人羊膜匀浆上清液抑制兔角膜新生血管的研究

Suppression of corneal neovascularization by human amniotic homogenate in rabbits

目的观察人羊膜匀浆上清液对家兔角膜新生血管(CNV)的抑制作用。方法63只新西兰白兔随机选取3只作为正常对照组,余60只制作双眼碱烧伤模型,随机分为4个组:A组为碱烧伤对照组滴用PBS液,B、C、D组为实验组分别滴用50、200、400μg/mL的羊膜匀浆上清液。术后1、5、7、14、28d每组分别随机处死3只兔子,行组织病理学检查,测量微血管数量、多形核白细胞(PMNL)含量、血管内皮生长因子(VEGF)的表达量。结果碱烧伤1、5、7、14、28d各时间点CNV的生长面积、微血管计数、PMNL浸润、VEGF的表达4个组差异均有统计学意义(P<0.01),其中D组均数最小。VEGF、PMNL、微血管计数三者呈正相关(P<0.01)。结论羊膜匀浆上清液有减轻角膜血管化的作用,呈质量浓度依赖性,质量浓度达到400μg/mL时抑制作用显著增强。

Objective Corneal neovascularization （CNV） is a long-lasting problem in clinic, and it is a major factor leading to blindness. Present study aimed to investigate the effects of human amniotic homogenate on corneal neovascularization in rabbits. Methods Intermediate CNV model was induced by putting the 1 mol/L NaOH filter in the central cornea for 1 minute and identified on the Hughes＇ s grade in 60 New Zealand white rabbits,and 3 normal New Zealand white rabbits were as control group. Sixty model rabbits were randomly assigned to four groups. The protein level in human amniotic homogenate was determined by Bradford method. 50,200 and 400 μg/mL amniotic supernatant was topically used for 6 times daily in model eyes of group B, C,D separately,and PBS was topically used at the same way in normal eyes and model eyes of group A. The area of CNV was calculated by computer image analysis system at 1,3,5,7,14 and 28 days. Three rabbits of every group were killed at 1,5,7,14 and 28 days for histopathological examination under the light microscope. Polymorphonuclear leukocyte（PMNL） ,capillary vessels and the expression of VEGF in corneas were detected with computer image analysis system. Results Areas of the CNV was gradually increased with time in PBS group. However, the CNV area was smaller after different dose of amniotic supernatant treated, showing statistically significant differences upon the drug dose and time （P 〈 0. 01 ）. The significant differences were seen in capillary vessels number, PMNL infiltration and VEGF expression in cornea between 1,5,7,14 and 28 days groups（P 〈 0.01 ） with the most significant difference in 400 μg/mL amniotic supernatant group. A positive correlation was found between VEGF expression and PMNL infiltration （ r = 0. 861 , P 〈 0. 01 ） or between VEGF expression and the number of capillary vessel （ r = 0. 656,P 〈 0.01 ）. Conclusion Supernatant of fresh homogenized amnion can inhibit and restrain the growth of corneal new blood vessel through inhibition of PMNL releasing VEGF,and the suppression effect is stronger in the dose of 400 μg/mL.