人口腔扁平苔藓角质形成细胞体外培养方法的改良及TLR7、TLR8、TLR9在其内的异常表达
发布时间：2018-11-07 12:11
【摘要】：研究目的:1、研究抗真菌治疗因素、年龄因素和包被条件对口腔扁平苔藓(oral lichen planus,OLP)角质形成细胞的培养成功率的影响,探索建立其最佳的体外培养方法和条件。2、研究TLR7、TLR8、TLR9在OLP患者上皮组织中及体外培养的角质形成细胞内的表达。研究方法:1、将48名网纹型OLP患者按照是否进行抗真菌治疗、年龄因素及包被条件进行分组。分别通过倒置相差显微镜和电子显微镜观察细胞形态学变化,Fisher确切概率法比较不同情况下的细胞培养成功率。免疫荧光法检测NF-kb p65在培养的角质形成细胞内的表达。酶联免疫吸附试验(ELISA)检测IL-6、TNF-α、IFN-β在细胞培养上清中的表达。2、通过实时定量聚合酶链式反应(QRT-PCR)及免疫组化法检测OLP患者上皮组织中TLR7(Toll样受体7)、TLR8(Toll样受体8)、TLR9(Toll样受体9)的表达。并用间接免疫荧光法比较TLR7、TLR8、TLR9在体外培养的角质形成细胞内的表达。研究结果:1、抗真菌治疗后角质形成细胞的培养成功率明显提高;年龄小于40岁的OLP患者,其角质形成细胞的培养成功率明显高于年龄大于40岁的患者;使用重组人I型胶原蛋白包被的细胞,贴壁成功率明显高于使用多聚赖氨酸或不使用包被材料的细胞,且差别均具有统计学意义。经鉴定,体外培养的是OLP患者的角质形成细胞,且高表达NF-κB p65及IL-1β、IL-6、TNF-α等细胞因子。2、QRT-PCR结果显示,TLR7、TLR8、TLR9在OLP患者上皮层的表达明显高于正常对照(p0.05)。免疫组化的结果显示,TLR7,TLR8,TLR9在OLP患者上皮层的表达与QRT-PCR的结果相一致。体外培养的细胞模型中,TLR7、TLR8、TLR9同样在OLP患者的角质形成细胞内表达较高,差别具有统计学意义。结论:1、本研究验证,黏膜上皮标本来自经过抗真菌治疗后或年龄较轻的OLP患者,且当细胞接种于重组人I型胶原蛋白包被后的培养板时,角质形成细胞体外培养的成功率更高,进一步完善了人OLP角质形成细胞系的体外建立方法。2、TLR7、TLR8、TLR9在OLP患者口腔黏膜上皮层及体外培养的角质形成细胞内表达均有升高,提示该疾病与机体固有免疫密切相关。
[Abstract]:Objective: 1. To study the effects of antifungal therapy factors, age factors and coating conditions on the success rate of keratinocyte culture in oral lichen planus (oral lichen planus,OLP). To investigate the expression of TLR7,TLR8,TLR9 in keratinocytes of OLP patients and in vitro. Methods: 1. 48 patients with reticulated OLP were divided into three groups according to antifungal therapy, age factors and envelope conditions. The morphologic changes of cells were observed by inverted phase contrast microscope and electron microscope respectively. The Fisher exact probability method was used to compare the success rate of cell culture under different conditions. The expression of NF-kb p65 in cultured keratinocytes was detected by immunofluorescence assay. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-6,TNF- 伪 and IFN- 尾 in the supernatant of cell culture. The expression of TLR7 (Toll like receptor 7), TLR8 (Toll like receptor 8) and TLR9 (Toll like receptor 9) in the epithelium of OLP patients was detected by real-time quantitative polymerase chain reaction (QRT-PCR) and immunohistochemistry. Indirect immunofluorescence assay was used to compare the expression of TLR7,TLR8,TLR9 in cultured keratinocytes. The results showed that: 1, the success rate of keratinocyte culture was significantly increased after antifungal therapy, and the success rate of keratinocyte culture in OLP patients aged less than 40 years was significantly higher than that in patients older than 40 years old. The adherent success rate of cells coated with recombinant human type I collagen was significantly higher than that with polylysine or without coating material, and the difference was statistically significant. The keratinocytes of OLP patients were identified as keratinocytes cultured in vitro, and NF- 魏 B p65, IL-1 尾, IL-6,TNF- 伪 and other cytokines were overexpressed. The expression of TLR9 in OLP patients was significantly higher than that in normal controls (p0. 05). Immunohistochemical results showed that the expression of TLR7,TLR8,TLR9 in OLP patients was consistent with that of QRT-PCR. The expression of TLR7,TLR8,TLR9 in keratinocytes of OLP patients was also higher than that of in vitro cultured cells, and the difference was statistically significant. Conclusion: 1. In this study, the mucosal epithelial specimens were obtained from patients with OLP after antifungal therapy or younger, and when the cells were inoculated with the culture plate of recombinant human type I collagen. The success rate of cultured keratinocytes in vitro was higher, which further improved the method of establishing human OLP keratinocytes in vitro. 2 TLR7 and TLR8, TLR8, The expression of TLR9 in the oral mucosal epithelium of OLP patients and the keratinocytes cultured in vitro were increased, suggesting that the disease is closely related to the innate immunity of the body.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781.5
本文编号：2316332
[Abstract]:Objective: 1. To study the effects of antifungal therapy factors, age factors and coating conditions on the success rate of keratinocyte culture in oral lichen planus (oral lichen planus,OLP). To investigate the expression of TLR7,TLR8,TLR9 in keratinocytes of OLP patients and in vitro. Methods: 1. 48 patients with reticulated OLP were divided into three groups according to antifungal therapy, age factors and envelope conditions. The morphologic changes of cells were observed by inverted phase contrast microscope and electron microscope respectively. The Fisher exact probability method was used to compare the success rate of cell culture under different conditions. The expression of NF-kb p65 in cultured keratinocytes was detected by immunofluorescence assay. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-6,TNF- 伪 and IFN- 尾 in the supernatant of cell culture. The expression of TLR7 (Toll like receptor 7), TLR8 (Toll like receptor 8) and TLR9 (Toll like receptor 9) in the epithelium of OLP patients was detected by real-time quantitative polymerase chain reaction (QRT-PCR) and immunohistochemistry. Indirect immunofluorescence assay was used to compare the expression of TLR7,TLR8,TLR9 in cultured keratinocytes. The results showed that: 1, the success rate of keratinocyte culture was significantly increased after antifungal therapy, and the success rate of keratinocyte culture in OLP patients aged less than 40 years was significantly higher than that in patients older than 40 years old. The adherent success rate of cells coated with recombinant human type I collagen was significantly higher than that with polylysine or without coating material, and the difference was statistically significant. The keratinocytes of OLP patients were identified as keratinocytes cultured in vitro, and NF- 魏 B p65, IL-1 尾, IL-6,TNF- 伪 and other cytokines were overexpressed. The expression of TLR9 in OLP patients was significantly higher than that in normal controls (p0. 05). Immunohistochemical results showed that the expression of TLR7,TLR8,TLR9 in OLP patients was consistent with that of QRT-PCR. The expression of TLR7,TLR8,TLR9 in keratinocytes of OLP patients was also higher than that of in vitro cultured cells, and the difference was statistically significant. Conclusion: 1. In this study, the mucosal epithelial specimens were obtained from patients with OLP after antifungal therapy or younger, and when the cells were inoculated with the culture plate of recombinant human type I collagen. The success rate of cultured keratinocytes in vitro was higher, which further improved the method of establishing human OLP keratinocytes in vitro. 2 TLR7 and TLR8, TLR8, The expression of TLR9 in the oral mucosal epithelium of OLP patients and the keratinocytes cultured in vitro were increased, suggesting that the disease is closely related to the innate immunity of the body.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781.5
【参考文献】
相关期刊论文 前5条
1 姚声;周杰;;口腔扁平苔藓相关危险因素的logistic回归分析[J];临床口腔医学杂志;2013年05期
2 张文怡;唐国瑶;;口腔扁平苔藓治疗的循证医学研究进展[J];临床口腔医学杂志;2011年02期
3 李琴;杜观环;唐国瑶;;口腔扁平苔藓相关细胞模型的建立[J];上海口腔医学;2011年01期
4 汪群;诸焕颖;孙红英;;人口腔扁平苔藓角质形成细胞的体外培养及生物学鉴定[J];复旦学报(医学版);2007年05期
5 周曾同,周海文,商庆新,曹谊林;体外连续培养人口腔粘膜角化细胞(英文)[J];Chinese Medical Journal;2001年07期
，本文编号：2316332
本文链接：https://www.wllwen.com/yixuelunwen/kouq/2316332.html
最近更新
教材专著
