A Preliminary Study On The Effect Of Lentivirus-mediated Gene Silencing Of Interleukin-1 Type Ⅰ Receptor In Osteoarthritic Rats

BackgroundOsteoarthritis (OA) is a chronic and degenerative disease of joint and is one of the most common joint disorders. According to statistics, the incidence of OA is the second in the people above 50 years old, only lower than that of heart disease. And the incidence is higher in the people suffering from limbs deformity or joint trauma. OA is becoming one of the principal diseases leading to disability with aging of population and high incidence of trauma. Previous research in the field of OA has characterized that OA is the result of disequilibrium between cartilage degeneration and synthesis controlled by the mechanic and biologic factors. The effect of inflammation mediators, such as interleukin-1 (IL—1), is necessary in the cartilage degeneration although cytokines is not enough. So, the study on IL—1 and IL—1 receptors is a hot point. IL-1 include IL-1αand IL-1βand the latter is principle in extracellular environment. IL-1βmay be synthesized by several kinds of cells, such as chondrocytes, synoviocytes and macrophages. According to the previous researches, IL—1βstimulates chondrocytes, fibroblasts and synoviocytes to synthesize prostaglandin E2 (PGE2), collagenases and matrix metalloproteinases (MMPs), and accelerates the degeneration of main compositions of cartilage, such as proteoglycan and type II collagen, depresses the mRNA expression of type II collagen, destroys the environment of chondrocytes and promotes their apoptosis.There are two kinds of IL-1 receptors, 80kD and 68kD in molecular weight respectively and the former is called IL-1RI, expressing extensively in organisms. When coupled with IL-1, serine and threonine residues in IL—1RI are phosphorylated and IL-1RI become activated to signal transduction. It has been certificated that mitogen-activated protein kinases (MAPKs)and nuclear factor-κB(NF-κB) signaling pathways participate in the process IL-1β- induced MMP-1 express and chondrocytes apoptosis. The primary goal of the present study is to observe, using lentivirus mediated RNA interference to repress the expression of IL-1RI, the effect on MMP-1 expression of IL—1 in both cultured rat chondrocytes and osteoarthritic rats. We also observed the effect of IL—1 on articular cartilage destruction in osteoarthritic rats and evaluated the validy of the treatment.Part I Design and Screening of Valid siRNA of Rat IL-1R IObjectives Design siRNA of rat IL-1R I and screen those valid for the following experiments.Material and methods Design 4 gene sequences and 1 non-sence control gene sequence consistent with the screening feature described in previous articles using open websites, and the sequences were synthesized with the help of Genechem Company. The sequences were amplificated in Bacterium coli after recombinated with pGCL-GFP plasmid. After PCR and sequencing identification, the positive clones were packaged into lentivirus. Then we screen the valid targets by western blot in cultured rat chondrocytes after detect the virus titer.Results PCR and DNA sequencing certificate that the oligonucleotides were correct. The protein level of IL—1R I in chondrocytes with transfection by siRNA-2# plasmid is depressed, while that of the chondrocytes with transfection by siRNA-NC plasmid is not influenced.Conclusions We designed and recombinated valid siRNA of rat IL-1R I , and packaged lentivirus vector.Part II The Effect of Lentivirus Mediated RNA Interference on MMP-1 Expression in Cultured Rat ChondrocytesObjectives Culture rat chondrocytes and observe the effect on IL-1R I and MMP-1 expression of lentivirus mediated IL-1R I RNA interference in cultured rat chondrocytes. Material and methods Harvest articular cartilage from 80～100g SD rats knee joints to carry out rat chondrocytes culture and identification. IL—1 stimulation up-regulates IL-1R I and lentivirus mediated siRNA transfection to the chondrocytes. If the infection ratio beyond 70% three days later through observing the expression of reporting gene GFP, Western blot is carried out to detect the expression of IL-1R I and MMP-1.Results Through morphologic and immunocytochemistry identification, cultured chondrocytes were identified. Compared with non-sence control goup, IL-1R I and MMP-1 expression is much lower in 2#-siRNA group after lentivirus mediated RNA interference.Conclusions We cultured rat chondrocytes successfully and lentivirus mediated IL-1R I RNA interference work well. IL-1R I RNA interference depress the expression of MMP-1 in cultured rat chondrocytes.Part III The Effect of Lentivirus Mediated RNA Interference on MMP-1 Expression and Cartilage Destruction in Osteoarthritic RatObjectives Observe MMP-1 expression and cartilage destruction in osteoarthritic rats and observe the effect on MMP-1 expression and cartilage destruction of lentivirus mediated IL-1R I RNA interference in osteoarthritic rat. Evaluate the validity of the gene therapy and make foundation for further studies.Material and methods Establish OA rat model by resecting of the anterior cruciate ligament (ACL) and anterior part of the right knee medial meniscus. Evaluate the validity of the model by observing the MMP-1 expression and cartilage destruction in osteoarthritic rats. Then repress IL-1R I expression by lentivirus mediated RNAi. Observe the cartilage destruction and detect MMP-1 expression in cartilage by Western blot.Results There was osteoarthritic change in articular cartilage from 4 weeks after operation by macroscopic and microscopic observe. While, MMP-1 expression was up-regulated in articular cartilage from 2 weeks after operation. There was significant decrease in articular cartilage destruction in both macroscopic and microscopic observing. However, the differences of MMP-1 expression among the IL-1R I RNAi group and negative control groups did not reach statistical significance.Conclusions It works to establish OA rat model by resecting of the anterior cruciate ligament (ACL) and anterior part of the right knee medial meniscus. Lentivirus mediated RNA interference on IL-1R I provide protect on articular cartilage in osteoarthritic rats. MMP-1 expression may be controlled by several inflammatory factors including IL-1.