Toll样受体介导的TSLP在过敏性结膜炎中的作用和调控机制研究

发布时间：2018-09-04 11:02

【摘要】：目的：探讨Toll样受体(TLR)介导的胸腺基质淋巴细胞生成素(TSLP)及其下游信号分子OX40L/OX40在过敏性结膜炎中的作用机制。方法：本研究建立了三种不同类型的过敏模型,据此,实验分以下三部分进行。1.花粉诱导的小鼠的过敏性结膜炎模型：取野生型BALB/C小鼠,TLR4基因缺陷鼠；基于C57BL/6的Myd88+/+和Myd88-/-小鼠；分别在建模第一天给予花粉的全身致敏,在第10-15天进行花粉的局部眼表直接刺激。应用qPCR,免疫组织化学染色,免疫荧光检测小鼠角膜上皮,结膜上皮,颈部致敏淋巴结中TSLP,其下游相关因子及Th2细胞因子的表达。2.花粉诱导的小鼠局部眼表TSLP诱发模型：取野生型BALB/C小鼠,TLR4基因缺陷鼠；基于C57BL/6的Myd88+/+和Myd88-/-小鼠；直接给予花粉进行眼表的局部刺激4-24小时。应用qPCR和ELISA检测小鼠的角膜和结膜上皮中TSLP的表达变化。3.花粉诱导的人角膜上皮细胞TSLP诱发模型：体外培养人的角膜上皮细胞,分别给予不同浓度的花粉刺激,以及TLR4抗体,NF-K b-I的预处理,应用qPCR和ELISA检测TSLP的表达变化。结果：1.花粉诱导的小鼠的过敏性结膜炎模型： ①在BALB/C小鼠的过敏性结膜炎模型中,qPCR显示：与PBS对照组相比,模型鼠角膜上皮,结膜上皮和颈部致敏淋巴结中TSLP, TSLP下游通路因子(TSLPR,OX40L,OX40, CD11C, CD4)以及Th2细胞因子(IL-4. IL-5, IL-13)表达增加,而Thl细胞因子IFNγ表达量则基本不变。免疫组织化学染色在蛋白水平证实了这一变化。 ②在BALB/C小鼠和TLR4基因缺陷鼠的过敏模型中,qPCR显示：与BALB/C小鼠过敏模型对照组相比,TLR4基因缺陷模型鼠角膜上皮,结膜上皮和颈部致敏淋巴结中TSLP, TSLP下游通路因子以及Th2细胞因子表达量降低。对模型鼠眼球的免疫组织化学染色和淋巴结的免疫荧光染色在蛋白水平证实了这一变化。 ③在Myd88+/+小鼠的过敏性结膜炎模型中,qPCR显示：与Myd88++PBS对照组相比,模型鼠角膜上皮,结膜上皮和颈部致敏淋巴结中TSLP, TSLP下游通路因子以及Th2细胞因子表达增加。与Myd88-/-过敏模型相比,这些细胞因子的表达也明显高于Myd88-/-小鼠的过敏性模型组。对模型鼠眼球的免疫组织化学染色和淋巴结的免疫荧光染色在蛋白水平证实了这些变化。 2.花粉诱导的小鼠局部眼表TSLP诱发模型： 1)与PBS对照组相比,BALB/C小鼠眼表接受花粉直接刺激后,其角膜上皮和结膜上皮中TSLP表达量增加。给予TLR4抗体的预处理可以抑制花粉对TSLP的刺激作用。TSLP表达量的ELISA检测在蛋白水平证实了这些变化。 2)与BALB/C小鼠花粉刺激组相比,TLR4基因缺陷刺激组角膜上皮,结膜上皮中TSLP表达量降低。与Myd88+/+小鼠花粉刺激组相比,Myd88-/-花粉刺激组角膜上皮,结膜上皮中TSLP表达量也同样是降低的。TSLP表达量的ELISA检测在蛋白水平证实了这些变化。 3.花粉诱导的人角膜上皮细胞TSLP诱发模型：在体外培养的人角膜上皮细胞中,花粉可以诱导上皮细胞中TSLP表达量增加,并呈现出浓度依赖的变化趋势。给予TLR4抗体或者NF-K b-I可以抑制花粉对TSLP的刺激作用。TSLP表达量的ELISA检测在蛋白水平证实了这些变化。结论：1.TSLP及其下游通路因子在过敏性结膜炎的发病机制中起重要作用。 2.在花粉诱导的过敏性结膜炎中,外界致敏原可以通过TLR4/Myd88这一信号通路,诱导上皮细胞中TSLP及其相关信号分子的表达,进而诱发Th2炎症反应。 3.人为阻断TLR4, Myd88等关键位点,可以抑制花粉诱导的过敏反应,降低TSLP及相关Th2细胞因子的表达,从而可能成为临床上新的治疗靶点。
[Abstract]:AIM: To investigate the role of Toll-like receptor (TLR)-mediated thymic stromal lymphopoietin (TSLP) and its downstream signal molecule OX40L/OX40 in allergic conjunctivitis.
Methods: Three different types of allergic models were established in this study. The experiment was divided into three parts. 1. Pollen-induced conjunctivitis in mice: wild-type BALB/C mice, TLR4 gene-deficient mice, Myd88+/+ and Myd88-/-mice based on C 57BL/6, and whole-body sensitization with pollen on the first day of the model, respectively. Pollen-induced TSLP expression in corneal epithelium, conjunctival epithelium and cervical sensitized lymph nodes was detected by qPCR, immunohistochemical staining and immunofluorescence. 2. Pollen-induced TSLP induction model of local ocular surface in mice: wild-type BALB/C mice, TLR4 Gene-deficient mice; Myd88+/+and Myd88-/-mice based on C57BL/6; local eye surface stimulation with pollen was performed for 4-24 hours directly. The expression of TSLP in corneal and conjunctival epithelium of mice was detected by qPCR and ELISA. 3. Pollen-induced TSLP induction model of human corneal epithelial cells: human corneal epithelial cells were cultured in vitro and given to the mice respectively. The expression of TSLP was detected by qPCR and ELISA after different concentrations of pollen stimulation and pretreatment with TLR4 antibody and NF-K b-I.
Results: 1. pollen induced allergic conjunctivitis models in mice:
(1) In BALB/C mice with allergic conjunctivitis, the expression of TSLP, TSLP downstream pathway factors (TSLPR, OX40L, OX40, CD11C, CD4) and Th2 cytokines (IL-4.IL-5, IL-13) in corneal epithelium, conjunctival epithelium and cervical sensitized lymph nodes were increased, while the expression of Thl cytokine IFN-gamma was increased, compared with PBS control group. The immunohistochemical staining confirmed this change at the protein level.
(2) In BALB/C mice and TLR4-deficient mice, the expression of TSLP, TSLP downstream pathway factors and Th2 cytokines in corneal epithelium, conjunctival epithelium and cervical sensitized lymph nodes of TLR4-deficient mice were decreased by qPCR compared with BALB/C mice. Immunofluorescence staining of chromatic and lymph nodes confirmed this change at protein level.
(3) The expression of TSLP, TSLP downstream pathway factors and Th2 cytokines in corneal epithelium, conjunctival epithelium and cervical sensitized lymph nodes in Myd88 + / + mice were increased by qPCR compared with Myd88 + + PBS control group. The expression of these cytokines was also significantly higher than that in Myd88 - / - allergic model. Immunohistochemical staining of the eyeballs and immunofluorescence staining of the lymph nodes of the model mice at protein level confirmed these changes.
2. pollen induced local ocular surface TSLP induced model in mice:
1) Compared with PBS control group, the expression of TSLP in corneal epithelium and conjunctival epithelium of BALB/C mice increased after direct pollen stimulation. Pretreatment with TLR4 antibody inhibited the stimulation of pollen to TSLP. These changes were confirmed at protein level by ELISA.
2) Compared with BALB/C mice, TSLP expression in corneal epithelium and conjunctival epithelium was decreased in TLR4 deficient stimulation group. Compared with Myd88+/+ mice, TSLP expression in corneal epithelium and conjunctival epithelium was also decreased in Myd88-/-pollen stimulation group. Change.
3. pollen induced TSLP model of human corneal epithelial cells:
In cultured human corneal epithelial cells, the expression of TSLP was induced by pollen in a concentration-dependent manner. TLR4 antibody or NF-K b-I inhibited the stimulation of pollen on TSLP. These changes were confirmed at protein level by ELISA.
Conclusion: 1.TSLP and its downstream pathway factors play an important role in the pathogenesis of allergic conjunctivitis.
2. In allergic conjunctivitis induced by pollen, the external allergens can induce the expression of TSLP and related signaling molecules in epithelial cells through the TLR4/Myd88 signaling pathway, and then induce Th2 inflammation.
3. Artificial blockade of TLR4, Myd88 and other key sites can inhibit pollen-induced allergic reactions, reduce the expression of TSLP and related Th2 cytokines, which may become a new therapeutic target in clinic.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R777.31

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