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S100A蛋白在角膜新生血管中的实验研究

发布时间:2018-08-16 14:04
【摘要】:目的S100A蛋白家族主要参与多种炎症的病理过程,其中的部分成员还可能参与肿瘤、新生血管等病理过程的发生、发展。本课题旨在研究S100A8/9蛋白是否参与炎症相关性角膜新生血管(CorNV)的发病过程,并进一步通过诱导形成新生血管的体外及体内模型,探讨S100A8/9蛋白在其中的变化规律及作用机制。 方法我们用10-0尼龙缝线或化学烧伤的方法在Balb/c或C57B1/6小鼠角膜诱导两种CorNV模型,进行基因表达谱的芯片分析。在缝线术后不同的时间点,取材做病理切片来检测角膜基质部炎性细胞的浸润情况。应用实时定量PCR(RT-QPCR)的方法对部分有代表性的基因进行验证性的测定,包括S100A家族中(S100A4、S100A6、S100A8、S100A9和S100A13),促炎症细胞因子(IL-1β、IL-6、转化生长因子β1和MIP-2),以及促血管生成因子(成纤维细胞生长因子和血管内皮生长因子)。用免疫荧光的方法检测长有新生血管的角膜中中性粒细胞或巨噬细胞浸润以及S100A8或S100A9蛋白的表达。在小鼠体内实施抗体介导的中性粒细胞或S100A8中和的实验能够有效证明中性粒细胞和S100A蛋白在缝线诱导角膜新生血管模型(S-CorNV)中发挥的作用。体外观察S100A8/9蛋白对人脐静脉内皮细胞(HUVEC细胞)增殖、成管、迁移能力的作用。体内采用Matrigel皮下注射的方法,通过对血红蛋白定量和HE组织切片的比较,从而验证S100A8/9蛋白对新生血管的促生长变化。 结果基因芯片分析显示,与正常小鼠相比,两种CorNV模型中的S100A4、S100A6、S100A8、S100A9和S100A13基因都有所上调,其中,S100A8和S100A9的变化最为显著。RT-QPCR检测结果表明在S-CorNV模型角膜中S100A和细胞因子表达的变化具有时间依赖性,在术后第5天达到顶点。免疫荧光显示角膜中的中性粒细胞和巨噬细胞能够分泌S100A8和S100A9蛋白。在诱导S-CorNV模型前一天进行中性粒细胞清除可以减轻发病的严重程度,并且减少了角膜新生血管中S100A8/9蛋白的产生,同时S100A基因和促炎症或促血管生成的基因上调的程度也有所降低。另外结膜下注射S100A8抗体也显着抑制S-CorNV模型中血管的生长和炎症的发展。在MTT实验中,与对照组相比S100A8/9蛋白在10ug/ml的浓度下可促进HUVEC细胞的增殖、迁移和成管。与单独应用Matrigel对照组比较,皮下注射含有S100A8/9蛋白的Matrigel中的新生血管生长明显,其中的血红蛋白含量高,局部浸润的炎性细胞较多,从而证明S100A8/9蛋白在体内能够促进新生血管的形成。 结论S100A蛋白参与了炎症性角膜新生血管的发病过程,尤其是S100A8或S100A9蛋白有可能作为有效控制该疾病病理过程的靶蛋白。同时,体内体外的实验也证明S100A8/9蛋白参与新生血管的形成过程,可能是通过直接作用于血管内皮细胞而实现的。
[Abstract]:Objective the S100A protein family is mainly involved in the pathological process of many kinds of inflammation, and some of its members may also be involved in the occurrence and development of tumor and neovascularization. The purpose of this study was to investigate whether S100A8/9 protein was involved in the pathogenesis of inflammatory related corneal neovascularization (CorNV), and to explore the mechanism of S100A8/9 protein in the process by inducing the formation of in vitro and in vivo models of neovascularization. Methods two kinds of CorNV models were induced by 10-0 nylon suture or chemical burn in the cornea of Balb/c or C57B1/6 mice. The microarray analysis of gene expression profile was performed. At different time points after suture, pathological sections were taken to detect the infiltration of inflammatory cells in corneal stroma. A real-time quantitative PCR (RT-QPCR) method was used to test the confirmatory properties of some representative genes. These include S100A family (S100A4, S100A6, S100A8, S100A9 and S100A13), pro-inflammatory cytokines (IL-1 尾 -IL-6, transforming growth factor 尾 1 and MIP-2), and angiogenic factors (fibroblast growth factor and vascular endothelial growth factor). The infiltration of neutrophil or macrophage and the expression of S100A8 or S100A9 protein were detected by immunofluorescence. Antibody-mediated neutrophils or S100A8 neutralization in mice can effectively demonstrate the role of neutrophils and S100A proteins in sewn induced corneal neovascularization (S-CorNV). To observe the effect of S100A8/9 protein on proliferation, tube formation and migration of human umbilical vein endothelial cells (HUVEC cells) in vitro. By subcutaneous injection of Matrigel, hemoglobin quantitative and HE tissue sections were compared to verify the effect of S100A8/9 protein on the growth of neovascularization. Results Gene chip analysis showed that S100A4, S100A6, S100A8, S100A9 and S100A13 genes were up-regulated in both CorNV models compared with normal mice. The changes of S100A8 and S100A9 were most significant. RT-QPCR results showed that the expression of S100A and cytokines in the cornea of S-CorNV model was time-dependent and reached its peak on the 5th day after operation. Immunofluorescence showed that neutrophils and macrophages in the cornea secreted S100A8 and S100A9 proteins. Neutrophil clearance on the day before induction of S-CorNV model reduced the severity of the disease and reduced the production of S100A8/9 protein in corneal neovascularization. At the same time, the S100A gene and pro-inflammatory or angiogenic genes were also reduced. In addition subconjunctival injection of S100A8 antibody also significantly inhibited the growth of blood vessels and inflammation in S-CorNV model. In MTT experiment, compared with the control group, S100A8/9 protein could promote the proliferation, migration and tube formation of HUVEC cells at the concentration of 10ug/ml. Compared with the control group treated with Matrigel alone, the neovascularization in Matrigel with subcutaneous injection of S100A8/9 protein was obvious, in which the hemoglobin content was high and the inflammatory cells infiltrated locally were more. It is proved that S100A8/9 protein can promote the formation of neovascularization in vivo. Conclusion S100A protein is involved in the pathogenesis of inflammatory corneal neovascularization, especially S100A8 or S100A9 protein may be the target protein to control the pathological process of the disease. At the same time, in vivo and in vitro experiments also proved that S100A8/9 protein involved in the process of angiogenesis, which may be realized by direct action on vascular endothelial cells.
【学位授予单位】:青岛大学
【学位级别】:博士
【学位授予年份】:2011
【分类号】:R77

【参考文献】

相关期刊论文 前2条

1 王泳;柳林;;角膜新生血管的调控[J];中国临床康复;2005年42期

2 邱培瑾 ,姚 克 ,裘世杰 ,朱丽君 ,周彩云;大鼠角膜碱烧伤后碱性成纤维细胞生长因子在角膜中的表达及意义[J];眼科研究;2002年02期



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