本文選題：表皮 + 角質��； 參考：《中國協(xié)和醫(yī)科大學》2008年博士論文

【摘要】： Toll樣受體(TLR)是近年來發(fā)現(xiàn)的一組新的模式識別受體,在天然免疫和獲得性免疫中均有重要作用。皮膚角質形成細胞(KC)是機體與外界環(huán)境接觸的首要屏障細胞,易受各種微生物的侵襲,TLR在其中發(fā)揮重要作用。TLR在很多炎癥性和感染性皮膚病中有一定作用。到目前為止,KC中TLR的表達仍未明確,KC的TLR調節(jié)和功能的研究不多。本課題擬檢測KC的TLR表達譜,研究金黃色葡萄球菌肽聚糖(PGN)對KC部分TLR表達的影響,以及對KC分泌趨化因子的影響及TLR在其中的作用,為今后研究TLR在某些感染性和炎癥性皮膚病中作用奠定基礎。全文共分三部分。 第一部分人表皮角質形成細胞Toll樣受體表達的研究 目的研究人表皮KC中TLR的表達譜。方法培養(yǎng)人永生化角質形成細胞系HaCaT細胞和正常人表皮角質形成細胞(NHEK)以及用分散酶分離表皮,分別用RT-PCR檢測10種TLRmRNA表達。流式細胞儀檢測HaCaT細胞和NHEK表面TLR2和TLR4蛋白的表達。結果HaCaT細胞、NHEK以及分離的表皮均有全部10種TLRmRNA表達,其表達強弱各不相同。流式細胞術檢測顯示HaCaT細胞和NHEK表面均有TLR4蛋白的表達,而TLR2無明顯表達。結論人表皮KC有TLR1～10mRNA的組成性表達,細胞表面有TLR4蛋白的表達。 第二部分金黃色葡萄球菌肽聚糖對HaCaT細胞Toll樣受體2和4表達的影響 目的研究病原體成分對HaCaT細胞TLR2和TLR4表達的調節(jié)。方法不同濃度的金黃色葡萄球菌胞壁成分PGN與HaCaT細胞共培養(yǎng)不同時間后,RT-PCR法檢測TLR2和TLR4mRNA表達變化。流式細胞術檢測HaCaT細胞表面TLR2和TLR4蛋白的表達。結果與30μg/ml PGN共培養(yǎng)6小時后,HaCaT細胞TLR2和TLR4mRNA表達明顯升高。與100μg/ml PGN共培養(yǎng)24小時后,HaCaT細胞表面TLR2和TLR4蛋白表達量最高。結論PGN可以上調HaCaT細胞TLR2和TLR4的表達。 第三部分金黃色葡萄球菌肽聚糖對正常人表皮角質形成細胞分泌趨化因子的影響及Toll樣受體2的作用 目的研究金黃色葡萄球菌PGN對NHEK分泌白介素—8(IL-8)、調節(jié)激活和正常T細胞表達和分泌的細胞因子(RANTES)以及巨噬細胞來源的趨化因子(MDC)的影響以及TLR2在其中的作用。方法不同濃度的金黃色葡萄球菌PGN與NHEK共培養(yǎng)不同時間后,采用酶聯(lián)免疫吸附實驗法(ELISA)檢測細胞培養(yǎng)上清液中IL-8、RANTES以及MDC的濃度。預先加用功能性TLR2單克隆抗體處理培養(yǎng)細胞,再以100μg/ml PGN與NHEK共培養(yǎng)12小時,檢測上述趨化因子的濃度。結果NHEK可以自發(fā)的分泌IL-8和RANTES。隨共培養(yǎng)的PGN濃度的升高,培養(yǎng)時間的延長,IL-8分泌量逐漸增多,RANTES分泌量逐漸降低。TLR2單克隆抗體可以明顯抑制PGN誘導的IL-8分泌,對RANTES分泌量無明顯影響。未檢測到NHEK自發(fā)或PGN刺激作用下產(chǎn)生MDC。結論PGN很可能通過激活TLR2途徑誘導NHEK分泌IL-8。PGN能夠抑制NHEK產(chǎn)生RANTES。
[Abstract]:Toll like receptor (TLR) is a new group of pattern recognition receptors found in recent years, which plays an important role in both innate and acquired immunity. Skin keratinocytes (KCCs) are the primary barrier cells in contact with the environment. TLR, which is susceptible to various microorganisms, plays an important role in many inflammatory and infectious skin diseases. Up to now, the expression of TLR in KC is still unclear, and the regulation and function of TLR in KC are not well studied. The aim of this study was to investigate the effect of Staphylococcus aureus peptidoglycan (PGNN) on the expression of partial TLR of KC and the effect of TLR on the secretion of chemokines in KC by detecting the TLR expression profile of KC. To lay a foundation for the future study of the role of TLR in some infectious and inflammatory dermatoses. The full text is divided into three parts. The expression of Toll like receptors in human epidermal keratinocytes Objective to study the expression of TLR in human epidermis KC. Methods the human immortalized keratinocytes (HaCaT) and normal human epidermal keratinocytes (NHEK) were cultured, and the epidermis was isolated by disperse enzyme. The expression of 10 kinds of TLRmRNA was detected by RT-PCR. The expression of TLR2 and TLR4 on HaCaT cells and NHEK was detected by flow cytometry. Results all 10 kinds of TLRmRNA were expressed in HaCaT cells and in isolated epidermis. Flow cytometry showed that TLR4 protein was expressed on both HaCaT cells and NHEK, but TLR2 was not. Conclusion Human epidermis KC has the constitutive expression of TLR1~10mRNA and the expression of TLR4 protein on the surface of human epidermis. The effect of Staphylococcus aureus peptidoglycan on the expression of Toll like receptors 2 and 4 in HaCaT cells Objective to study the effect of pathogen components on the expression of TLR2 and TLR4 in HaCaT cells. Methods different concentrations of staphylococcus aureus cell wall PGN and HaCaT cells were co-cultured for different time, then the expression of TLR2 and TLR4mRNA were detected by RT-PCR. The expression of TLR2 and TLR4 on HaCaT cells was detected by flow cytometry. Results after co-culture with 30 渭 g/ml PGN for 6 hours, the expression of TLR2 and TLR4mRNA in HaCaT cells increased significantly. After co-cultured with 100 渭 g/ml PGN for 24 hours, the expression of TLR2 and TLR4 on the surface of HaCaT cells was the highest. Conclusion PGN can up-regulate the expression of TLR2 and TLR4 in HaCaT cells. The effect of Staphylococcus aureus peptidoglycan on the secretion of chemokines in normal human epidermal keratinocytes and the effect of Toll like receptor 2 Objective to study the effects of Staphylococcus aureus (PGN) on the secretion of interleukin-8 (IL-8), a cytokine (RANTES) and a chemokine derived from macrophages (macrophage) from NHEK, and the role of TLR2 in the secretion of IL-8, a cytokine that regulates the expression and secretion of activated and normal T cells. Methods different concentrations of Staphylococcus aureus PGN co-cultured with NHEK for different time were used to detect the concentration of IL-8 RANTES and MDC in the supernatant of cell culture by enzyme linked immunosorbent assay (Elisa). The cultured cells were pretreated with functional TLR2 monoclonal antibody, then co-cultured with 100 渭 g/ml PGN and NHEK for 12 hours to detect the concentration of the above chemokines. Results NHEK could secrete IL-8 and RANTES spontaneously. With the increase of PGN concentration in co-culture, the secretion of IL-8 increased with the prolongation of culture time. The secretion of RANTES decreased gradually. TLR2 monoclonal antibody could significantly inhibit the secretion of IL-8 induced by PGN, but had no obvious effect on the secretion of RANTES. No NHEK spontaneous or PGN stimulated MDC was detected. Conclusion PGN may induce NHEK to secrete IL-8.PGN by activating TLR2 pathway, which can inhibit the production of RANTESby NHEK.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R363

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