Toll样受体信号对角膜天然免疫影响的实验研究

发布时间：2018-05-17 09:36

本文选题：Toll样受体 + 氢化可的松　；参考：《浙江大学》2007年博士论文

【摘要】： 第一部分糖皮质激素对角膜上皮细胞和角膜成纤维细胞TLRs的表达和功能的调控目的：评估糖皮质激素对人角膜上皮细胞和角膜成纤维细胞的Toll样受体(Toll-like receptors，TLRs)表达和功能的影响。方法：使用三种不同浓度的氢化可的松(1，10，100μg／ml)去刺激培养的人角膜上皮细胞系和角膜成纤维细胞，用RT-PCR和Real-time PCR来检查TLR1-10的mRNA的表达，以及这种表达在不同浓度氢化可的松干预下的变化情况。用酶联免疫吸附实验(enzyme-linked immunosorbent assays，ELISA)来检测两种培养细胞在正常状态下和氢化可的松干预下的IL-6，8的释放。TLR2，3，4，9蛋白水平的表达使用荧光免疫组织化学和western blot来检测。结果：人角膜上皮细胞系和角膜成纤维细胞均表达TLR1-10 mRNA，但是两种细胞表达的TLRs的量存在差异性，其中人角膜上皮细胞系TLR1，3，4高表达，TLR7，8，9，10低表达；而在人角膜成纤维细胞TLR4高度表达，TLR7，8，9，10，2表达量相当低。在氢化可的松的干预下这两种细胞表达TLRs明显不同，在人角膜上皮细胞系氢化可的松能促进大多数TLRs mRNA的表达，特别是TLR2，3，4，9；而在人角膜成纤维细胞却表现为明显抑制TLRs mRNA的表达。荧光免疫组织化学和western blot也表明氢化可的松能促进人角膜上皮细胞系TLR2，3，4，9在蛋白水平的表达，但是在人角膜成纤维细胞却表现为抑制TLR2，3，4，9在蛋白水平的表达。ELISA结果表明氢化可的松能促进人角膜上皮细胞系IL-6在培养液中的释放，同时抑制IL-8的释放；而在人角膜成纤维细胞IL-6，8均表现为明显的抑制。结论：TLR1-10在人角膜上皮细胞系和角膜成纤维细胞均有表达，不同TLRs受体在同一细胞表达量不同，而各受体在不同细胞的表达也不同；氢化可的松能调控人角膜上皮细胞系和角膜成纤维细胞TLRs在mRNA和蛋白水平的表达，也能调控TLRs下游细胞因子IL-6，8的释放，但是对人角膜上皮细胞系TLRs表现为轻度促进，而对角膜成纤维细胞TLRs表现为抑制，提示了角膜的这两种细胞在角膜天然免疫和角膜炎症过程中发挥不同的作用；氢化可的松促进人角膜上皮细胞系TLRs的表达和功能暗示了糖皮质激素在天然免疫方面的一个新的功能，提示糖皮质激素在角膜不同细胞发挥不同的免疫作用，其中对角膜上皮可能具有免疫增强功能，，因此局部使用糖皮质激素在角膜上皮完整时可能有降低角膜机会性感染的可能，也可能会有益于某些特定的角膜感染，研究结果为糖皮质激素在临床合理应用提供了新的依据。第二部分茄病镰刀菌灭活菌丝对角膜上皮细胞TLRs的表达和功能的影响目的：通过研究人角膜上皮细胞系在接受灭活的茄病镰刀菌菌丝刺激后Toll样受体(toll-like receptors，TLRs)的表达和功能变化，探讨角膜上皮及其表达的TLRs在角膜真菌感染中的作用方法：茄病镰刀菌的灭活菌丝片断按照1∶1比例来刺激培养的角膜上皮细胞系，细胞的TLR1-10 mRNA表达用Real-time PCR来分析，用ELISA来检测培养液中的细胞因子IL-6，8；然后10μg／ml的氢化可的松被用来和灭活菌丝一起来刺激培养的细胞，再次分析TLR1-10 mRNA的表达和IL-6，8释放；TLR2，4蛋白水平表达的变化用荧光免疫组织化学和western blot进行检查。另外用TLR2，4的单克隆抗体来阻断TLR2，4观察菌丝对IL-6，8释放的影响。结果：茄病镰刀菌的灭活菌丝能增加上皮细胞TLR2，3，4，6 mRNAs的表达和IL-6，8的释放，糖皮质激素能轻度增加角膜上皮细胞TLR2和TLR4的表达和IL-6的释放。荧光免疫组织化学和western blot结果显示灭活菌丝片断能促进TLR2和TLR4蛋白水平的表达，而且这种表达能进一步被氢化可的松上调。预先用anti-TLR2或anti-TLR4抗体封闭HCF后这两种抗体发现能显著抑制IL-6和IL-8的释放，但是对照的同型的IgG却没有抑制作用，联合使用两种抗体能达到最大的抑制效果：IL-6被抑制78％；IL-8被抑制了82％。单独TLR2抗体能抑制灭活菌丝诱导的IL-6，8分别约为68％和62％；而TLR4抗体抑制IL-6，8分别约为53％和56％。结论：角膜上皮细胞除自身的屏障作用外，其表达的TLRs与角膜抵御真菌的天然免疫密切相关；角膜上皮的天然免疫受体TLR2，4可能在角膜上皮防御真菌感染的过程中发挥重要作用；糖皮质激素可能增加角膜上皮TLRs表达而使角膜对真菌的侵袭力具有防御作用，研究结果对理解真菌在角膜的感染机制和进一步研究真菌的药物治疗提供理论依据。第三部分人角膜成纤维细胞TLRs在茄病镰刀菌灭活菌丝刺激后的表达和功能目的：通过研究人角膜成纤维细胞在接受灭活的茄病镰刀菌菌丝刺激后Toll样受体(toll-like receptors，TLRs)的表达和功能变化，探讨TLRs在角膜真菌感染中的作用。方法：在培养的人角膜成纤维细胞中加入灭活的茄病镰刀菌菌丝片断，按照1∶1比例以菌丝片断刺激细胞，使用Real-time PCR来分析成纤维细胞TLR1-10 mRNA的表达，用ELISA来检测培养液中的细胞因子IL-6，8；然后使用三种不同浓度(1，10，100μg／ml)的氢化可的松和灭活菌丝一起和成纤维细胞共培养，再次分析TLR1-10 mRNA的表达和IL-6，8释放；TLR2，4蛋白水平表达的变化用荧光免疫组织化学检查。另外用TLR2，4的单克隆抗体来阻断TLR2，4观察菌丝对IL-6，8释放的影响。结果：茄病镰刀菌灭活菌丝能增加成纤维细胞的TLRs mRNA表达和上调HCF IL-6，8的释放，而氢化可的松能抑制灭活菌丝诱导的TLRs mRNA的表达和IL-6，8的释放。预先用anti-TLR2或anti-TLR4抗体封闭HCF后这两种抗体发现能显著抑制IL-6和IL-8的释放，但是对照的同型的IgG却没有抑制作用，联合使用两种抗体能达到最大的抑制效果，IL-6抑制80％；IL-8抑制了92％。单独TLR2抗体能抑制灭活菌丝诱导的IL-6，8分别约为72％和82％；而单用TLR4抗体对IL-6，8的抑制分别为64％和68％。免疫荧光组化也显示灭活菌丝能增加轻度增加成纤维细胞TLR2，4蛋白水平的表达。结论：角膜成纤维细胞在角膜抵御真菌感染的过程发挥重要作用，角膜成纤维细胞的天然免疫受体TLR2，4可能在角膜成纤维细胞对抗茄病镰刀菌感染的过程中起关键作用，糖皮质激素可能是通过抑制了TLRs表达而增加了真菌在角膜的侵袭力，研究结果提供了一个理解真菌性角膜炎免疫机制的重要信息，有望为真菌性角膜炎的找到一个新的免疫治疗途径第四部分 Toll样受体在健康角膜和单疱病毒性角膜炎的病变角膜的表达目的：调查Toll样受体(toll-like receptors，TLRs)在健康角膜和单疱病毒感染的活动期和非活动期基质型角膜炎病变角膜的表达，探讨TLRs在单疱病毒性角膜炎中可能的作用。方法：8个单疱病毒性角膜炎(herpetic stromal keratitis，HSK)的病变角膜(包括5个活动期和3个稳定期)和8个健康的角膜被用来做本研究，首先我们用RT-PCR来分析TLR1-10 mRNA在健康角膜和病变角膜的表达情况，然后用相对定量的Real-time PCR来比较TLR1-10 mRNA在健康角膜和病变角膜的相对表达差异，最后我们用免疫荧光组织化学来分析在的蛋白表达在健康角膜和活动期HSK和稳定期HSK病变角膜的表达，数据的统计使用SPSS11.5来分析。结果：使用RT-PCR和Real-time PCR分析发现健康角膜和HSV感染角膜均表达TLR1-10 mRNA，在健康角膜Real-time PCR显示的了LR1，2，3，4，6 mRNA表达较高，而TLR7，8，9较低，TLR5，10居于中间。在活动性HSK的角膜TLR1-10的均上调，特别是TLR4，7，8，9；非活动性HSK的病变角膜除了TLR7明显上调外，其他TLRs均表现为下降，特别是TLR5，6，3。荧光免疫组织化学显示出在健康角膜和HSV感染角膜均有表达，TLR9的表达在活动性HSV感染的角膜要稍强于非活动性HSV感染角膜和健康角膜。结论：TLRs在健康角膜和HSV感染角膜表达的差异可能在角膜的天然免疫和角膜感染免疫中发挥，TLR7可能在角膜HSK中发挥重要作用，而TLR4，8，9可能仅与HSV的活动性感染有关，进一步的研究TLRs可能阐明角膜HSV感染的免疫控制机制
[Abstract]:the first portion

Regulation of glucocorticoid on expression and function of TLRs in corneal epithelial cells and corneal fibroblasts

Objective : To evaluate the effect of glucocorticoid on the expression and function of Toll - like receptors ( TLRs ) in human corneal epithelial cells and corneal fibroblasts .

Methods : Three different concentrations of hydrocortisone ( 1 , 10 , 100 渭g / ml ) were used to stimulate cultured human corneal epithelial cells and corneal fibroblasts . The expression of TLR1 - 10 mRNA was examined by RT - PCR and Real - time PCR . The expression of IL - 6 , 8 was detected by enzyme - linked immunosorbent assay ( ELISA ) .

Results : TLR1 - 10 mRNA was expressed in human corneal epithelial cells and corneal fibroblasts , but there was a difference in the amount of TLRs expressed in both cells . The expression of TLR1 , 3 , 4 in human corneal epithelial cells was higher than that of TLR7 , 8 , 9 and 10 .
The expression of TLRs was very low in human corneal fibroblasts . TLRs were significantly different in TLR7 , 8 , 9 , 10 and 2 , and the expression of TLRs mRNA was promoted in human corneal epithelial cells , especially TLR2 , 3 , 4 , 9 .
The results showed that hydrocortisone can promote the expression of TLR2 , 3 , 4 , 9 in human corneal epithelial cell line TLR2 , 3 , 4 , 9 at protein level .
In human corneal fibroblasts , IL - 6 and 8 showed significant inhibition .

Conclusion : TLR1 - 10 is expressed in human corneal epithelial cell line and corneal fibroblasts , and the expression of different TLRs is different in the same cell , and the expression of each receptor in different cells is different .
hydrocortisone can regulate the expression of TLRs of human corneal epithelial cells and corneal fibroblasts at mRNA and protein levels and regulate the release of cytokines IL - 6 , 8 downstream of TLRs , but the expression of TLRs in human corneal epithelial cells is mild , and the expression of TLRs in corneal fibroblasts is inhibited , suggesting that the two cells of the cornea play different roles in the course of corneal natural immunity and keratitis .
hydrocortisone promotes the expression and function of human corneal epithelial cell line tlrs and suggests a new function of glucocorticoid in the aspect of natural immunity , which suggests that the glucocorticoid can play a different immune function in different cells of the cornea .

the second part

The effect of Fusarium moniliforme inactivated mycelium on the expression and function of TLRs in corneal epithelial cells

Objective : To investigate the expression and functional changes of Toll - like receptors ( TLRs ) in human corneal epithelial cells after stimulation of inactivated solanacoccus , and to investigate the role of TLRs in corneal fungal infections .

Methods : The expression of TLR1 - 10 mRNA in cultured corneal epithelial cells was stimulated with 1 : 1 ratio of inactivated mycelium of Fusarium moniliforme . The expression of TLR1 - 10 mRNA was analyzed by Real - time PCR , and IL - 6 and 8 were detected by ELISA .
Then 10.mu . g / ml hydrocortisone was used to stimulate cultured cells together with inactivated mycelium , and the expression of TLR1 - 10 mRNA and IL - 6 , 8 release were analyzed again .
In addition , TLR2 and 4 were used to block TLR2 and 4 to observe the effect of mycelium on the release of IL - 6 and 8 .

Results : The expression of TLR2 , 3 , 4 , 6 mRNAs and the release of IL - 6 , 8 in epithelial cell TLR2 , 3 , 4 , 6 were increased . The expression of TLR2 and TLR2 and IL - 6 in corneal epithelial cells could be significantly increased by fluorescence immunohistochemical and western blot .
IL - 8 inhibited IL - 6 , 8 % and 62 % , respectively .
The inhibition of IL - 6 , 8 was 53 % and 56 % respectively .

Conclusion : The expression of TLRs in corneal epithelial cells is closely related to the natural immunity of the cornea .
The natural immune receptor TLR2 , 4 of corneal epithelium may play an important role in corneal epithelial defense fungal infection .
Glucocorticoids may increase the expression of TLRs in the corneal epithelium , which can provide a theoretical basis for understanding the mechanism of fungal infection in the cornea and the further study of fungal drug therapy .

PART III

Expression and function of human corneal fibroblasts TLRs following stimulation of Fusarium moniliforme

Objective : To investigate the role of TLRs in corneal fungal infections by investigating the expression and functional changes of toll - like receptors ( TLRs ) in human corneal fibroblasts .

Methods : In the cultured human corneal fibroblasts , the inactivated eggplant mycelium fragment was added . The expression of TLR1 - 10 mRNA was analyzed by Real - time PCR according to the ratio of 1 : 1 , and the cytokines IL - 6 and 8 were detected by ELISA .
The expression of TLR1 - 10 mRNA and the release of IL - 6 and 8 were analyzed by using hydrocortisone and inactivated mycelium of three different concentrations ( 1 , 10 , 100 渭g / ml ) together with fibroblasts .
In addition , TLR2 , 4 monoclonal antibodies were used to block TLR2 and 4 to observe the effect of mycelium on IL - 6 , 8 release .

Results : The expression of TLRs mRNA in fibroblasts and the release of IL - 6 and 8 were inhibited by Fusarium moniliforme . The release of IL - 6 and IL - 8 was inhibited by hydrocortisone .
IL - 8 inhibited 92 % . TLR2 alone inhibited IL - 6 , 8 % and 82 % , respectively .
The inhibition of IL - 6 and 8 was 64 % and 68 % , respectively . Immunofluorescence also showed that the expression of IL - 6 and 4 - protein was increased with the increase of IL - 6 and 8 % .

Conclusion : Corneal fibroblasts play an important role in corneal defense against fungal infections . The natural immune receptor TLR2 , 4 of corneal fibroblasts play a key role in the process of corneal fibroblasts . The results provide an important information to understand the immune mechanism of fungal keratitis , which is expected to find a new way to treat fungal keratitis .

PART IV

Expression of Toll - like receptor in cornea of healthy cornea and herpes simplex keratitis

Objective : To investigate the expression of toll - like receptors ( TLRs ) in cornea of healthy cornea and herpes simplex virus and to investigate the possible role of TLRs in herpes simplex keratitis .

Methods : The expression of TLR1 - 10 mRNA in healthy cornea and lesion cornea was analyzed by RT - PCR , and the expression of TLR1 - 10 mRNA in healthy cornea and lesion cornea was analyzed by RT - PCR .

Results : TLR1 - 10 mRNA was expressed by RT - PCR and Real - time PCR . The mRNA expression of TLR1 , 2,3,4,6 mRNA was higher in healthy cornea Real - time PCR . TLR7 , 8 , 9 were lower , TLR5 and 10 were intermediate .
In addition to TLR7 , TLRs showed a decrease , especially TLR5 , 6 , 3 . The expression of TLR9 in the cornea of active HSV - infected cornea was slightly stronger than that of non - active HSV - infected cornea and healthy cornea .

Conclusion : TLRs may play an important role in the immunity of cornea and corneal infection . TLR7 may play an important role in corneal transplantation , while TLR7 may be related to the activity of HSV , and further study of TLRs may clarify the mechanism of immune control of corneal HSV infection .
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R772.2;R392

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