外源性肿瘤坏死因子α对碱烧伤诱导角膜新生血管的影响及机制

Effect of exogenous TNF-α on corneal neovascularization induced by alkali burn

目的探讨外源性肿瘤坏死因子α（TNF—α）局部干预对碱烧伤诱导角膜新生血管（CNV）形成的影响及内在机制。方法BALB／c小鼠45只，利用碱烧伤法制作左眼CNV模型。根据实验设计．分3组实验进行研究。①CNV模型小鼠15只，随机分为3组，每组5只。自伤后分别给予100、500μg／ml TNF—α（实验组）或0-2％透明质酸钠（HA，对照组）点眼1周。于碱烧伤后第14天处死小鼠取眼球，免疫组织化学法检测各组角膜组织中CD31的表达，计数微血管密度（MVD），分析TNF-α对碱烧伤CNV形成的作用。②CNV模型小鼠20只．随机分为2组，每组10只。实验组给予100μg／mlTNF一仅点眼，对照组给予0．2％HA点眼。分别于伤后第2天和第4天随机取5只小鼠处死取角膜．RT—PCR法检测角膜组织中血管内皮生长因子（VEGF）mRNA的表达情况，分析TNF—α对碱烧伤角膜中VEGF表达的影响。③利用二氯亚甲二磷酸脂质体（Cl2MDP—lip）射制作小鼠巨噬细胞特异剔除模型，10只BALB／c小鼠随机分为Cl2MDP—lip实验组及PBS—lip对照组，每组5只。所有小鼠自碱烧伤后均给予100μg／mlTNF-α滴眼1周于.伤后第14天拍照记录CNV形成情况，图像分析软件测算CNV长度及面积，分析巨噬细胞在TNF—α调控碱烧伤后CNV形成中的作用。结果①碱烧伤后第14天，100μg／mlTNF-α组CNV形成较对照组明显增多．而500μg／ml TNF-α组CNV形成与对照组差异不明显。整张切片和热点区域MVD值，100μg／ml TNF-α组为63．25±9．75和114．94±12．93，500μg／ml TNF-α组为39．53±10．41和108．04±23．55．0．2％HA对照组为30．99±7．08和73．81±13．80和100μg／mlTNF—α组与对照组比较，差异有统计学意义（P〈0．05），而500μg／mlTNF-α组与对照组相比差异无统计学意义（19〉0．05）。②碱烧伤后第2天和第4天VEGFmRNA与β—actin的比值，100μg／mlTNF-α组为0．40±0．12和1，51±0．21，0．2％HA对照组为0，31±0．10和0．58±0．30。两组差异均有统计学意义（P〈0．05）。⑧碱烧伤后第14天，Cl2MDP—lip组CNV形成较对照组明显减少，Cl2MDP—lip组的CNV长度和面积分别为（0．84±0．10）mm和（6．64±1．19）mm^2,PBS—lip对照组为f1．37±0．24）mm和（12．25±1．33）mm^2，两组差异有统计学意义（P〈0．01）.结论外源性TNF-α可通过上调碱烧伤角膜组织中VEGF表达来促进CNV的形成，其促进碱烧伤CNV形成机制主要是通过巨噬细胞起作用．

Objective To explore the effect and mechanism of exogenous TNF-α on corneal neovascularization （CNV） in BALB/c mice injured by alkali burn. Methods①Fifteen BALB/c mice were burned on central cornea of left eye by 1 mol/L NaOH for 40 s combine with epithelial denudation, then divided into 3 groups （5 mice each group）： 0.2% sodium hyaluronate control group, 100 μg/ml TNF-α treated group, 500 μg/ml TNF-α treated group. The eye drops were applied twice daily for one week. CNV was observed by ophthalmic microscope. Corneal microvessel densities of each group were detected at day 14 after alkali injury by CD31 immunohistochemical analysis. The effect of recombinant murine TNF-α on alkali injury induced CNV was observed. ②Afier alkali injury, 20 BALB/c mice were divided into 2 groups （10 mice each group）： 0.2% sodium hyaluronate control group, 100 μg/ml TNF-α treated group. The eye drops were applied twice daily. At day 2 and day 4 after injury, 5 randomly selected mice of each group were euthanized, vascular endothelial growth factor （VEGF） expressions in the corneal tissue were detected by RT-PCR.③Effect of exogenous TNF-α on macrophage depleted mice. Macrophages were depleted by Cl2MDP-lip. 10 BALB/c mice were divided into 2 groups （5 mice each group）： Cl2MDP-lip treated group and PBS-Iip control group. After alkali injury, all mice were given 100 μg/ml TNF-α eye drops twice daily for one week. Macroscopic CNV was observed by ophthalmic microscope, and the length and area of CNV on day 14 after injury were evaluated and compared between ClMDP-lip experimental and control group. Results ①Corneal microvessel density in 100 μg/ml TNF-α treated group were significantly greater than those in control group （P〈0.05）, and no significant difference was found between 500 μg/ml TNF-α treated group and control group （P〉0.05）. ②The intra-corneal VEGF mRNA expression in the early phase （day 2, day 4） after alkali injury in 100 μg/ml TNF-α treated group were significantly higher compared to those in control group （P〈0.05）. ③Length and area of CNV in the ClMDP-lip group were significantly less than those in control group （P〈0.01）. Conclusion Exogenous TNF-αcan promote the development of alkali injury induced CNV by enhancing intra-corneal VEGF mRNA expression. Macrophage play a critical role in the mechanism of TNF-α promote alkali injury induced CNV.