人表皮角质形成细胞Toll样受体的表达以及Toll样受体2功能的初步研究

发布时间：2018-05-06 23:43

本文选题：表皮 + 角质　；参考：《中国协和医科大学》2008年博士论文

【摘要】： Toll样受体(TLR)是近年来发现的一组新的模式识别受体,在天然免疫和获得性免疫中均有重要作用。皮肤角质形成细胞(KC)是机体与外界环境接触的首要屏障细胞,易受各种微生物的侵袭,TLR在其中发挥重要作用。TLR在很多炎症性和感染性皮肤病中有一定作用。到目前为止,KC中TLR的表达仍未明确,KC的TLR调节和功能的研究不多。本课题拟检测KC的TLR表达谱,研究金黄色葡萄球菌肽聚糖(PGN)对KC部分TLR表达的影响,以及对KC分泌趋化因子的影响及TLR在其中的作用,为今后研究TLR在某些感染性和炎症性皮肤病中作用奠定基础。全文共分三部分。第一部分人表皮角质形成细胞Toll样受体表达的研究目的研究人表皮KC中TLR的表达谱。方法培养人永生化角质形成细胞系HaCaT细胞和正常人表皮角质形成细胞(NHEK)以及用分散酶分离表皮,分别用RT-PCR检测10种TLRmRNA表达。流式细胞仪检测HaCaT细胞和NHEK表面TLR2和TLR4蛋白的表达。结果HaCaT细胞、NHEK以及分离的表皮均有全部10种TLRmRNA表达,其表达强弱各不相同。流式细胞术检测显示HaCaT细胞和NHEK表面均有TLR4蛋白的表达,而TLR2无明显表达。结论人表皮KC有TLR1～10mRNA的组成性表达,细胞表面有TLR4蛋白的表达。第二部分金黄色葡萄球菌肽聚糖对HaCaT细胞Toll样受体2和4表达的影响目的研究病原体成分对HaCaT细胞TLR2和TLR4表达的调节。方法不同浓度的金黄色葡萄球菌胞壁成分PGN与HaCaT细胞共培养不同时间后,RT-PCR法检测TLR2和TLR4mRNA表达变化。流式细胞术检测HaCaT细胞表面TLR2和TLR4蛋白的表达。结果与30μg/ml PGN共培养6小时后,HaCaT细胞TLR2和TLR4mRNA表达明显升高。与100μg/ml PGN共培养24小时后,HaCaT细胞表面TLR2和TLR4蛋白表达量最高。结论PGN可以上调HaCaT细胞TLR2和TLR4的表达。第三部分金黄色葡萄球菌肽聚糖对正常人表皮角质形成细胞分泌趋化因子的影响及Toll样受体2的作用目的研究金黄色葡萄球菌PGN对NHEK分泌白介素—8(IL-8)、调节激活和正常T细胞表达和分泌的细胞因子(RANTES)以及巨噬细胞来源的趋化因子(MDC)的影响以及TLR2在其中的作用。方法不同浓度的金黄色葡萄球菌PGN与NHEK共培养不同时间后,采用酶联免疫吸附实验法(ELISA)检测细胞培养上清液中IL-8、RANTES以及MDC的浓度。预先加用功能性TLR2单克隆抗体处理培养细胞,再以100μg/ml PGN与NHEK共培养12小时,检测上述趋化因子的浓度。结果NHEK可以自发的分泌IL-8和RANTES。随共培养的PGN浓度的升高,培养时间的延长,IL-8分泌量逐渐增多,RANTES分泌量逐渐降低。TLR2单克隆抗体可以明显抑制PGN诱导的IL-8分泌,对RANTES分泌量无明显影响。未检测到NHEK自发或PGN刺激作用下产生MDC。结论PGN很可能通过激活TLR2途径诱导NHEK分泌IL-8。PGN能够抑制NHEK产生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.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

【参考文献】