整合素α9β1对角膜缝线术后新生淋巴管生成及血管内皮生长因子C表达的影响

发布时间：2018-08-14 10:36

【摘要】：目的因新生淋巴管在炎症性角膜病变的发生发展中起重要作用，本研究通过构建角膜缝线炎症模型的方法，诱导小鼠角膜产生新生淋巴管，研究外源性的整合素α9β_1及其特异性抗体Y9A2对小鼠角膜缝线术后新生淋巴管的生成及角膜中血管内皮生长因子C(Vascular endothelialgrowth factor C，VEGF-C)表达的影响，并探讨其意义。方法 80只健康雌性BALB/c小鼠，随机分为正常对照组、缝线对照组、α9β_1组、α9β_1抑制组，每组20只。正常对照组不处理；缝线对照组、α9β_1组、α9β_1抑制组小鼠制作双眼角膜缝线模型，术后分别予以左氧氟沙星、左氧氟沙星+α9β_1、左氧氟沙星+α9β_1单抗Y9A2点双眼，所有药物均为每日两次点眼，，连续14天。术后每天在手术显微镜下观察小鼠角膜及缝线的变化和新生血管生长情况。在角膜缝线术后第3、5、7、14、21天分别随机取每组4只小鼠，被处死后取双眼角膜组织进行检测。其中2只小鼠，左眼角膜进行切片后免疫组织化学淋巴管内皮细胞染色，观察角膜切片新生淋巴管在不同时间点的动态变化并行淋巴管计数；右眼角膜进行免疫荧光淋巴管内皮细胞染色观察全角膜淋巴管的大体形态。另2只小鼠，左眼角膜用于进行RT-PCR检测VEGF-C mRNA的表达；右眼角膜组织采用Western blotting法检测不同时间点角膜中VEGF-C蛋白的表达。最后，采用SPSS13．0统计学软件进行统计分析。各组数据资料以mean±S.D.表示，经Levene检验方差齐，各组测试指标的总体均数比较采用单因素方差分析，各组均数间两两比较采用SNK-q检验。结果一、小鼠角膜缝线术后，角膜的大体变化及新生血管生长情况：手术显微镜下观察，正常组小鼠角膜透明，角膜缘存在血管网，但角膜中无新生血管。缝线对照组小鼠角膜缝线模型建立后第3天，角膜缘处可见毛刷状新生血管发出；第5天角巩膜缘处新生血管侵入角膜；第7天角膜新生血管交织成网状，部分血管已达缝线处；第14天角膜新生血管超过缝线处并侵入角膜中央，末端呈细小分枝状。第21天，垂柳状侵入并围绕在缝线周围。α9β_1组和α9β_1抑制组角膜和缝线对照组比较，新生血管产生和变化趋势肉眼下未见明显差别。二、免疫组织化学检测发现：正常对照组角膜无新生淋巴管；另3组小鼠缝线后有新生淋巴管由角膜缘发出，在第14天淋巴管计数（LVC）达峰值。缝线对照组术后第3天开始出现新生淋巴管，第14天LVC是3.27±0.25；与缝线对照组比较，在各观察时间点时α9β_1组LVC均增多，第14天LVC4.93±0.38（P0.05），α9β_1抑制组角膜LVC明显降低，第14天为2.25±0.34（P0.05）。三、角膜淋巴管免疫荧光染色：正常对照组在角膜缘内可见点状红色荧光，角膜内无荧光。缝线对照组、α9β1组、α9β1抑制组在缝线术后个观察时间点均可见除角膜缘绿色免疫荧光外，自角膜缘发出的芽状或管道样荧光，通过调节荧光倒置像差显微镜，可发现这些管样荧光分布于角膜基质的不同层面。四、RT-PCR和Western blotting检测结果示,正常对照组仅少量VEGF-C表达，缝线对照组VEGF-C表达较正常对照组显著增多且在第14天达峰值（P0.05）；与缝线对照组比较，α9β_1组VEGF-C表达增高（P0.05），而α9β_1抑制组VEGF-C表达降低（P0.05）。结论小鼠角膜缝线模型，在诱导角膜产生新生血管的同时产生新生淋巴管；整合素α9β_1可促进角膜新生淋巴管发生；通过Y9A2抑制整合素α9β_1可下调VEGF-C的表达和显著减少角膜新生淋巴管。
[Abstract]:objective
Because the new lymphatic vessels play an important role in the development of inflammatory keratopathy, this study established a corneal suture inflammation model to induce new lymphatic vessels in the cornea of mice. The expression of C Vascular endothelialgrowth factor C (VEGF-C) was investigated and its significance was discussed.
Method
Eighty healthy female BALB/c mice were randomly divided into normal control group, suture control group, alpha 9 beta 1 group and alpha 9 beta 1 inhibitory group with 20 mice in each group. The changes of cornea and suture and neovascularization in mice were observed under operating microscope every day after operation. Four mice in each group were randomly selected on the 3rd, 5th, 7th, 14th and 21st days after suture surgery. The corneal tissues in both eyes were taken for examination after death. Immunohistochemical staining of lymphatic endothelial cells was performed on the cornea of the left eye and the dynamic changes of lymphatic vessels were observed at different time points. Immunofluorescent staining of lymphatic endothelial cells was performed on the cornea of the right eye to observe the gross morphology of the whole corneal lymphatic vessels. The expression of VEGF-C mRNA was detected by RT-PCR and the expression of VEGF-C protein was detected by Western blotting in right cornea at different time points. One way ANOVA was used. SNK-q test was used to compare 22 of the average in each group.
Result
1. The general changes of cornea and the growth of neovascularization after corneal suture in mice: under the operating microscope, the cornea of normal mice was transparent and there was a vascular network in the corneal limbus, but no new blood vessels were found in the cornea. The corneal neovascularization invaded the cornea at the limbus of cornea and sclera; on the 7th day, the corneal neovascularization interlaced into a network and some of the vessels reached the suture; on the 14th day, the corneal neovascularization exceeded the suture and invaded the central cornea with a small branch at the end. On the 21st day, the willow-shaped invasion and surrounded the suture. Compared with the control group, there was no significant difference in the production and the trend of neovascularization.
2. Immunohistochemistry showed that there was no new lymphatic vessel in the normal control group, and the new lymphatic vessel originated from the limbus of cornea after suture in the other three groups. LVC reached its peak on the 14th day. LVC increased at the time point, LVC4.93 (+0.38) (P 0.05) at the 14th day, and decreased significantly at the 14th day, 2.25 (+0.34) (P 0.05).
Three, corneal lymphatic immunofluorescence staining: normal control group in the limbus of the cornea can see red fluorescence, no fluorescence in the cornea. The tube fluorescence can be found at different levels of the corneal stroma by means of an aberration microscope.
Fourthly, the results of RT-PCR and Western blotting showed that only a small amount of VEGF-C was expressed in the normal control group, and the expression of VEGF-C in the suture control group was significantly increased and peaked at the 14th day (P 0.05); compared with the suture control group, the expression of VEGF-C in the alpha-9 beta-1 group was increased (P 0.05), while that in the alpha-9 beta-1 inhibition group was decreased (P 0.05).
conclusion
The mouse corneal suture model induced corneal neovascularization and lymphangiogenesis; integrin alpha 9 beta 1 promoted corneal neovascularization; Y9A2 inhibited integrin alpha 9 beta 1 down-regulated the expression of VEGF-C and significantly reduced corneal neovascularization.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.6

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