碱烧伤诱导小鼠角膜新生血管中miR-21与VEGF表达相关性

目的观察microRNA-21（miR-21）在碱烧伤诱导小鼠角膜新生血管（CorNV）模型中的变化规律及其与血管内皮生长因子-A（VEGF-A）的关系。方法实验研究。68只BALB/c小鼠，右眼制备碱烧伤诱导CorNV模型作为实验组，左眼作为正常对照组不予任何处理。碱烧伤后第4、7、14、21、28天行眼前节照相测量CorNV面积。分别在碱烧伤后第4、7、14、28天分别随机处死3只小鼠取眼球制石蜡切片，通过苏木素-伊红（HE）染色观察角膜的组织病理学改变，免疫组织化学（IHC）染色检测角膜VEGF-A表达；各时间点随机处死14只小鼠行实时荧光定量聚合酶链式反应（Real-time PCR）检测角膜miR-21及VEGF-A mRNA的表达。数据分析采用单因素方差分析、配对样本t检验、直线相关分析。结果小鼠CorNV于碱烧伤后第4天开始出现，14天时生长达高峰，21天后逐渐消退。HE染色显示实验组角膜第14天呈现较多粗大CorNV，28天时明显减少。同期IHC染色显示实验组角膜在第4、7、14天时VEGF-A阳性表达均高于对照组（t=19.47、61.86、44.18，P<0.05），28天时差异无统计学意义（t=2.52，P>0.05）。实验组第7天时角膜VEGF-A阳性表达明显，14天后表达相对减弱，差异具有统计学意义（F=649.4，P<0.05）。实验组角膜在第4、7、14、28天时miR-21表达均高于对照组（t=4.60、14.74、9.78、5.69，P<0.01）；VEGF-A mRNA实验组角膜在第4、7、14天时高于对照组（t=5.82、14.11、3.41，P<0.05），28天时差异无统计学意义（t=0.76，P>0.05）；且两者表达均于第7天时达高峰，差异均具有统计学意义（F=62.1、64.5，P<0.05）。miR-21与VEGF-A mRNA及其蛋白表达均呈正相关（r=0.62、0.66，P<0.05）。结论碱烧伤诱导小鼠角膜CorNV形成中miR-21、VEGF-A表达均升高，且miR-21、VEGF-A mRNA和蛋白表达高峰较CorNV形成高峰早，提示miR-21可能通过VEGF信号通路参与CorNV形成。

Relationship between miR-21 and VEGF in mice corneal neovascularization induced by alkali burn

ObjectiveTo observe the changes that occur as a rule in microRNA-21 （miR-21） expression in a mouse corneal neovascularization （CorNV） model induced by alkali burn and its relationship with vascular endothelial growth factor-A （VEGF-A）. MethodsThis was an experimental study. All right eyes of sixty-eight BALB/c mice were used to create models of CorNV induced by alkali burn and were included as the experimental group； the untreated left eyes were included as the control group. Biomicroscopic neovascularization was observed and photographed on days 4， 7， 14， 21 and 28. The areas of CorNV were then calculated. On days 4， 7， 14， 28 after alkali-induced injury， 3 mice were randomly euthanized and six eye balls were embedded in paraffin. Sections were cut for hematoxylin-eosin （HE） staining and immunohistochemical （IHC） staining to observe the corneal histopathological changes and to detect the protein expression of VEGF-A. Fourteen mice were randomly sacrificed for testing the expression of miR-21 and VEGF-A mRNA in corneal tissues by quantitative real-time polymerase chain reaction （real-time PCR）. The results were analyzed statistically with an analysis of variance of repeated measurement data. Linear correlation analysis was used for correlation analysis. ResultsAfter alkali-induced injury， slit lamp biomicroscopy was used to detect CorNV. It was first detected on day 4， peaked on day 14， and started to regress after day 21. The maximum area of CorNV appeared on day 14. HE staining showed that CorNV was most obvious on day 14 after alkali injury and less obvious on day 28. IHC showed that expression of VEGF-A positive staining was higher than in the control group （t=19.47， 61.8， 44.18， P<0.05） on days 4， 7， 14. Positive staining of VEGF-A was obvious on day 7 and started to decrease after day 14. The difference was statistically significant （F=649.4， P<0.05）. Real-time PCR analysis showed that on days 4， 7， 14， 28 after injury， the relative expression of miR-21 increased in the alkali-burn group compared to the control group （t=4.60， 14.74， 9.78， 5.69， P<0.01） and VEGF-A mRNA levels were higher than levels in the control group on days 4， 7， 14 after injury （t=5.82， 14.11， 3.41， P<0.05）. Their expression levels both reached a maximum on day 7 （F=62.1， 64.5， P<0.05）. There were positive correlations in miR-21 expression with VEGF-A mRNA and VEGF protein at each investigative time point （r=0.62 and r=0.66， respectively， P<0.05）. ConclusionmiR-21 and VEGF-A were upregulated during the CorNV course in a mouse model induced by alkali burn. The peak expression of miR-21， VEGF-A mRNA and protein preceded CorNV formation. miR-21 may play a potential role through the VEGF signal pathway in the course of CorNV formation.