【作者】 郎莉莉；

【导师】 柳林；

【作者基本信息】 第二军医大学 ， 眼科学， 2010， 博士

【摘要】 细菌性角膜炎(bacterial keratitis)是指由细菌感染引起的角膜化脓性炎症,至今仍是世界性的常见致盲眼病。在许多国家和地区,铜绿假单胞菌是首位的细菌性角膜炎致病菌,铜绿假单胞菌属于革兰氏阴性的条件致病菌,该菌可以产生多种致病因子,脂多糖(lipopolysaccharide, LPS)是其最主要的致病因子,LPS通过诱导角膜细胞产生TNF-α、IL-1、IL-6、IL-8和IL-12等,发挥关键的致炎作用。哺乳动物细胞对LPS刺激产生应答,需要通过一系列蛋白辅助,其中产生应答的起始为LPS受体复合物。目前认为LPS受体复合物至少包括四个组成部分：LPS结合蛋白(LPS binding protein, LBP)、CD14、髓样分化蛋白2(myeloid differentiation-2, MD-2)以及Toll样受体4(Toll-like receptor 4, TLR4). MD-2分子的发现是近些年来LPS受体复合物研究方面最重要的进展之一,MD-2关联LPS与TLR4,为LPS/TLR4信号途径提供必需的桥接。然而,人角膜上皮细胞对LPS刺激是否发生应答,可能与人角膜上皮细胞MD-2的表达与否,以及TLR4的分布位置有关。研究目的：本课题以人角膜上皮细胞SDHCEC1(spontaneously derived human corneal epithelial cell linel, SDHCEC1)作为实验对象,体外探讨人角膜上皮细胞对LPS的刺激应答机制。研究方法：传代培养来源于中山大学眼科中心王智崇教授团队连续培养成功的人角膜上皮细胞SDHCEC1,通过光学显微镜观察细胞形态,免疫组化法鉴定角膜上皮细胞的特异蛋白CK3/CK12的表达。不同浓度的LPS刺激SDHCEC1细胞不同时间后,Real-time PCR检测TLR4、CD14、IL-8和MD-2 mRNA; ELISA检测细胞培养基IL-8; Western Blot检测IκB-α和p-IκB-α的活化；流式细胞术检测SDHCEC1细胞膜表面和细胞内TLR4、CD14的表达。构建MD-2克隆质粒并鉴定,构建重组MD-2表达质粒并鉴定。重组MD-2表达质粒转染293T细胞,Western Blot检测重组MD-2表达。将293T细胞分泌的重组MD-2蛋白加入SDHCEC1细胞培养基中,Real-time PCR检测TLR4、CD14和IL-8；流式细胞术检测TLR4、CD14; ELISA检测细胞培养基IL-8; Western Blot检测IκB-α和p-IκB-α。结果：光学显微镜下,SDHCEC1细胞呈典型角膜上皮细胞形态；SDHCEC1细胞内CK3/CK12阳性表达。不同浓度的LPS刺激SDHCEC1细胞不同时间长度后,Real-time PCR结果显示TLR4、CD 14弱阳性表达,统计学无显著差异,IL-8和MD-2则未见表达；ELISA检测培养基无IL-8的分泌；Western Blot检测未见活化的p-IκB-α;流式细胞术发现SDHCEC1细胞膜表面未见TLR4、CD 14表达,而细胞质中TLR4、CD 14弱阳性表达。MD-2克隆质粒和重组MD-2表达质粒构建成功。MD-2表达质粒转染293T细胞后,Western Blot检测重组MD-2蛋白表达。与正常细胞相比较,添加重组MD-2蛋白培养的SDHCEC1细胞对LPS产生应答,表现为：Real-time PCR检测TLR4、CD 14和IL-8 mRNA表达上调；流式细胞术检测TLR4、CD 14在细胞表面和细胞内均阳性表达；ELISA检测细胞培养基IL-8含量增高；Western Blot检测到NF-κB的活化。结论：人角膜上皮细胞SDHCEC1 MD-2表达缺失,细胞膜表面缺乏功能性的TLR4表达,LPS/TLR4/MD-2信号途径中,NF-κB不活化,细胞未见分泌促炎细胞因子IL-8,这可能是在正常情况下,人角膜上皮细胞通过调控MD-2与细胞膜表面的TLR4表达,抑制对LPS刺激的先天免疫反应,避免炎症细胞在角膜中聚集和角膜结构被炎症破坏,维持角膜局部的免疫自稳状态。但当出现外源性或内源性MD-2的时候,角膜上皮细胞可能在MD-2的诱导下,TLR4重新分布在细胞膜表面,并且募集下游分子,NF-κB活化,分泌促炎细胞因子,激活LPS/TLR4信号途径,参与炎症的发生。更多还原

【Abstract】 Bacterial keratitis is a kind of suppurative keratitis caused by bacterial infection and can often lead to blindness. In many countries and areas,pseudomonas aeruginosa is the main pathogen which belongs to gram-negative opportunistic bacteria group.This strain can generate many pathogenic factors,among which, lipopolysaccharide(LPS)is the most important one.LPS plays a critical role in inflammation by producing TNF-α, IL-1, IL-6, IL-8 and IL-12 from corneal epithelial cells.The mammal’s cells need some accessory proteins to response to LPS. It is currently known that LPS receptor complex is composed of 4 parts at least, which are LPS binding protein(LBP), CD 14, myeloid differentiation-2(MD-2)and toll-like receptor 4(TLR4). MD-2 is one of the most important progresses in this field until now and it acts as a necessary bridge in the signaling pathway associated with LPS and TLR4.But now it is still unclear whether the human corneal epithelial cells can response to LPS, if yes, are expression of MD-2 and location of TLR4 involved in the signaling pathway?Objects:To study the LPS responding mechanisms of human corneal epithelial cells in vitro.Methods:Spontaneously derived human corneal epithelial cell linel(SDHCEC1), donated by professor Wang Zhichong from Zhongshan Ophthalmic Center, were subcultured and specific protein CK3/CK12 were identified by immunohistochemistry. TLR4, CD 14, IL-8 and MD-2 expression in SDHCEC1 were analyzed by real-time PCR after incubating with different concentrations of LPS at different times. IL-8 in the supernatant was tested by ELISA. The activation of NF-κB was analyzed by western blot. The expression of TLR4 and CD 14 were tested by flow cytometry. MD-2 recombinant cloning and expression plasmids were constructed and identified. 293T cells were transfected with MD-2 recombinant expression plasmids.Western blot was used to test the MD-2 expression. SDHCEC1 was co-cultured with supernatant secreted by 293T cells transfected with MD-2 recombinant plasmids. TLR4, CD 14, IL-8, and NF-κB in SDHCEC1 were tested as the previous. Results:SDHCECl showed typical characters of corneal epithelial cells with the positive staining of CK3/CK12. SDHCEC1 cultured with different concentrations of LPS at different times only came up with weak expression of TLR4 and CD 14 (P＜0.05). IL-8 and MD-2 expression were negative. No IL-8 secretion was seen in the supernatant. Activation of NF-κB did not show up. TLR4 and CD 14 were not expressed at the cell surface with only weak expression within cytoplasm. MD-2 cloning plasmids and recombinant plasmids were successfully constructed and expressed in 293T cells. TLR4, CD 14 and IL-8 mRNA were up-regulated in the SDHCEC1 cells co-cultured with supernatant secreted by 293T cells transfected with MD-2 recombinant plasmids. TLR4 and CD 14 were also positive at the cell surface and within cell cytoplasm. IL-8 concentration was also up-regulated with the activation of NF-κB at the same time.Conclusions:MD-2 expression is absent in SDHCEC1, which may lead to the deficiency of TLR4 at the cell surface, inactivation of NF-κB and no secretion of IL-8 in LPS/TLR4/MD-2 signaling pathway. According to this, we speculate that under normal conditions, in order to maintain corneal immunosilence, corneal epithelial cells may use this way to inhibit the innate immune response induced by LPS and avoid corneal epithelial cells’destruction caused by invasive inflammatory cells. But with endogenous or exogenous MD-2, TLR4 may express at the cell surface with the activation of NF-κB and secretion of IL-8, which activates the whole signaling pathway and plays an important role in sequent inflammation.更多还原