大鼠角膜移植后角膜新生淋巴管的动态变化  

Dynamic aspects of corneal lymphatic vessels after keratoplasty in rats

作　　者：凌士奇[1] 黎韦华[2] 梁凌毅[2] 徐建刚[2] 邝文辉[2] 

机构地区：[1]中山大学附属第三医院眼科,广州510630 [2]中山大学中山眼科中心

出　　处：《中华眼科杂志》2012年第4期337-343,共7页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金（30700927;81070711）;广东省科技计划项目（93068;20108060200008）;广东省自然科学基金（$2011010006061）

摘　　要：目的研究大鼠角膜移植后角膜新生淋巴管的动态变化及其与血管内皮生长因子c（VEGF—C）、炎症指数间的关系。方法实验研究。制备大鼠角膜移植模型，于移植后第1、3、7、10、14、30、60天通过免疫组织化学染色和全角膜免疫荧光法标记角膜新生淋巴管，并进行淋巴管计数（LVC），检测淋巴管的动态变化；运用免疫组织化学染色、酶联免疫吸附测定（ELISA）、实时荧光定量PCR法检测角膜组织中VEGF-C的蛋白和mRNA的变化；同时记录炎症指数以评估角膜移植后的炎症反应。运用配对t检验比较角膜移植后3d和14d时VEGF—CmRNA的差异，Pearson相关分析检验LVC和炎症指数、LVC和VEGF—C之间的关联。结果免疫组织化学染色提示角膜移植后3d角膜基质中出现了新生淋巴管，LVC为（1．8±0．3）个，14d时达到高峰，为（9．1±1．5）个。角膜移植后VEGF．C的分泌迅速增加，3d时达到第1个高峰，VEGF．CmRNA为（1．62±0．08）copies／g，以后逐渐减少，7d时角膜基质中几乎无VEGF．C的表达，继而VEGF—C的分泌再次增加，14d时达到第2个高峰，VEGF-CmRNA为（2．48±0．03）copies／g，与3d时比较，两者之间差异有统计学意义（t=4．296，P=0．020）。LVC与VEGF—C、炎症指数间相关系数有统计学意义（r=0．51，P=0．003和r=0．55，P=0．003）。结论角膜移植后角膜新生淋巴管与VEGF—C及炎症指数间密切相关。Objective To study the development of lymphatic vessels after keratoplasty and to explore the molecular mechanisms of corneal lymphangiogenesis in transplanted corneas. Methods Experimental research. The development of corneal lymphangiogenesis was examined by LYVE-1 immunohistochemistry and whole mount immunofluorescence 1,3,7,10, 14,30 and 60 days after corneal transplantation, then lymphatic vessels counting （LVC） was evaluated. The expression of vascular endothelial growth factor-C （ VEGF-C ） in transplanted corneas was examined by immunohistochemistry, enzyme-linked immunosorbent assay （ELISA）, and real time-PCR at same time. In addition, the inflammatory index （IF） was recorded at each time point. The association of VEGF-C and IF with LVC in transplanted corneas was examined. Analysis of the significance of differences between two groups was performed using paired Student＇s t-test. Pearson＇s analysis was used to analyze the correlation between VEGF-C, IF and LVC. Results Corneal lymphangiogenesis occurred in the stroma with LVC （ 1.8 ±0. 3） on Day 3, then developed and reached the peak with LVC （9. 1±1.5） on Day 14 after corneal transplantation. Both VEGF-C protein and mRNA up-regulated dramatically in rat transplanted corneas. The immunoreactivity reached the peak on the 3rd day and the 14th day after keratoplasty. Compared with the expression of VEGF-C mRNA （ 1.62± 0. 08 copies/g） on Day 3, the expression of VEGF-C mRNA （2. 48 ±0.03 copies/g） significantly increased 14 days after the transplantation （ t = 4. 296, P = O. 02 ）. LVC was strongly and positively correlated with IF （ r = 0. 55, P = 0. 003 ） and the expression of VEGF-C mRNA （ r = 0. 51, P = 0. 003 ）. Conclusions Corneal lymphangiogenesis correlates closely with corneal inflammation. The increased expression of VEGF-C in the cornea may be one of the important molecular mechanisms in the occurrence of corneal lymphangiogenesis after keratoplasty.

关 键 词：角膜移植 角膜 淋巴管生成 大鼠 

分 类 号：R73-37[医药卫生—肿瘤]