【摘要】 目的：探讨血管紧张素II(angiotensin II, Ang II)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在视网膜新生血管发生发展中的作用。方法：随机对照实验研究。选取7d龄C57BL/6J新生小鼠40只，随机分为2组：正常对照组和高氧模型组，每组20只，高氧模型组建立氧诱导视网膜病变模型。采用荧光素血管灌注视网膜铺片观察视网膜血管形态学改变;制作视网膜组织切片并进行HE染色计数突破视网膜内界膜的血管内皮细胞核数;采用Western blot检测视网膜Ang II和VEGF蛋白的表达。采用独立样本t检验和Pearson相关分析进行统计学分析，以P<0.05作为差异具有统计学意义。结果：荧光素血管灌注视网膜铺片：高氧模型组较正常对照组可见大量新生血管丛，伴明显荧光渗漏。突破视网膜内界膜的血管内皮细胞核数：高氧模型组(43.23±2.57)个较正常对照组(1.37±0.93)个明显增多，差异具有统计学意义(P=0.00)。视网膜Ang II蛋白表达水平：高氧模型组(0.365 ±0.004)较正常对照组(0.035 ± 0.003)明显上调，差异具有统计学意义(P=0.00)。视网膜VEGF蛋白表达水平：高氧模型组(0.372 ± 0.004)较正常对照组(0.049 ± 0.007)明显上调，差异具有统计学意义(P=0.00)。结论：Ang II促血管生成的作用与VEGF存在明确的联系，Ang II通过上调VEGF参与视网膜新生血管的形成。
【关键词】 视网膜新生血管;肾素血管紧张素系统;血管紧张素II;血管内皮生长因子
Expression and significance of angiotensin II and vascular endothelial growth factor in retinal neovascularization
HeNan Liu，XiaoLong Chen
Foundation items: Natural Science Foundation of Liaoning Province，China(No.20052089);Scientific Research Fund of Liaoning Provincial Education Department，China(No. 20060994)
Department of Ophthalmology，Shengjing Hospital，China Medical University，Shenyang 110004，Liaoning Province，China
AbstractAIM: To investigate a possible role for the angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) in the development of retinal neovascularization.METHODS: It was a random control experimental study. Forty sevendayold C57BL/6J mice were divided into two groups randomly including normal control group and hyperoxia model group. Hyperoxia model group were exposed to 75% oxygen to establish a model of oxygeninduced retinopathy. Fluorescent angiography was used to assess the vascular pattern of retina. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in crosssections. Ang II and VEGF protein levels in retinas were measured by Western blot. Significant differences between groups were evaluated by independentsamples t test and Pearson correlation analysis. Statictical difference was considered significant at a P value less than 0.05.RESULTS： Fluorescent angiography presented increasing neovascular tufts with fluorescein leakage in hyperoxia model group compared to the normal control group. The number of endothelial cells of new vessels extending from retina to vitreous were increased significantly in hyperoxia model group (43.23±2.57) as compared with normal control group (1.37±0.93) (P=0.00). The expression of Ang II protein in retinas were increased significantly in hyperoxia model group (0.365±0.004) compared with normal control group (0.035±0.003) (P=0.00). The expression of VEGF protein in retinas were increased significantly in hyperoxia model group (0.372±0.004) compared with normal control group (0.049±0.007) (P=0.00).CONCLUSION: There is specific relation between Ang II and VEGF in angiogenesis promoting effect. Ang II participate in the occurrence and development of retinal neovascularization via the upregulation the expression of VEGF.
KEYWORDS：retinal neovascularization;reninangiotensin system;angiotensin II;vascular endothelial growth factor
0引言
视网膜新生血管是糖尿病视网膜病变和早产儿视网膜病变等增生性视网膜病理改变的主要特征[1]。在这些增生性视网膜病变中，视网膜新生血管形成导致视网膜水肿、出血、脱离等严重影响视力的并发症[1]。尽管刺激视网膜新生血管生长的因素还未完全明确，但有证据显示参与此过程的不仅有血管源性的细胞因子如血管内皮细胞生长因子(vascular endothelial growth factor，VEGF)，还包括血管活性激素如血管紧张素II(angiotensin II，Ang II)[2]。在增生性视网膜病变的发生、发展过程中，肾素血管紧张素系统(reninangiotensin system，RAS)中主要效应介质Ang II可能与特异性生长因子特别是VEGF相互作用来影响视网膜的发育和功能，最终共同导致视网膜新生血管形成。本研究的目的在于探讨视网膜新生血管发生发展过程中，Ang II和VEGF影响视网膜新生血管形成的机制。
1材料和方法
1.1材料 实验动物：鼠龄7d的健康C57BL/6J清洁级新生小鼠(中国医科大学动物部)40只，体质量4.0～5.0g，性别不限，与哺乳母鼠共同饲养。药品试剂：卡托普利(美国BristolMyers Squibb公司)、缬沙坦(瑞士Novartis公司)、BCA蛋白定量试剂盒(美国Pierce公司)、ECL发光试剂盒(美国Pierce公司)、异硫氰酸葡聚糖荧光素(美国Sigma公司)、兔抗小鼠Ang II多克隆抗体(美国Sigma公司)、兔抗小鼠VEGF多克隆抗体(美国Sigma公司)、羊抗小鼠IgGHRP(美国Sigma公司)。
1.2方法 动物分组和模型建立：将40只7d龄C57BL/6J清洁级新生小鼠随机分为两组：正常对照组和高氧模型组，每组20只。参照Smith等[3]的方法建立氧诱导视网膜病变模型，高氧模型组小鼠及哺乳母鼠置于密闭氧箱中，控制氧浓度为(75±2)%，5 d(鼠龄12 d)后回到正常空气环境中，正常对照组一直置于正常空气环境中。所有动物保持温度(21±1)°C，光照黑暗循环/12h。荧光素血管灌注视网膜铺片：两组鼠龄17d的小鼠各取2只，将异硫氰酸葡聚糖荧光素(分子量：2×106)溶于1mL的40g/L多聚甲醛中，经左心室灌注(30mL/kg)，摘取眼球，于40g/L多聚甲醛中固定1h，游离视网膜，以视盘为中心呈放射状对称切开，用抗荧光衰退封片剂将视网膜封片，采用荧光显微镜(日本Olympus公司)观察视网膜血管形态的变化。突破视网膜内界膜的血管内皮细胞核计数：两组鼠龄17d的小鼠各取3只，摘除眼球后，于40g/L多聚甲醛中固定24h，常规脱水，石蜡包埋，平行于角膜至视盘的矢状位连续6μm切片，苏木素伊红染色。每只眼球取10张切片，每组30张，由同一操作者采用随机、盲法在光学显微镜(日本Olympus公司)下对切片样本计数每张切片突破视网膜内界膜的血管内皮细胞核数。选取切片时注意避开视盘周围，计数血管内皮细胞核时仅计数与内界膜有紧密联系的细胞核，不包括玻璃体腔内其它与内界膜无联系的血管内皮细胞核。Western blot检测Ang II及VEGF蛋白表达：两组鼠龄17d的小鼠各取15只，每组取5只新生鼠双眼完整的视网膜，提取视网膜组织蛋白质，用BCA蛋白定量试剂盒定量视网膜组织蛋白质含量，每个样品上样含量50μg，以100g/L SDSPAGE电泳(4℃，120V，2h)分离蛋白质，电转移法将蛋白质转移至硝酸纤维素滤膜上，用40g/L脂奶粉，于4℃下过夜封闭硝酸纤维素滤膜的非特异性蛋白质结合位点，将滤膜于兔抗小鼠Ang II多克隆抗体(1∶400)、兔抗小鼠VEGF多克隆抗体(1∶400)在37℃下孵育2h，TBST缓冲液冲洗滤膜3次后，将滤膜转移至二抗羊抗小鼠IgGHRP(1∶1000)，37℃下孵育1h，TBST缓冲液再次冲洗滤膜后，ECL化学发光显影。采用βactin(1∶400)作为内参照。每组样品重复检测3次。应用Quantity One 4.2软件分析，分别比较各组Ang II，VEGF条带灰度值与βactin条带灰度值的比值，表示各组蛋白相对表达水平。
统计学分析：所有数据应用SPSS 16.0 for Windows进行统计学分析，结果以±s表示，计量资料组间比较采用独立样本t检验，两变量间相关性采用Pearson相关分析，以P<0.05作为差异有统计学意义。
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