Effect Of TLR4 On Expression Of IL-1β And TNF-α In Synovial Fibroblasts From Temporomandibular Joint Exposed To Lipopolysaccharide

Part one:Isolation and culture of synovial fibroblast in Temporomandibular Joint in ratsObjectives:Tocollect the temporomandibular joint synovial tissue of rats, culturing synovial fibroblasts, and to establish a stable system of synovial fibroblasts in vitro.Methods:Collect the synovial membrane from rat temporomandibular joint. Use enzyme digestion method to get the original generation of synovial cells. The cells were observed cell morphological changes under inverted phase contrast microscope. The cells were identified by immunofluorescence chemistry methods detecting vimentin and the expression of CD68.Results:The cells were in spindle, uniform appearance, with the morphology of fibroblasts under inverted microscope. All synovial cellswere detected to be negative for expression of CD68, and positive for vimentin expression.Conclusions:The method of enzyme digestion method in this experiment can get cultured synovial fibroblast successfully from temporomandibular joint in rat, which laid a foundation for subsequent experimental study.Part one:Isolation and culture of synovial fibroblast in Temporomandibular Joint in ratsObjectives:Tocollect the temporomandibular joint synovial tissue of rats, culturing synovial fibroblasts, and to establish a stable system of synovial fibroblasts in vitro.Methods:Collect the synovial membrane from rat temporomandibular joint. Use enzyme digestion method to get the original generation of synovial cells. The cells were observed cell morphological changes under inverted phase contrast microscope. The cells were identified by immunofluorescence chemistry methods detecting vimentin and the expression of CD68.Results:The cells were in spindle, uniform appearance, with the morphology of fibroblasts under inverted microscope. All synovial cellswere detected to be negative for expression of CD68, and positive for vimentin expression.Conclusions:The method of enzyme digestion method in this experiment can get cultured synovial fibroblast successfully from temporomandibular joint in rat, which laid a foundation for subsequent experimental study.Part two:Effect of TLR4 on Expression of IL-1β and TNF-α in Synovial Fibroblasts from Temporomandibular Joint Exposed to LipopolysaccharideObjectives:To investigatethe effect of TLR4 on expression of IL-1β and TNF-α in synovial fibroblasts from temporomandibular joint in ratsinduced by lipopolysaccharide, to explore the role of TLR4 in the temporomandibular joint disease pathogenesis.Methods:Stimulate synovial fibroblastsusing LPS with concentration of 0,0.1, 1,10,100 ng/mL for 24 h, or with LPS (final concentration of 100 ng/mL)for 0,1,6,12,24h. Detect the correlationofthe expression of IL-1β and TNF-α with the stimulationdurationandthe stimulation concentration, and choose the best stimulation of concentration and duration. Stimulated cellsusing LPS, and detect the expression of TLR4 and MyD88 by Western blot, and detect the expression of TLR4, MyD88,IL-1β and TNF-αmRNAby real time PCR, and detect the expression IL-1β and TNF-α in cell culture supernatants using enzyme-linked immunoassay (ELISA). Pretreated the cells with specific inhibitors of TLR4 (TAK-242) before the LPS stimulation, detect the expression IL-1β and TNF-αmRNA using Realtime PCR, and detect the expressionIL-1β and TNF-αusing ELISA.Results:The expression IL-1β and TNF-α mRNA and protein increased significantly with LPS stimulation.The results showed a dose-dependent increase of IL-1β and TNF-α proteins and mRNA expression in SFs with treated by LPS (0.1,1, 10,100 ng/mL) for 12 h, with the peak expression at 100 ng/mL of the stimulation.The proteins expression of IL-1β and TNF-α showed a time-course dependent increase, with the peak expression at 12 h of the stimulation. Consistent with their proteins increasing, their mRNA expression was also enhanced, with the peak expressionat 6 h of the stimulation in a time-dependentmanner. LPS can significantly enhance TLR4, MyD88 mRNA and protein expression, the pretreatment of TAK-242 can significantly inhibit theexpressionof IL-1β and TNF-α in synovial fibroblasts at mRNA and protein levels.Conclusions:TLR4 play an important role in the expression ofIL-1β and TNF-α in synovial fibroblasts induced by LPS. The pretreatment of TAK-242 can significantly inhibit theexpressionof IL-1β and TNF-α in synovial fibroblasts at mRNA and protein levels.TLR4 may participate in the development process of the temporomandibular joint disorders.