[1]郑嘉琳,贾育蓉,郭星艺,等.肿瘤抑素Tum5对人脐静脉血管内皮细胞血管生成活性以及碱烧伤诱导大鼠角膜新生血管的抑制作用[J].眼科新进展,2018,38(5):434-439.[doi:10.13389/j.cnki.rao.2018.0101]
　ZHENG Jia-Lin,JIA Yu-Rong,GUO Xing-Yi,et al.Inhibitory effect of Tum5 on angiogenic activity of human umbilical vein endothelial cells and corneal neovascularization induce by alkali burn in rats[J].Recent Advances in Ophthalmology,2018,38(5):434-439.[doi:10.13389/j.cnki.rao.2018.0101]
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肿瘤抑素Tum5对人脐静脉血管内皮细胞血管生成活性以及碱烧伤诱导大鼠角膜新生血管的抑制作用/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:
434-439
栏目:
实验研究
出版日期:
2018-05-05

文章信息/Info

Title:
Inhibitory effect of Tum5 on angiogenic activity of human umbilical vein endothelial cells and corneal neovascularization induce by alkali burn in rats
作者:
郑嘉琳; 贾育蓉; 郭星艺; 张琰; 漆晨; 孙靖; 张红
300384　天津市，天津医科大学眼科医院 天津医科大学眼科研究所 天津医科大学眼视光学院
Author(s):
ZHENG Jia-Lin; JIA Yu-Rong; GUO Xing-Yi; ZHANG Yan; QI Chen; SUN Jing; ZHANG Hong
Tianjin Medical University Eye Hospital，Tianjin Medical University Eye Institute，School of Optometry and Ophthalmology of Tianjin Medical University，Tianjin 300384，China
关键词:
血管内皮生长因子; 抗血管生成活性; 角膜新生血管; 肿瘤抑素; 转染
Keywords:
vascular endothelial growth factor; anti-angiogenic activity; corneal neovascularization; tumstatin; transfection
分类号:
R772
DOI:
10.13389/j.cnki.rao.2018.0101
文献标志码:
A
摘要:
目的　研究肿瘤抑素Tum5对人脐静脉血管内皮细胞血管生成活性以及碱烧伤诱导的大鼠角膜新生血管的抑制作用。方法　取对数生长期人脐静脉血管内皮细胞分为4组：正常对照组：无任何处理的人脐静脉血管内皮细胞；rAd-GFP组：人脐静脉血管内皮细胞经rAd-GFP感染并培养；rAd-Tum5组：人脐静脉血管内皮细胞经rAd-Tum5感染并培养；rAd-Tum5+VEGF组：人脐静脉血管内皮细胞经rAd-Tum5感染后经外源性VEGF处理。通过CCK-8活性检测试剂盒、Transwell实验、Matrigel实验分别检测细胞增殖率、迁移细胞数及管腔形成情况。取雄性健康清洁级SD大鼠64只采用随机数字表法将大鼠分为：正常对照组、碱烧伤组、碱烧伤+rAd-GFP组、碱烧伤+rAd-Tum5组，每组16只，除正常对照组外其余各组建立角膜碱烧伤模型。其中正常对照组无任何处理，碱烧伤组、碱烧伤+rAd-GFP组、碱烧伤+rAd-Tum5组于烧伤后分别经结膜下注射等量无菌生理盐水、rAd-GFP、rAd-Tum5病毒，记录碱烧伤后第1天、第7天、第14天角膜新生血管相对面积及浸润炎性细胞数。结果　CCK-8实验结果显示rAd-Tum5组较正常对照组、rAd-GFP组细胞增殖率降低（均为P<0.01）；而rAd-Tum5+VEGF组细胞增殖率较rAd-Tum5组显著升高（P=0.004），rAd-Tum5+VEGF组与正常对照组和rAd-GFP组相比差异均无统计学意义（均为P>0.05）。Transwell实验结果显示rAd-Tum5组相对于正常对照组、rAd-GFP组每个视野细胞迁移数显著降低（均为P<0.01）；而rAd-Tum5+VEGF组相对于rAd-Tum5组显著升高（P=0.000），但其迁移能力尚未恢复到正常水平，与正常对照组和rAd-GFP组相比差异均有统计学意义（均为P<0.05）。Matrigel实验结果显示rAd-Tum5组相对于正常对照组、rAd-GFP组每个视野细胞成管数显著降低（均为P<0.01）；而rAd-Tum5+VEGF组相对于rAd-Tum5组显著升高（P=0.001）。大鼠角膜碱烧伤后第1-14天，各组角膜新生血管密度及数量逐渐增高，但是碱烧伤+rAd-Tum5组第7天、第14天角膜新生血管相对面积均显著低于碱烧伤组和碱烧伤+rAd-GFP组，提示Tum5能降低角膜新生血管生长速率并减少新生血管密度，抑制大鼠碱烧伤诱导的新生血管的形成。碱烧伤后第7天、第14天，正常对照组角膜完整、细胞排列有序，无炎性细胞浸润；碱烧伤后第7天，碱烧伤组、碱烧伤+rAd-GFP组上皮结构紊乱，角膜水肿，炎性细胞浸润明显，而碱烧伤+rAd-Tum5组每个视野炎性细胞浸润数较碱烧伤组、碱烧伤+rAd-GFP组明显减少，差异均有统计学意义（均为P<0.01）。碱烧伤后第14天，碱烧伤组、碱烧伤+rAd-GFP组和碱烧伤+rAd-Tum5组每个视野炎性细胞浸润数较第7天显著下降，同时碱烧伤+rAd-Tum5组角膜上皮结构整齐，水肿消退，每个视野炎性细胞浸润数显著低于碱烧伤组以及碱烧伤+rAd-GFP组，差异均有统计学意义（均为P<0.01）。结论　Tum5可能经VEGF通路抑制人脐静脉血管内皮细胞血管生成活性；Tum5可显著抑制碱烧伤诱导的角膜新生血管生成及炎性细胞浸润。
Abstract:
Objective To investigate the inhibitory effects of Tum5 on the angiogenesis of human umbilical vein endothelial cells(HUVECs)andalkali-induced corneal neovascularization.Methods HUVECs in logarithmic growth phase were divided into 4 groups,cells with untreated as normal control group,cells with the infection of rAd-GFP virus as rAd-GFP group,cells with the infection of rAd-Tum5 virus as rAd-Tum5 group,andcells with the infection of rAd-Tum5 virus followed by VEGF treatment as rAd-Tum5+VEGF group.Then cell proliferation,migration,andtube formation of HUVECs were examined by CCK-8,Transwell andMatrigel assays,respectively.64 healthy male SD rats were randomly divided into 4 groups(n=16)by using random number table,andthey were normal control group,alkali-burn group,alkali-burn+rAd-GFP group,alkali-burn+rAd-Tum5 group.The alkali-burn rat model was then established except normal control group,andthe normal control group received no treatment,whereas the alkali-burn,alkali-burn+rAd-GFP,alkali-burn+rAd-Tum5 groups received subconjunctival injection of equal volumes of sterilized saline,rAd-GFP virus,andrAd-Tum5 virus,respectively following the alkaline burn.The relative area of corneal neovascularization andthe number of infiltrating inflammatory cells were recorded on day 1,7and14 after injection.Results The CCK-8 assay showed that the proliferative rate of rAd-Tum5 group was lower than that of the normal control group andrAd-GFP group(both P<0.01),while rAd-Tum5+VEGF group exhibited a significantly greater cell proliferative capability than rAd-Tum5 group(P=0.004).There were no statistical differences between rAd-Tum5+VEGF group,normal control group andrAd-GFP group(all P>0.05).Transwell assay showed the significantly lower number of migrating cells in the rAd-Tum5 group than those in the normal control group andrAd-GFP group(both P<0.01).The number of migrating cells in rAd-Tum5+VEGF group was higher than those in rAd-Tum5 group(P=0.000);however,the migration capacity had not been restored to normal level,andrAd-Tum5+VEGF group had significant difference with normal control group andrAd-Tum5 group(both P<0.05).Matrigel assay showed that the number of meshes in rAd-Tum5 group was lower than that in the normal control group andrAd-GFP group(both P<0.01);while the number of meshes in rAd-Tum5+VEGF group was significantly increased compared with rAd-Tum5 group(P=0.001).The density andnumber of corneal neovascularization increased gradually from day 1 to day 14 after alkali burn,while the relative neovascularization area in the alkali-burn+rAd-Tum5 group was significantly reduced as compared to those in the alkali-burn group andalkali-burn+rAd-GFP group on day 7 andday 14,suggesting that Tum5 could reduce the growth rate anddensity of corneal neovascularization,so as to inhibit corneal neovascularization induced by alkali burn.On day 7 and14 after alkali burn,in the normal control group,the corneas were intact,no infiltrating inflammatory cells andcells were arranged in an orderly manner.On day 7 after alkali burn,there were disordered epithelial structure,corneal edema andinfiltrating inflammatory cells in alkali-burn group andalkali-burn+rAd-GFP group.The number of infiltrating inflammatory cells in alkali-burn+rAd-Tum5 group was significantly lower than that in alkali-burn group andalkali-burn+rAd-GFP group(both P<0.01).On day 14 after alkali burn,the number of infiltrating inflammatory cells in alkali-burn,alkali-burn+rAd-GFP andalkali-burn+rAd-Tum5 group were significantly lower than those on day 7,while the corneal epitheliums were intact anddropsy was alleviated,while the number of inflammatory cells was significantly lower than that in alkali burn group andalkali-burn+rAd-GFP group,with significant difference(both P<0.01).Conclusion Tum5 can inhibit the angiogenic capability of HUVECs by VEGF pathway,as well as suppress the alkali-burn-induced corneal neovascularization andinflammatory cell infiltration in rats.

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备注/Memo

备注/Memo:
天津市高等学校科技发展计划项目（编号：20120128）；天津市自然科学基金重点项目（编号：14JCZDJC36200）
更新日期/Last Update: 2018-05-09