The Research On Inhibit Of IL-1R â…  Gene Expression In RA Synovial Fibroblast-like Cells Using RNAi Technology

Objective:Construct the RNAi system of IL-1RI which can inhibit the expression of IL-1RI in human rheumatoid arthritis fibroblast-like synovial cell significantly.Maybe this experiment is a beneficial research for curing the human rheumatoid arthritis.Method:1.Culture and identify the fibroblast-like synovial cell of human rheumatoid arthritis in vitro..In arthroplasty and arthroscopy,we get synovial tissue from the knee of the patients troubled with rheumatoid arthritis.Two methods are used to culture the cells in vitro:tissue culture and enzyme digestion.The synovial tissue will be processed into the small volume in both methods.In tissue culture,the small tissue will be tiled in the bottle directly.In digestion method the small tissue was digested with typeâ…¡collagen enzyme and trypsin.Use the FCM to detect the purity of fibroblast-like synovial cell in the fourth generation.Do the experiment of indirect immunofluorescence with anti-human CD3mAb,anti-human CD19 mAb,anti-human CD16 mAb, anti-human CD56 mAb,anti-human CD14 mAb,anti-human CD55 mAb,anti-h uman cell CD9OmAb.set up the same type of negative control at the same time. After washing,do the machine detection and using Cell Quest software analysize.2.Design,synthesize and screen for the special IL-1RIsiRNA and Use the technology of RNAi to inhibit the expression of IL-1RI gene in FLS:According to the selected gene sequence of IL-1RI,use online software to design three specific interference gene sequence fragment.After the synthesis of the dsOligo,connect U6 vector with the synthesized dsOligo, transformed the connective into One Shot Stb13 Competent E.coli to cloning it.Then use ammonia Bian kanamycin resistance to screen the positive clone.Do the experiment of transient tranfection with Lipofectamine2000 reagent and construct RNAi system of IL-1RI which use Lentiviral Vector as mediator to tranfect the fibroblast-like synovial cell.Extract IL-1RI RNA from the cell and do the experiment of RT-PCR to identify the inhibitory effect.Result:1.We have successfully developed a kind synovial fibroblast cells using the two methods of tissue culture and enzyme digestion.The cell can spread to eighth generation at average and its condition is very good. The fourth generation cell' s purity is higher than 99%when we use the FCM to test the cell' grow condition.CD90~+ can be used as FLS' surface markers.2.Design three pair specific interfere IL-1RI gene sequences.We constructed a lentiviral vector system with using the second and the third fragment successfully,In RT-PCR detection,the third pair can inhibit the expression of IL-1RI in rheumatoid arthritis fibro-blast cells.Conclusion:1.We have established the technology platform to cultivate the fibroblast-like synovial cell of human rheumatoid arthritis in vitro successfully.2.We have constructed the RNAi system of IL-1RI which can inhibit the expression of IL-1RI in human rheumatoid arthritis fibroblast-like synovial cell significantly.It suggests that this experiment is a beneficial research for curing the human rheumatoid arthritis.