Composition for preventing or treating diseases caused by overexpression of chemokine cx3cll, containing death receptor inhibitor as active ingredient
A chemokine, overexpression technology, applied in the field of compositions containing a death receptor inhibitor as an active ingredient for the prevention or treatment of diseases caused by the overexpression of the chemokine CX3CL1, can solve problems such as insignificant research results
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0078] Example 1. Identification of membrane proteins that bind to sFas ligand (DR5)
[0079] So far, it is known that FasL binds to its receptor Fas, induces apoptosis of target cells, and thus causes joint inflammation. In addition, in previous studies by the present inventors, it has been revealed that FasL controls the expression of chemokines with a new mechanism different from conventional inflammation-induced apoptosis of Fas-FasL, and it has been determined that FasL itself functions as a chemokine that attracts inflammatory cells role. However, in previous studies, it has not been specifically revealed how FasL controls the occurrence of inflammation by binding to this membrane protein in a novel mechanism of FasL-induced inflammation.
[0080] Therefore, the present inventors conducted protein identification experiments to find proteins that bind to sFasL (soluble Fas ligand) in a novel mechanism of FasL-induced inflammation.
[0081] First, the synovium of an arth...
Embodiment 2
[0084] Example 2. Confirmation of expression of DR5 surface protein in hFLS and joint synoviocytes of arthritis-induced mice
[0085] To confirm the expression of DR5 surface protein in human FLS (hFLS) and joint synoviocytes of arthritis-induced mice, flow cytometry and real-time PCR were performed. In the real-time fluorescent quantitative PCR, use the Applied Biosystems7500 real-time PCR system, use F:GGGCCACAGGGACACCTT / R:GCATCTCGCCCGGTTTT as primers, and make them react at 50°C for 2 minutes, 95°C for 2 minutes, 95°C for 15 seconds, and then 60°C for 1 minute. 40 times.
[0086] Then, synoviocytes (adherent cells / Adh.) and normal immune cells (supernatant cells / Sup.) were isolated from joints obtained from normal mice (Normal) and arthritis-induced mice (RA), respectively. Afterwards, the expression of DR genes was compared in each cell. The experimental results are shown in figure 1 .
[0087] Such as figure 1 As confirmed in (A), the relative expression of DR5 surfa...
Embodiment 3
[0089] Example 3. Confirmation of mutual binding of FasL-DR5
[0090] 3-1. DR5 antibody treatment
[0091] To confirm that FasL is actually bound to the membrane protein DR5, hFLS were treated with an anti-DR5 antibody (R&D systems AF631) to interrupt the binding of FasL to cell surface DR5.
[0092] Then, IgG-bound FasL was processed, and the IgG signal intensity on the surface was confirmed by flow cytometry, the results are shown in figure 2 (A). as in figure 2 In (A) it can be seen that the signal intensity of IgG is reduced when treated with anti-DR5 antibody, while FasL-DR5 binding is reduced when anti-DR5 antibody inhibits DR5 activity.
[0093] 3-2. Knockdown using siRNA against DR5
[0094] To confirm whether FasL and the membrane protein DR5 were truly bound, hFLS were treated with siRNA (Sigma Aldrich MISSION siRNA; SASI_Hs01_00040567) and the expression of DR5 was knocked down using an electroporator (Neon Transfection System, Thermo Fisher Scientific).
[0...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More