The Research Of Synergistic Action Of NOD And TLR4 In Keratocyte

Background:Mycotic keratitis is a capital eye diseases caused blindness. especially infectious corneal diseases has become a major eye diseases caused blindness of the world. In recent years, the incidence of mycotic keratitis has risen to the first of infectious corneal diseases. Therefore, the prevention and research of mycotic keratitis has become an important scientific problems of eye diseases caused to blindness in our country.Normally, eumycete don't encroach normal cornea, but when the corneal epithelium damaged, cornea propria exposed, eumycete can invade into propria lamina and substantia propria layer through the lesions, caused to mycotic keratitis. Corneal epithelium and stromal cells participate in the natural immune response of the host to oppose the invading of eumycete. TLR is on the surface of the cell which is responsible for sensing danger signals from outside of the cells, and NOD is located in the cytoplasm, which is responsible for identifying dangerous signal inside of the cells. Studies show that the TLR-mediated signaling pathway in innate immune plays an important role in resistance to fungal infection of corneal cells, and takes part in immune tolerance of corneal cells ect. But the role of NOD in these processes and its relationship with the TLR is little known currently.This study discusses the expression of NOD in corneal cells and researches the role of the process in corneal cells to produce pro-inflammatory cytokines induced by the innate immune signaling pathways mediated by NOD, and then explores its relationship with the TLR-mediated innate immune signaling pathways process.Objective:To study the expression of cellular pattern recognition receptors nucleotide-binding oligomerization domain containing in the corneal cell and the role of activate the innate immune of corneal cell, to explore the mutual relations with the Toll-like receptor TLR4 in this process.Method:Cultured immortalized human corneal epithelial cells (THCE) and immortalized human corneal stromal cells (THSF), experimental cells were divided into 6 groups,1 to 6 groups were replaced DMEM medium, LPS stimulation solution (1μg/ml), DAP stimulation solution (10μg/ml), MDP stimulation solution (10μg/ml), LPS (1μg/ml) and DAP (10μg/ml) the joint stimulation solution, LPS (1μg/ml) and MDP (10μg/ml) the joint stimulation solution, the stimulation 1h,3h,6h, 12h,24h, and cell supernatants were collected. ELISA detect the TNF-αand IL-6 secretion in Supernatant, and RT-PCR detect the mRNA of NOD1, NOD2 and TLR4, as well as the mRNA of antimicrobial peptides hBD2 and LL37 in corneal cells,. Application SPSS13.0 statistical analysis software, data are mean standard deviation of that group compared with t test was used to compare between groups analysis of variance, P<0.05 considered significant difference. Result:DAP and MDP can increased the expression of mRNA of NODI and NOD2 in corneal cells as time-dependent manner, and the IL-6 and TNF-αsecretion increased in cell culture supernatants, the expression of antimicrobial peptides LL37 and hBD2 also be increased.The co-stimulation of DAP, MDP and LPS expression of a certain synergy.Conclusion:All these indicated that congenital immunity signal conducting pathway mediated by NOD1, NOD2 and TLR4 played an important role in anti-fungous infection.DAP, MDP and LPS can regulate the expression of NOD1, NOD2 and TLR4, and participate in synthesis process of proinflammatory factor and antibacterial peptide.