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中华眼科医学杂志(电子版)

中华眼科医学杂志(电子版) ›› 2019, Vol. 09 ›› Issue (02) : 90 -95. doi: 10.3877/cma.j.issn.2095-2007.2019.02.005

论著

组织蛋白酶B与血管内皮生长因子影响高氧诱导小鼠视网膜新生血管形成的实验研究
王永瑞1, 孔丽2, 王文娟3, 李宸宇1, 周国宏3,†()   
  1. 1. 030001　太原，山西医科大学第一临床医学院2016级硕士研究生
    2. 030001　太原，山西医科大学解剖教研室
    3. 030002　太原，山西省眼科医院泪道病科
  • 收稿日期:2018-12-18 出版日期:2019-04-28
  • 通信作者: 周国宏
  • 基金资助:
    山西省卫生厅科技攻关计划项目(2011057); 山西省留学回国人员科技活动择优资助项目(2017-31)

Effects of cathepsin B and vascular endothelial growth factor on the retinal neovascularization induced by hyperoxia in mice

Yongrui Wang1, Li Kong2, Wenjuan Wang3, Chenyu Li1, Guohong Zhou3,†()   

  1. 1. Master′s Graduate Students of 2016, First Clinical College of Shanxi Medical University, Taiyuan 030001, China
    2. Department of Anatomy, Shanxi Medical University, Taiyuan 030001, China
    3. Department of Lacrimal Duct Diseases, Shanxi Eye Hospital, Taiyuan 030002, China
  • Received:2018-12-18 Published:2019-04-28
  • Corresponding author: Guohong Zhou
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王永瑞, 孔丽, 王文娟, 李宸宇, 周国宏. 组织蛋白酶B与血管内皮生长因子影响高氧诱导小鼠视网膜新生血管形成的实验研究[J]. 中华眼科医学杂志(电子版), 2019, 09(02): 90-95.

Yongrui Wang, Li Kong, Wenjuan Wang, Chenyu Li, Guohong Zhou. Effects of cathepsin B and vascular endothelial growth factor on the retinal neovascularization induced by hyperoxia in mice[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2019, 09(02): 90-95.

目的

探讨组织蛋白酶B(cathepsin B)与血管内皮生长因子(VEGF)在小鼠视网膜新生血管形成中的影响。

方法

选用清洁级C57BL/6J小鼠共44只，7~8日龄，雌雄不限。应用随机数字表法将其分为正常对照组和高氧处理组。其中，正常对照组11只小鼠(22只眼)，高氧处理组33只小鼠(66只眼)。正常对照组小鼠在标准环境中生长；高氧处理组小鼠为建立视网膜新生血管动物模型，其环境除氧体积分数为(75±2)%，其他均相同，氧箱内饲养5 d后，将其返回到标准环境中。以视网膜表面出现新生血管簇、无灌注区，判定为造模成功。再应用随机数字表法将成模的小鼠随机分为模型对照组、实验对照组和实验组，每组11只小鼠(22只眼)。正常对照组和模型对照组不予任何药物干预，实验对照组和实验组分别给予玻璃体腔注射二甲基亚砜和溶于二甲基亚砜的CA-074 Me(cathepsin B抑制剂)，继而在标准环境中饲养5 d。采用心腔注射荧光素标记葡聚糖，视网膜铺片法观察新生血管生成的情况；分别采用实时聚合酶链反应法和蛋白质印迹法检测小鼠视网膜组织中cathepsin B、VEGF信使核糖核酸(mRNA)相对表达量及蛋白的表达量。所有数据均以均数±标准差(±s)表示。采用单因素方差分析比较各组总体差异，当差异有统计学意义时，再用SNK-q检验进行组间多重比较分析。

结果

在荧光显微镜下，实验组较模型对照组和实验对照组视网膜血管走行相对清晰，未见明显的缺血无灌注区。各组cathepsin B与VEGF mRNA及蛋白的表达量总体存在差异，差异有统计学意义(F＝25.23，22.98，43.86，67.92；P<0.05)。模型对照组和实验对照组中cathepsin B与VEGF mRNA及蛋白的表达量均增高，而实验组中cathepsin B与VEGF的mRNA及蛋白的表达量则降低。实验组与正常对照组比较、实验对照组与模型对照组比较，差异均无统计学意义(P>0.05)。

结论

CA-074 Me可抑制cathepsin B和VEGF的mRNA及蛋白的表达。cathepsin B和VEGF表达减少可抑制小鼠视网膜新生血管的形成，且cathepsin B与VEGF之间可能存在着双向调节的关系。

关键词: 组织蛋白酶B, 视网膜新生血管化, 血管内皮生长因子
Objective

The aim of this study was to investigate the correlation between cathepsin B and VEGF in the retinal neovascularization of mice.

Methods

44 clean 7-day-old C57BL/6J mice were randomly divided into the control group (22 eyes of 11 mice) and hyperoxia treatment group (66 eyes of 33 mice). The mice in the control group were feed under the standard condition. Retinal neovascularization animal model was established in mice of the hyperoxic treatment group, which was placed in an airtight oxygen chamber with an oxygen volume fraction of (75±2)% for 5 days, and then transferred to the standard condition. After 5 days, the retinal surface was successfully modeled with new vascular clusters and non-perfusion areas. The model mice were randomly assigned into model control group, experimental control group and experimental group with 11 mice (22 eyes) in each group. The mice in the normal control group and the model control group were without any drug intervention. While the mice in the experimental control group and the experimental group received intravitreal injection of DMSO and CA-074 Me dissolved in DMSO (the inhibitor of cathepsin B), respectively. After 5 days for treatment, the neovessels were observed by FITC-dextran retinal angiography in 17-day-old mice. The mRNA expression levels of cathepsin B and VEGF were performed by real-time PCR; the protein expression levels were detected by Western blot. All data were presented with mean±standard deviation. Univariate ANOVA was used to compare the overall difference among all groups. When the difference was statistically significant, SNK-q test was further used for multiple comparisons between each group.

Results

Under fluorescence microscope, the retinal vascular in the experimental group was relatively clear compared with the model control group and the experimental control group, and the obvious ischemia or perfusion area were not found. There was a significant difference in the total mean of each group(F=25.23, 22.98, 43.86, 67.92; P<0.05). Compared with the control group, the expression levels of mRNA and protein of cathepsin B and VEGF in the control group and the experimental control group were significantly increased; the expression levels in the experimental group were significantly decreased compared with the control group and the experimental control group. While there was not shown significant difference between the control group and the experimental group and between the control group and the experimental control group (P>0.05).

Conclusions

The CA-074 Me could inhibit the expression levels of mRNA and protein content of cathepsin B and VEGF; the reduction of cathepsin B and VEGF could be inhibited the expression of retinal neovascularization in mice; and thus there may be a two-way regulation mechanism between cathepsin B and VEGF.

Key words: Cathepsin B, Retinal neovascularization, Vascular endothelial growth factor
图1 各组小鼠视网膜新生血管的荧光显微镜图像　A图示正常对照组视网膜血管形态清晰，走行清楚，无新生血管簇、血管渗漏，无缺血无灌注区；B和C图分别示模型对照组和实验对照组视网膜血管走行迂曲，较多新生血管簇和视网膜缺血无灌注区；D图示实验组视网膜血管走行相对清晰，未见明显的缺血无灌注区(荧光素标记的葡聚糖　×50)
图2 蛋白质印迹法法检测各组小鼠视网膜中组织蛋白酶B和血管内皮生长因子蛋白的表达量比较
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