The Roles And Significance Of IL-24and CXCL13Gene In The Knee Osteoarthritis

Objective:To investigate roles and mechanism of IL-24and CXCL13Gene in the knee osteoarthritis, and to provide scientific basis for the knee osteoarthritis in early diagnosis, development of related drug and gene therapy.Methods:Synovium specimens from patients of older than50years in Yanbian area who were diagnosed as primary osteoarthritis of the knee were choosed, extracted total RNA, analized the differently expressed gene in primary osteoarthritis of the knee using NimbleGen mRNA expression microarray detection. Gene and protein expression of IL-24and CXCL13in Knee osteoarthritis Synovium were detected by real time RT-PCR, RT-PCR, Western blot and immunohistochemistry. Treated the knee joint synovium cells With TNF-a which was the recognized pathogenic factor of the knee osteoarthritis, detected the inflammatory factor expression changes of synovium cells after treatment by ELISA, confirmed the establishment of knee osteoarthritis synovium cell model,detected IL-24and CXCL13gene and protein expression using RT-PCR, Western blot analysis and laser scanning confocal microscopy analysis. Treated knee osteoarthritis synovium cells by celecoxib which were widely used in osteoarthritis, detected the gene and protein expression of IL-24and CXCL-13, in knee osteoarthritis synovium cells after treatment. Results:1. There was obvious difference of genetic expressionbetween osteoarthritis synovium tissue and normal synovium tissue:Compared with the normal knee joint synovium, in the knee osteoarthritis synovium,451genes of22885genes which mRNA gene chip contained were up-regulated(The expression of HOXD10,NGEF, TNFSF7,IGSF10,TNFSF9,ITGA11changed obviously),810genes were down-regulated(The expression of CD79A、CXCL13、IL-1Ra、IL-24、 IGF-2、FGF-7changed Obviously), IL-24and CXCL13gene expression of knee osteoarthritis synovium was decreased obviously than normal synovium by the results of real time RT-PCR, the changes of expression had statistically significant difference (P<0.001).2. Expand the number of cases for the verification of the changes and significance of IL-24and CXCL13expression in the knee normal and osteoarthritis synovium:IL-24and CXCL13mRNA of30cases knee osteoarthritis synovium tissues and22cases of normal knee synovium tissues was detected by qRT-PCR, qRT-PCR results showed, osteoarthritis group compared with the normal group,IL-24and CXCL13mRNA expression decreased significantly, there was statistically significant difference (P<0.001).the expression of IL-24and CXCL13protein was decreaseed in the knee osteoarthritis synovium by Western blot results, this results were consistent with the results of gene chip detection,real time RT-PCR and qRT-PCR.IL-24and CXCL13protein was expressed in the cytoplasm of synovium cells by the results of immunohistochemical detection,IL-24and CXCL13protein was significantly decreased in the knee osteoarthritis synovium compared with normal.3. Establish the model of osteoarthritis in human synovium cells HS by TNF-α:100U/ml of TNF-α can induce inflammation in human synovium cells HS,the results of ELISA showed, the expression of IL-6and TGF-β1was increased and the expression of IL-2and IL-10was decreased along with the extension of processing time by TNF-a, It’s showed that TNF-a can induce inflammation model of human synovium cells in vitro.4. In the inflammatory model of human synovium cells HS,IL-24, CXCL13expression was significantly lower than normal human synovium cells HS:RT-PCR results showed that,3h,6h,12h,24h after treated the knee synovium cells by TNF-a, IL-24and CXCL13mRNA levels significantly decreased along with the extension of processing time,it decreased by a time dependent manner,it showed that with the osteoarthritis inflammation reaction increased, the levels of IL-24and CXCL13mRNA got down. Western blot analysis showed that treated the knee synovium cells by TNF-a, with the extension of time, the levels of IL-24and CXCL13protein decreased significantly, it decreased by a time dependent manner, it showed that with the osteoarthritis inflammation reaction increased, the levels of IL-24and CXCL13protein got down, it was consistent with the results of RT-PCR.5. Celecoxib. could inhibit the inflammation reaction through up-regulation of IL-24and CXCL13:RT-PCR results showed that,24h after treated by TNF-a, treated the knee synovium cells with celecoxib for24h and48h, IL-24and CXCL13mRNA expression rised again. Western blot analysis showed,24h after treated by TNF-a, treated the knee synovium cells with celecoxib for24h and48h, IL-24and CXCL13protein expression rised again. Laser scanning confocal microscopy analysis showed that the fluorescence signal of IL-24and CXCL13protein significantly reduced after treated the knee synovium cells by TNF-a, showed that the expression of IL-24and CXCL13protein significantly decreased, it confirmed the results of gene chip detection, RT PCR and Western blot, treated the knee synovium cells with celecoxib for48h,the fluorescence signal of IL-24and CXCL13protein rised again.Conclusions:1. Compared with normal knee joint synovium, there were obvious differences of gene expression in the knee osteoarthritis synovium.2. In the knee osteoarthritis synovium and synovium cells which were treated by TNF-a,the expression of IL-24gene and protein were obviously decreased, it decreased by a time dependent manner with the inflammatory stimulation time.3. In the knee osteoarthritis synovium and synovium cells which were treated by TNF-a, the expression of CXCL13gene and protein were obviously decreased, it decreased by a time dependent manner with the inflammatory stimulation time.4. Celecoxib had strong anti-inflammatory activity, in knee osteoarthritis celecoxib produce anti-inflammatory activity through inhibition of cyclooxygenase and enhancing IL-24and CXCL13.