S100A蛋白在角膜新生血管中的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-16 14:04
【摘要】：目的S100A蛋白家族主要參與多種炎癥的病理過(guò)程,其中的部分成員還可能參與腫瘤、新生血管等病理過(guò)程的發(fā)生、發(fā)展。本課題旨在研究S100A8/9蛋白是否參與炎癥相關(guān)性角膜新生血管(CorNV)的發(fā)病過(guò)程,并進(jìn)一步通過(guò)誘導(dǎo)形成新生血管的體外及體內(nèi)模型,探討S100A8/9蛋白在其中的變化規(guī)律及作用機(jī)制。 方法我們用10-0尼龍縫線或化學(xué)燒傷的方法在Balb/c或C57B1/6小鼠角膜誘導(dǎo)兩種CorNV模型,進(jìn)行基因表達(dá)譜的芯片分析。在縫線術(shù)后不同的時(shí)間點(diǎn),取材做病理切片來(lái)檢測(cè)角膜基質(zhì)部炎性細(xì)胞的浸潤(rùn)情況。應(yīng)用實(shí)時(shí)定量PCR(RT-QPCR)的方法對(duì)部分有代表性的基因進(jìn)行驗(yàn)證性的測(cè)定,包括S100A家族中(S100A4、S100A6、S100A8、S100A9和S100A13),促炎癥細(xì)胞因子(IL-1β、IL-6、轉(zhuǎn)化生長(zhǎng)因子β1和MIP-2),以及促血管生成因子(成纖維細(xì)胞生長(zhǎng)因子和血管內(nèi)皮生長(zhǎng)因子)。用免疫熒光的方法檢測(cè)長(zhǎng)有新生血管的角膜中中性粒細(xì)胞或巨噬細(xì)胞浸潤(rùn)以及S100A8或S100A9蛋白的表達(dá)。在小鼠體內(nèi)實(shí)施抗體介導(dǎo)的中性粒細(xì)胞或S100A8中和的實(shí)驗(yàn)?zāi)軌蛴行ёC明中性粒細(xì)胞和S100A蛋白在縫線誘導(dǎo)角膜新生血管模型(S-CorNV)中發(fā)揮的作用。體外觀察S100A8/9蛋白對(duì)人臍靜脈內(nèi)皮細(xì)胞(HUVEC細(xì)胞)增殖、成管、遷移能力的作用。體內(nèi)采用Matrigel皮下注射的方法,通過(guò)對(duì)血紅蛋白定量和HE組織切片的比較,從而驗(yàn)證S100A8/9蛋白對(duì)新生血管的促生長(zhǎng)變化。 結(jié)果基因芯片分析顯示,與正常小鼠相比,兩種CorNV模型中的S100A4、S100A6、S100A8、S100A9和S100A13基因都有所上調(diào),其中,S100A8和S100A9的變化最為顯著。RT-QPCR檢測(cè)結(jié)果表明在S-CorNV模型角膜中S100A和細(xì)胞因子表達(dá)的變化具有時(shí)間依賴性,在術(shù)后第5天達(dá)到頂點(diǎn)。免疫熒光顯示角膜中的中性粒細(xì)胞和巨噬細(xì)胞能夠分泌S100A8和S100A9蛋白。在誘導(dǎo)S-CorNV模型前一天進(jìn)行中性粒細(xì)胞清除可以減輕發(fā)病的嚴(yán)重程度,并且減少了角膜新生血管中S100A8/9蛋白的產(chǎn)生,同時(shí)S100A基因和促炎癥或促血管生成的基因上調(diào)的程度也有所降低。另外結(jié)膜下注射S100A8抗體也顯著抑制S-CorNV模型中血管的生長(zhǎng)和炎癥的發(fā)展。在MTT實(shí)驗(yàn)中,與對(duì)照組相比S100A8/9蛋白在10ug/ml的濃度下可促進(jìn)HUVEC細(xì)胞的增殖、遷移和成管。與單獨(dú)應(yīng)用Matrigel對(duì)照組比較,皮下注射含有S100A8/9蛋白的Matrigel中的新生血管生長(zhǎng)明顯,其中的血紅蛋白含量高,局部浸潤(rùn)的炎性細(xì)胞較多,從而證明S100A8/9蛋白在體內(nèi)能夠促進(jìn)新生血管的形成。 結(jié)論S100A蛋白參與了炎癥性角膜新生血管的發(fā)病過(guò)程,尤其是S100A8或S100A9蛋白有可能作為有效控制該疾病病理過(guò)程的靶蛋白。同時(shí),體內(nèi)體外的實(shí)驗(yàn)也證明S100A8/9蛋白參與新生血管的形成過(guò)程,可能是通過(guò)直接作用于血管內(nèi)皮細(xì)胞而實(shí)現(xiàn)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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R77
本文編號(hào)：2186203
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R77
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 王泳;柳林;;角膜新生血管的調(diào)控[J];中國(guó)臨床康復(fù);2005年42期
2 邱培瑾 ,姚 克 ,裘世杰 ,朱麗君 ,周彩云;大鼠角膜堿燒傷后堿性成纖維細(xì)胞生長(zhǎng)因子在角膜中的表達(dá)及意義[J];眼科研究;2002年02期
，本文編號(hào)：2186203
本文鏈接：http://sikaile.net/yixuelunwen/yank/2186203.html
最近更新
教材專著
