Study Of The Role Of PI3K/Akt Signaling Pathway In Temporomandibular Joint Synovitis Induced By TNF-α

Part one Cultivation and identification of synovial fibroblastsObject:To isolated and cultured the temporomandibular joint synovial fibroblasts of rat, the tissue block method is adopted.Our aim was to establish synovial fibroblasts culture system in vitro.Methods:We detached the temporomandibular joint synovial tissue of rats in aseptic conditions, the tissue block method is adopted to obtain the original generation synovial cells in vitro. To determine the cultured cells for synovial fibroblasts (SFs), the third generation of synovial cells were selected to use immunofluorescence staining method and morphology observation.Results:The third generation of cells were observed under the inverted microscope, the cells have the same cell shape, fusiform, abundant cytoplasm, oval nucleus, located in the central, cell nucleolus is clear. We took the third generation of synovial cells to immunofluorescence staining, it can be observed that in all synovial cells are positive for waveform protein (Vimentin) expression, however cell expression of CD68 protein were negative, positive staining areas within the cytoplasm, staining for CD68 only observe the nucleus aizen, it illustrates that the cells derived from mesoderm, and have the characteristics of the fibroblasts, synovial fibroblasts.Conclusion:1. The tissue block method can produce high purity and stability of the temporomandibular joint synovial fibroblasts in vitro culture system;2.The third generation of synovial cells are consistent, uniform distribution,it can be used for subsequent experiment.Part two The study of the influence of PI3K/Akt signal pathway on the level of IL-1β、COX2 secreted induced by TNF-αObject:Study of PI3K/Akt signaling pathway in the role of the temporomandibular joint synovial fibroblasts induceded by TNF-α and to explore its molecular mechanism.Methods:Real-time fluorescent quantitative PCR (RT-PCR) technique to detect SFs PI3K, and Akt, IL-1β and COX2 mRNA expression; the ELISA detection of the supernatant of IL-1β, COX2 expression induced by TNF-α.Results:RT-PCR results showed that compared with the TNF-α group, the expression of IL-1β,COX2 mRNA of each group were statistically significant (P<0.05); and LY294002 group compared with control group, IL-1β, COX2 mRNA expression quantity has no statistical difference; LY294002+TNF-α group compared with control group, IL-1β, COX2 mRNA expression has no statistical difference. According to the results of ELISA, the amount of IL-1β,COX2 from the supernatant fluid of LY294002 group has no statistical differences compared with the control group; in LY294002+ TNF-α group, the expression of IL-1β,COX2 compared with the control group had no statistical difference; compared with the TNF-α group, the amount of IL-1β and COX2 of other groups were statistically significant (P<0.05). Immunocytochemistry staining results showed that the mean optical density value of PI3K and Akt of TNF-α group has no statistical difference compared with the other three groups. Mean optical density value of P-PI3K, P-Akt of TNF-α group is significantly higher than the other three groups with statistical difference (P<0.05), LY294002+ TNF-α group compared with control group, the expression of P-PI3K and P-Akt quantity had no statistical difference.Conclusion:1. After induced by TNF-α, the IL-1β and COX2 mRNA expression increased, in the supernatant fluid, IL-1β, COX2 content also increased markedly. TNF-α induced SFs can be used as a cell model method of the temporomandibular joint synovitis.2. TNF-α induced SFs produce inflammatory factors such as IL-1β, COX2 because of the activation of PI3K/Akt signal pathway, LY294002 can block this effect.3. PI3K/Akt signaling pathway plays an important regulating role in the SFs secretion of IL-1β, COX2 induced by TNF-α, the pathway may be closely related to the happening and developing of the temporomandibular joint synovitis.