[1]吕洋,王雨生,窦国睿,等.整合素αvβ3在SDF-1/CXCR4介导的脉络膜新生血管中的作用[J].眼科新进展,2018,38(5):401-406.[doi:10.13389/j.cnki.rao.2018.0094]
　LV Yang,WANG Yu-Sheng,DOU Guo-Rui,et al.Roles of integrin αvβ3 in SDF-l/CXCR4-induced choroidal neovascularization[J].Recent Advances in Ophthalmology,2018,38(5):401-406.[doi:10.13389/j.cnki.rao.2018.0094]
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整合素αvβ3在SDF-1/CXCR4介导的脉络膜新生血管中的作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
38卷
期数:
2018年5期
页码:
401-406
栏目:
实验研究
出版日期:
2018-05-05

文章信息/Info

Title:
Roles of integrin αvβ3 in SDF-l/CXCR4-induced choroidal neovascularization
作者:
吕洋; 王雨生; 窦国睿; 李曼红; 常天芳; 孙嘉星; 徐文芹; 胡至察; 程子芳
710032　陕西省西安市，第四军医大学西京医院眼科、全军眼科研究所
Author(s):
LV Yang; WANG Yu-Sheng; DOU Guo-Rui; LI Man-Hong; CHANG Tian-Fang; SUN Jia-Xing; XU Wen-Qin; HU Zhi-Cha; CHENG Zi-Fang
Department of Ophthalmology，Eye Institute of Chinese PLA，Xijing Hospital，the Fourth Military Medical University，Xi’an 710032，Shaanxi Province，China
关键词:
脉络膜新生血管; 内皮细胞; SDF-1/CXCR4; 整合素αvβ3
Keywords:
choroidal neovascularization; endothelial cells; SDF-1/CXCR4; integrin αvβ3
分类号:
R773.4
DOI:
10.13389/j.cnki.rao.2018.0094
文献标志码:
A
摘要:
目的　探讨内皮细胞表达的整合素αvβ3在基质细胞衍生因子-1（stromal derived factor-1，SDF-1）/趋势化因子受体4（chemokine receptor 4，CXCR4）调控脉络膜新生血管（choroidal neovascularization，CNV）生成过程中的作用。方法　（1）体内动物实验：采用激光诱导C57BL/6J小鼠CNV模型，实验分为4组：单纯CNV组（对照组）、CNV后立即玻璃体内注射SDF-1组（SDF-1组）、CNV后立即玻璃体内注射SDF-1+CXCR4抑制剂AMD3100组（SDF-1+AMD3100组）和CNV后立即玻璃体内注射SDF-1+αvβ3抑制剂SB273005组（SDF-1+SB273005组）。实时定量PCR观察小鼠激光诱发CNV后1 d、3 d、5 d、7 d、10 d、14 d CXCR4和αvβ3的表达；免疫荧光染色观察4组处理后CNV局部及内皮细胞上αvβ3的表达；OCT观察4组处理后CNV中视网膜色素上皮（retinal pigment epithelial，RPE）层隆起高度。（2）体外细胞实验：Western blot检测正常对照组、Si-CXCR4敲减组和Si-NC模拟敲减组RF/6A细胞中CXCR4蛋白的表达，同时检测对照组、SDF-1组、SDF-1+AMD3100组、SDF-1+Si-NC组和SDF-1+Si-CXCR4组中整合素αvβ3蛋白的表达。Transwell检测正常对照组、SDF-1组、SDF-1+AMD3100组和SDF-1+SB273005组的细胞迁移能力。结果　（1）体内动物实验：实时定量PCR显示随着CNV诱导后时间的增加，CXCR4 mRNA和整合素亚基β3 mRNA表达开始增加，CXCR4 mRNA在CNV后3 d表达量最高（4.263±0.464），β3 mRNA在CNV后7 d表达量最高（3.678±0.364），随后开始下降。免疫荧光结果显示：SDF-1组β3荧光强度显著增加，β3/CD31比率也明显增加，显著高于CNV对照组（P<0.01）；而SDF-1+AMD3100组和SDF-1+SB273005组β3荧光强度和β3/CD31比率显著减弱（P<0.05）。OCT显示SDF-1组RPE层的隆起明显升高（135.503±10.301）μm，显著高于CNV对照组（94.443±12.156）μm（P<0.05）；而SDF-1+AMD3100组和SDF-1+SB273005组RPE隆起的高度显著降低（95.283±20.062）μm和（99.807±10.403）μm（P<0.05）。（2）体外细胞实验：SDF-1组和SDF-1+Si-NC组整合素亚基β3蛋白的表达均升高（1.301±0.043）、（1.273±0.077），显著高于对照组（0.244±0.069）（P<0.01）；SDF-1+Si-CXCR4组和SDF-1+AMD3100组整合素亚基β3蛋白的水平显著下降（0.322±0.042）、（0.336±0.077）（P<0.01）。Transwell检测显示SDF-1组细胞迁移量增多显著高于对照组，而SDF-1+AMD3100组和SDF-1+SB273005组迁移细胞的数量明显减少。结论　整合素αvβ3在内皮细胞中通过介导SDF-1/CXCR4信号转导，促进CNV的发生发展。
Abstract:
Objective　To explore the role of integrin αvβ3 in the promotion of the development of choroidal neovascularization （CNV） by SDF-1/CXCR4.Methods　This study was divided into two parts in vitro and in vivo.As for the in vivo study，a CNV model was induced by laser on C57BL/6J mice，and then assigned into 4 groups：mice with solely CNV modeling as control group，with intravitreal injection of SDF-1 after immediate CNV modeling as SDF-1 group，with intravitreal injection of SDF-1+CXCR4 inhibitor （AMD3100） after CNV modeling as SDF-1+AMD3100 group，and mice with intravitreal injection of SDF-1+αvβ3 inhibitor （SB273005） after modeling as SDF-1+SB273005 group.CXCR4 and αvβ3 expression levels in laser-induced eyes were quantified by qRT-PCR at time points of day 1，3，5，7，10 and 14 after modeling，and immunofluorescence staining was applied to detect αvβ3 expression in regional CNV and its endothelial cells in the four groups.Finally，OCT was used to observe the height of retinal pigment epithelial （RPE） layers in CNV after treatment in the four groups.Moreover，in the experiment in vitro，Western blot was used to measure the expression of CXCR4 protein of RF/6A cells in normal control group，Si-CXCR4 knockdown group and Si-NC knockdown model group.Meanwhile，the expression of integrin subunit β3 protein was determined in the normal control group，SDF-1 group，SDF-1+AMD3100 group，SDF-1+Si-NC group and SDF-1+Si-CXCR4 group.Transwell assay was conducted to detect the migration ability of RF/6A cells in the normal control group，SDF-1 group，SDF-1+AMD3100 group，SDF-1+SB273005 group.Results　On the one hand，the study in vivo，qRT-PCR showed that the expression of CXCR4 and integrin subunit β3 mRNA was up-regulated at first，and then down-regulated with time passed after CNV induction，with the highest expression level of CXCR4 mRNA （4.263±0.464） on day 3，and the peak expression of β3 mRNA （3.678±0.364） on day 7 after CNV modeling.The results of immunofluorescence staining showed that the β3 fluorescence intensity of SDF-1 group was significantly enhanced，and the ratio of β3/CD31 was also significantly increased，which both were significantly higher than those of the control group （P<0.01）.However，the β3 fluorescence intensity and β3/CD31 ratio of SDF-1+AMD3100 group and SDF-1+SB273005 group were significantly weakened and decreased，respectively （P<0.05）.OCT showed that the elevation level of RPE layer in SDF-1 group was significantly higher than that in the control group ［（135.503±10.301） μm vs.（94.443±12.156）μm］ （P<0.05）.The height of RPE uplift in SDF-1+AMD3100 group ［（95.283±20.062）μm］ and SDF-1+SB273005 group ［（99.807±10.403）μm］ was significantly decreased （P<0.05）.On the other hand，in experiment in vitro，Western blot showed that the expression levels of integrin β3 in SDF-1 group and SDF-1+Si-NC group were significantly higher than those in the control group［（1.301±0.043） and （1.273±0.077） vs.（0.244±0.069）］ （P<0.01）.The levels of integrin subunit β3 protein in SDF-1+si-CXCR4 group （0.322±0.042） and SDF-1+AMD3100 group （0.336±0.077） were significantly down-regulated （P<0.01）.Transwell assay showed that the amount of migrating cells in SDF-1 group increased，which was significantly higher than that of the control group （P<0.01），while the number of migrating cells in SDF-1+AMD3100 group and SDF-1+SB273005 group was significantly decreased.Conclusion　Integrin αvβ3 can promote the development of CNV by mediating SDF-1/CXCR4 signaling in endothelial cells.

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备注/Memo

备注/Memo:
国家自然科学基金资助（编号：81770936、81570856、81670863、81500748）
更新日期/Last Update: 2018-05-09