目的观察重组人血管内皮抑素(ES)对链脲佐菌素(STZ)诱导的早期糖尿病大鼠视网膜屏障相关蛋白表达的影响。
方法采用STZ诱导制作糖尿病大鼠模型，取36只造模成功后2周的糖尿病大鼠按照随机数字表法分为模型组、ES 1.0 μl组、ES 2.5 μl组、ES 5.0 μl组、贝伐单抗2.5 μl组和联合治疗组，每组各6只，并根据分组情况右眼玻璃体腔分别注射不同剂量的重组人ES和贝伐单抗。同时选取6只正常大鼠作为空白对照组。于玻璃体腔注药后4周，采集各组大鼠右眼视网膜组织，采用Western blot法检测视网膜组织中claudin-5、occludin、细胞间黏附分子-1(VCAM-1)、血管细胞黏附分子-1(ICAM-1)、血管内皮生长因子(VEGF)蛋白的表达水平。
结果糖尿病模型大鼠均相继表现出多饮、多尿、多食等典型糖尿病表现，体质量明显下降，造模成功率为100%。Western blot检测结果显示，空白对照组、ES 2.5 μl组、ES 5.0 μl组、贝伐单抗2.5 μl组、联合治疗组VCAM-1、ICAM-1、VEGF相对表达量均较模型组下降，claudin-5、occludin的相对表达量均较模型组升高，差异均有统计学意义(均 P<0.05)。ES 1.0 μl组occludin蛋白相对表达量为0.23±0.02，明显高于模型组的0.13±0.02，ES 1.0 μl组ICAM-1、VEGF蛋白相对表达量分别为0.53±0.01和0.57±0.00，明显低于模型组的0.81±0.01和0.86±0.00，差异均有统计学意义(均 P<0.05)。随着ES给药剂量的增加，claudin-5和occludin蛋白相对表达量有升高的趋势，VCAM-1、ICAM-1、VEGF蛋白相对表达量有下降趋势。
结论重组人ES可能通过直接或间接减少炎性因子VCAM-1、ICAM-1的释放、抑制VEGF的表达从而减少视网膜紧密连接蛋白claudin-5和occludin的缺失。
ObjectiveTo observe the effect of recombinant human vascular endostatin(ES) on retinal barrier related proteins in early streptozotocin (STZ)-induced diabetic rats.
MethodsDiabetes rat model was induced by STZ.Two weeks after the model was successfully constructed, 36 diabetic model rats were randomly divided into the model group, 1.0 μl ES group, 2.5 μl ES group, 5.0 μl ES group, 2.5 μl bevacizumab group, and the combination therapy group, with 6 rats in each group.Different doses of recombinant human ES and 2.5 μl bevacizumab were injected into the vitreous cavity of the right eye according to the grouping.Six normal rats were selected as the blank control group.At 4 weeks after intravitreal injection, the retinal tissue of the right eye in each group was collected, and the expression levels of inter cellular cell adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), occludin, claudin-5, vascular endothelial growth factor(VEGF) and other proteins in retinal tissue were detected by Western blot assay.The use and care of animals was in accordance with the regulations for the administration of experimental animals.
ResultsThe diabetic model rats showed polydipsia, polyuria, polyphagia and other typical diabetic symptoms, the body quality decreased significantly, and the success rate of modeling was 100%.Western blot results showed that the expression levels of VCAM-1, ICAM-1 and VEGF in the blank group, the 2.5 μl ES group, the 5.0 μl ES group, the bevacizumab group, the combination therapy group were significantly lower than those in the model group, while the expression levels of claudin-5 and occludin were significantly higher than those in the model group, the differences were statistically significant (all at P<0.05). The relative expression of occludin protein in the 1.0 μl ES group was significantly higher than that in the model group(0.23±0.02 vs.0.13±0.02), while the relative expression of ICAM-1 and VEGF was significantly lower than that in the model group(0.53±0.01 vs.0.81±0.01; 0.57±0.00 vs.0.86±0.00), the differences were statistically significant (all at P<0.05). As the dose of ES increased, the relative expressions of claudin-5 and occludin protein tended to increase, while the relative expressions of VCAM-1, ICAM-1 and VEGF tended to decrease.
ConclusionsRecombinant human vascular endostatin can directly or indirectly reduce the release of inflammatory factors VCAM-1 and ICAM-1 and inhibit the expression of VEGF, thereby reduce the loss of retinal tight junction.
周进华,解正高. 重组人血管内皮抑素对早期糖尿病大鼠视网膜屏障相关蛋白的影响[J]. 中华实验眼科杂志,2020,38(09)：740-745.
DOI:10.3760/cma.j.cn115989-20190228-00092版权归中华医学会所有。
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