In Vitro Cell Culture System Optimization And Abnormal Expression Of TLR-7, TLR8, TLR9 Of Keratinocytes From OLP Patients

Objectives: 1. The aim of this study was to optimize the culture system for keratinocytes obtained from the lesion of patients with oral lichen planus(OLP) in vitro. 2. The purpose was to determine the expression of TLR7, TLR8 and TLR9 in epithelium and keratinocytes obtained from the lesion of patients with OLP.Materials and methods: 1. A total of 48 OLP patients were included in this study and were divided into different groups according to the antifungus treatment, age of patients and the precoating factors of the culture. The morphological changes and ultrastructure of the cultured OLP keratinocytes were observed via inverted phase contrast microscopy and electron microscopy, respectively. The successful rates were analysed by Fisher's exact test. Indirect immunofluorescence staining was used to detect NF–?B p65. IL-1?, IL-6 and TNF-? in the supernatant were measured by ELISA. 2. We compared the expression of Toll-like receptor 7(TLR7), Toll-like receptor 8(TLR8) and Toll-like receptor 9(TLR9) in oral mucosa epithelium from oral lichen planus(OLP) lesion areas against healthy control(HC) by quantitative real-time PCR and indirect immunohistochemical staining. We also characterized expression of the three TLRs in keratinocytes obtained from OLP lesion areas by indirect immunofluorescence staining in vitro.Results: 1. Keratinocytes obtained from OLP patients with anti-fungus treatment who were aged below 40 years old and cultured with recombinant human Type-1 collagen precoating exhibited much higher success rate. Almost all cultured OLP keratinocytes expressed the characteristic protein of cytokeratin. Compared with normal oral keratinocytes, the cultured OLP keratinocytes demonstrated much stronger expression of NF-?B p65 and more production of inflammatory cytokines like IL-1?, IL-6, and TNF-?. 2. Both QRT-PCR and immunohistochemical staining revealed that TLR7, TLR8 and TLR9 expressed significantly higher in OLP epithelium than HC. The three TLRs also over expressed in keratinocytes obtained from OLP patients in the cell model in vitro(p<0.05).Conclusions: 1. Our research suggested that antifungus treatments, optimal ages of the patients and recombinant human Type-1 collagen precoating could enhance the culture success rate of OLP keratinocytes in vitro. The cultured OLP keratinocytes mimicked the local inflammatory environment of OLP to some extent and may be used as a cell model of OLP. 2. Our finding demonstrated that high expression of TLR7, TLR8 and TLR9 in both OLP mucosa epithelium and OLP keratinocytes cultured in vitro, which may represent a new approach to explore the pathogenesis of OLP in inherent immunology.