内皮抑素对小鼠脉络膜新生血管抑制作用的信号通路分析

Analysis of signaling pathway of inhibitory effect of endostatin on mouse choroidal neovascularization

目的探讨内皮抑素对C57BL/6J小鼠脉络膜新生血管抑制作用的基因表达及作用机制。方法应用532 nm固体激光机,制造C57BL/6J小鼠脉络膜新生血管模型,分为空白组、对照组和实验组。其中空白组不做任何处理,对照组玻璃体腔内注射生理盐水2μL;实验组玻璃体腔内注射内皮抑素2μL(0.01 mg)。将对照组和实验组小鼠组织选取4个样本进行杂交及基因芯片检测。比较实验组和对照组基因表达的差异,选取其中表达差异大于1.5倍且P≤0.05的基因。结果免疫组织化学染色可见实验组新生血管表达明显低于对照组。实验组与对照组相比上调的基因有116个,下调的基因有106个。通过KEGG(Kyoto Encyclopedia of Genes and Genomes)数据库对上述差异基因经行分析后发现,实验组与对照组相比下调处于前10位的通道有:细胞骨架激动蛋白的校准、白细胞穿越内皮细胞层、金黄色葡萄球菌感染、Fc epsilon RI信号通路、Fc gamma R信号调理吞噬作用、Toll样受体信号通路、2型糖尿病、子宫内膜癌、吞噬体、非小细胞肺癌;处于上调前几位的通道有:细胞外基质受体相互作用、叶酸的一碳单位、肥厚性心肌病、扩张型心肌病、组氨酸新陈代谢、黏着斑、心肌细胞收缩。结论由通道功能分析可以推测,内皮抑素可能通过抑制内皮细胞的活性及移行并协同抑制免疫系统,进而抑制新生血管的生成。

Objective To study the gene expression and mechanism of the inhibitory effect of endostatin on choroidal neovascularization of C57BL /6J mice. Methods The 532 nm diode laser machine was performed to create choroidal neovascularization（ CNV） model of C57BL /6J mice. The model mice were randomly divided into bland group,control group and experiment group,with ten mice in each group. The blank group received no treatment; the control group was injected saline 2 μL in vitreous body; the experiment group was injected endostatin 2 μL（ 0. 01 mg） in vitreous body. Four samples were selected respectively from mice of control group and experiment group,then the samples were hybridized and detected by gene chips. The differences of genes expression of the experiment group and control group were compared,then the genes which expressive differences were ≥1. 5 times and P≤0. 05 were chosen. Results The immunohistochemistry result showed that the expression of choroidal neovascularization in the experiment group was significantly lower than that in control group. The 116 genes were up-regulated and 106 genes were down-regulated in the experiment group compared with control group. The different genes were analyzed by Kyoto Encyclopedia of Genes and Genomes（ KEGG） database. The result showed that the down-regulated pathways in the top ten were regulation of actin cytoskeleton,leukocyte transendothelial migration,staphylococcus aureus infection,Fc epsilon RI signaling pathway,Fc gamma R-mediated phagocytosis,Toll-like receptor signaling pathway,type Ⅱ diabetes mellitus, endometrial cancer,phagosome,non-small cell lung cancer; the up-regulated pathways in the first were extracellular matrix（ ECM）-receptor interaction,one carbon pool by folate,hypertrophic cardiomyopathy（ HCM）,dilated cardiomyopathy,histidine metabolism,focal adhesion,cardiac muscle contraction. Conclusion The endostatin can suppress the growth of choroidal neovascularization by suppress the endothelial cell activity and migration and suppress the immune system.