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BACKGROUND: Fibroblast-like synoviocytes play an important role in the pathological process of rheumatoid arthritis. When inflammatory arthritis occurs, fibroblast-like synoviocytes in the synovial lining layer will proliferate in large quantities, and the release of inflammatory factors during this period can cause joint inflammation and cartilage damage. OBJECTIVE: To establish methods for isolation, culture, purification, and identification of tree shrew knee joint fibroblast-like synoviocytes in vitro, and explore their biological characteristics; to establish methods for detection of TLR8 pathway related molecules in fibroblast-like synoviocytes from tree shrews, and preliminarily explore the activation of TLR8 pathway in the cells. METHODS: Fibroblast-like synoviocytes from tree shrews were isolated and purified using tissue block method and continuous passage method. Cell proliferation was detected using cell counting kit-8. Cell morphology was observed using hematoxylin-eosin staining. Immunocytochemical staining was used to detect the expression of vimentin in synoviocytes. Before and after stimulation with human TLR8 ligand R848, the expression of TLR8 and its signaling pathway proteins in tree shrew fibroblast-like synoviocytes and human fibroblast-like synoviocytes was detected using western blot assay, while the expression of TLR8 pathway and its downstream mRNAs in tree shrew fibroblast-like synoviocytes were detected using RT-PCR. RESULTS AND CONCLUSION: Under an inverted microscope, tree shrew fibroblast-like synoviocytes after 3 generations of subculture were mostly spindleshaped cells with similar size, high purity, good growth and proliferation in vitro. Hematoxylin-eosin staining revealed that the cells were mostly spindle-shaped, similar in size. Immunocytochemical staining results showed that Vimentin positively expressed in fibroblast-like synoviocytes. Cell counting kit-8 testing results showed an “S”-shaped growth curve of tree shrew fibroblast-like synoviocytes. Western blot results revealed that at 48 hours after R848 stimulation, the expression level of nuclear factor-κB protein significantly increased (P < 0.05), the expression level of p-P38 protein decreased (P < 0.05), and there was no change in the expression of TLR8, MyD88, P38, p-Erk1/2, and RANKL proteins (in tree shrew fibroblast-like synoviocytes (P > 0.05). After R848 stimulation, the expression of p-P38 in human fibroblast-like synoviocytes also significantly decreased, and no changes were found in the expression of TLR8, MyD88, P38, p-Erk1/2, and RANKL proteins (P > 0.05). Findings from RT-PCR and agarose gel electrophoresis detection showed that the mRNA expression of tumor necrosis factor ɑ, interleukin 6, interferon β, TLR9-2, TLR9-1, TLR8-2, TLR8-1, TLR7-2, and TLR7-1 mRNA expression had no significant change after 48 hours of stimulation with R848 (P > 0.05). To conclude, the tissue block adherence method can be used to isolate tree shrew fibroblast-like synoviocytes, and TLR8 pathway-related molecular detection method for tree shrew fibroblast-like synoviocytes is successfully established. [ABSTRACT FROM AUTHOR]