人口腔扁平苔蘚角質(zhì)形成細胞體外培養(yǎng)方法的改良及TLR7、TLR8、TLR9在其內(nèi)的異常表達
發(fā)布時間：2018-11-07 12:11
【摘要】：研究目的:1、研究抗真菌治療因素、年齡因素和包被條件對口腔扁平苔蘚(oral lichen planus,OLP)角質(zhì)形成細胞的培養(yǎng)成功率的影響,探索建立其最佳的體外培養(yǎng)方法和條件。2、研究TLR7、TLR8、TLR9在OLP患者上皮組織中及體外培養(yǎng)的角質(zhì)形成細胞內(nèi)的表達。研究方法:1、將48名網(wǎng)紋型OLP患者按照是否進行抗真菌治療、年齡因素及包被條件進行分組。分別通過倒置相差顯微鏡和電子顯微鏡觀察細胞形態(tài)學變化,Fisher確切概率法比較不同情況下的細胞培養(yǎng)成功率。免疫熒光法檢測NF-kb p65在培養(yǎng)的角質(zhì)形成細胞內(nèi)的表達。酶聯(lián)免疫吸附試驗(ELISA)檢測IL-6、TNF-α、IFN-β在細胞培養(yǎng)上清中的表達。2、通過實時定量聚合酶鏈式反應(QRT-PCR)及免疫組化法檢測OLP患者上皮組織中TLR7(Toll樣受體7)、TLR8(Toll樣受體8)、TLR9(Toll樣受體9)的表達。并用間接免疫熒光法比較TLR7、TLR8、TLR9在體外培養(yǎng)的角質(zhì)形成細胞內(nèi)的表達。研究結(jié)果:1、抗真菌治療后角質(zhì)形成細胞的培養(yǎng)成功率明顯提高;年齡小于40歲的OLP患者,其角質(zhì)形成細胞的培養(yǎng)成功率明顯高于年齡大于40歲的患者;使用重組人I型膠原蛋白包被的細胞,貼壁成功率明顯高于使用多聚賴氨酸或不使用包被材料的細胞,且差別均具有統(tǒng)計學意義。經(jīng)鑒定,體外培養(yǎng)的是OLP患者的角質(zhì)形成細胞,且高表達NF-κB p65及IL-1β、IL-6、TNF-α等細胞因子。2、QRT-PCR結(jié)果顯示,TLR7、TLR8、TLR9在OLP患者上皮層的表達明顯高于正常對照(p0.05)。免疫組化的結(jié)果顯示,TLR7,TLR8,TLR9在OLP患者上皮層的表達與QRT-PCR的結(jié)果相一致。體外培養(yǎng)的細胞模型中,TLR7、TLR8、TLR9同樣在OLP患者的角質(zhì)形成細胞內(nèi)表達較高,差別具有統(tǒng)計學意義。結(jié)論:1、本研究驗證,黏膜上皮標本來自經(jīng)過抗真菌治療后或年齡較輕的OLP患者,且當細胞接種于重組人I型膠原蛋白包被后的培養(yǎng)板時,角質(zhì)形成細胞體外培養(yǎng)的成功率更高,進一步完善了人OLP角質(zhì)形成細胞系的體外建立方法。2、TLR7、TLR8、TLR9在OLP患者口腔黏膜上皮層及體外培養(yǎng)的角質(zhì)形成細胞內(nèi)表達均有升高,提示該疾病與機體固有免疫密切相關(guān)。
[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- 偽 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- 偽 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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R781.5
本文編號：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- 偽 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- 偽 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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R781.5
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