Experimental Study Of Toll Like Receptor 4 On Inflammatory Response Of Keratocytes Induced By Fusarium Solani In Vitro

Objective: To detect the expression of Toll 1ike receptor 4 (TLR4) in the cultured mouse keratocytes in vitro stimulated by Fusarium Solani suspension, research the relationship between TLR4 expression and inflammatory factors secrection, and investigate its role of fungal keratitis.Methods: Mouse keratocytes were cultured in vitro by enzyme digestion method. The cells in generation 3 were identified by immunocytochemistry staining using monoclonal anti-vimentin and anti-cytokeratin 12 polyclonal antibodies. Growth characteristic of cells was observed by inverted phase contrast microscope in vitro,growth curve was drawing by MTT colorimetry. Fusarium Solani suspension (Spore density was 1×106CFU/ml) was self-made. Cultured cells were stimulated with Fusarium Solani suspension. MTT colorimetry was used for assaying the proliferation of the cells induced by Fusarium Solani suspension within 48h. The techniques of immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to examine the expression of TLR4 protein and mRNA in the cultured mouse keratocytes at 0h, 3h, 6h, 12h post stimulation. The release of tumor necrosis factorα(TNF-α) and interleukin 8 (IL-8) was also measured using enzyme-linked immunosorbent assays (ELISA) in the presence and absence of specific blocking antibodies to TLR4 at 1h, 3h, 6h, 12h post stimulation.Results: Mouse keratocytes were cultured by enzyme digestion after 1~2d. Culured cells were positive for anti-vimentin immunofluorescence stain. Cell growth curve was approximate S-shaped. MTT colorimetry showed that Fusarium Solani can inhibit growth of the cells. The results of immunohistochemical technique and RT-PCR showed that TLR4 protein and mRNA were weakly expressed on cultured mouse keratocytes at 0h, significant increased at 3h, reached the peak at 6h, and decreased at 12h. Secrection of TNF-αand IL-8 were gradually increased, reached the peak at 6h, and then decreased gradually. The effects could be inhibited by treatment with TLR4 antibodies. There was positive correlation between TLR4 mRNA expression and inflammatory factors secrection at 3h, 6h, 12h (P<0.05). The diference of interclass has statistical significanc (P<0.05).Conclusion: TLR4 may play a critical role in recognizing fungi and mediating inflammatory defense response of cornea.