外源性小鼠干扰素诱导蛋白-10在碱烧伤诱导角膜新生血管中的作用研究

发布时间：2018-05-06 21:42

本文选题：干扰素诱导蛋白-10 + 角膜碱烧伤　；参考：《苏州大学》2012年硕士论文

【摘要】：背景与目的角膜作为重要的屈光介质，生理情况下透明、无血管，但在炎症、外伤、缺氧和水肿等病理状态下，保持角膜无血管状态的促血管形成因子与抑血管形成因子之间的平衡被打破，周围毛细血管就会由角膜缘血管网侵入角膜，形成角膜新生血管（cornealneovascularization, CNV），从而导致视力减退甚至失明。干扰素诱导蛋白-10（interferon-inducible protein-10, IP-10）可作为趋化因子介导Th1型炎症反应，也可作为血管新生抑制剂抑制血管生成，抑制肿瘤生长和转移，参与血管重塑等过程。有关IP-10在CNV中的作用国内外尚未见报道。本实验通过外源性小鼠IP-10蛋白对碱烧伤后新生血管形成的不同阶段进行局部干预，初步探讨其对CNV形成的作用及机制，以期对新生血管性角膜疾病的研究提供理论依据及治疗靶点。材料与方法 1、采用浸润1mol/L NaOH的2×2mm滤纸贴附左眼角膜中央40s的方法制作小鼠角膜碱烧伤诱导CNV模型，角膜碱烧伤后1d及7d开始每日三次局部滴用IP-10蛋白10mg/L共7d分别作为早期实验组（10眼）和中晚期实验组（5眼），，各自的对照组均予质量分数0.2%透明质酸钠点眼（分别为11眼和6眼）。早期干预组于造模后2w，中晚期干预组于造模后3w分离角膜组织，以CD31免疫荧光标记法标记新生血管，比较对照组及实验组CNV面积。 2、收集早期干预第2d及第4d小鼠的角膜组织，用RT-PCR法检测并比较各组小鼠角膜组织中趋化因子受体3（chemokine receptor3, CXCR3）、血管内皮生长因子（vascular endothelial growth factor, VEGF）及转化生长因子-β1（transforminggrowth factor-β1, TGF-β1）mRNA的表达情况。 3、分离早期干预第2d及第4d小鼠的角膜组织，制作铺片，免疫组织化学法以F4/80标记巨噬细胞，比较其在角膜组织中的浸润情况。结果 1、早期干预对照组小鼠CNV占角膜总面积的比例为（88.67±3.23）%，IP-10实验组小鼠CNV面积为（70.06±3.68）%，两组比较差异有统计学意义（t=3.77，P=0.0013）。中晚期干预对照组小鼠CNV占角膜总面积的比例为（87.33±5.50）%，IP-10实验组CNV面积为（86.56±5.58）%，两组比较差异无统计学意义（t=1.26，P0.05）。 2、 IP-10早期干预第2d及第4d的小鼠角膜组织中CXCR3表达较对照组同期值明显升高（t=3.13、3.07，P0.05），VEGF表达降低（t=5.99、6.27，P0.01），TGF-β1表达降低（t=8.50，P0.01；t=4.53，P0.05）。 3、 IP-10早期干预第2d及第4d的小鼠角膜组织中巨噬细胞的浸润实验组与对照组相比差异无统计学意义（t=1.65，P0.05；t=0.59，P0.05）。结论 1.角膜碱烧伤后外源性IP-10蛋白早期干预可抑制CNV的形成，中晚期干预对角膜碱烧伤诱导的CNV无明显作用； 2.角膜碱烧伤后外源性IP-10蛋白早期干预可通过上调CXCR3和下调VEGF及TGF-β1的表达抑制CNV的形成，但对巨噬细胞的浸润并无改变。
[Abstract]:Background and purpose The cornea, as an important refractive medium, is transparent and vascular free under physiological conditions, but in pathological conditions such as inflammation, trauma, hypoxia and edema. When the balance between angiogenic factors and angiogenic factors, which maintain the anangiogenic state of the cornea, is broken, the surrounding capillaries will invade the cornea by the limbal vascular network, forming the corneal neovascularization, CNV, which leads to the loss of vision and even blindness. Interferon-inducible protein-10 (IP-10) can act as a chemokine to mediate Th1 type inflammation and as a angiogenic inhibitor to inhibit angiogenesis, inhibit tumor growth and metastasis, and participate in vascular remodeling. The role of IP-10 in CNV has not been reported at home and abroad. In this experiment, exogenous mouse IP-10 protein was used to intervene in different stages of neovascularization after alkali burn, and to explore its effect on CNV formation and its mechanism. In order to provide theoretical basis and therapeutic targets for the study of neovascular corneal diseases. Materials and methods 1. The CNV model was induced by alkali burn of mouse cornea by applying 2 脳 2mm filter paper of infiltrating 1mol/L NaOH to the center of left cornea for 40 s. 10 eyes of the early experimental group and 5 eyes of the middle and late stage group were treated with IP-10 protein 10mg/L three times a day from 1 d and 7 d after alkali burn. The control groups were given 0.2% sodium hyaluronate (11 eyes and 6 eyes) respectively. Corneal tissue was isolated from the early intervention group at 2 weeks after modeling and the middle and late intervention group at 3 weeks after modeling. The neovascularization was labeled with CD31 immunofluorescence labeling method. The area of CNV in the control group and experimental group was compared with that in the control group and experimental group. 2. The corneal tissues of mice at the 2nd and 4th day after early intervention were collected. The expression of chemokine receptor 3, CXCR3, vascular endothelial growth factor (endothelial growth factor, VEGF) and transforming growth factor-尾 1(transforminggrowth factor- 尾 1, TGF- 尾 1)mRNA were detected and compared by RT-PCR method. 3. The cornea tissues of mice treated with early intervention on day 2 and day 4 were separated and prepared. Macrophages were labeled with F4 / 80 by immunohistochemical method to compare the infiltration of macrophages in cornea tissue. Result 1. The ratio of CNV to total corneal area in the early intervention control group was 88.67 卤3.23 and the area of CNV in the IP-10 group was 70.06 卤3.68. The difference between the two groups was statistically significant. The ratio of CNV to the total corneal area in the control group was 87.33 卤5.50 and the area of CNV in the IP-10 experimental group was 86.56 卤5.58. There was no significant difference between the two groups. 2. Compared with the control group, the expression of CXCR3 in the cornea of mice treated with IP-10 on the 2nd and 4th day was significantly higher than that of the control group. The expression of TGF- 尾 1 in the cornea of mice treated with IP-10 at the early stage was significantly higher than that in the control group (P 0.05). 3. There was no significant difference between the experimental group and the control group in the infiltration of macrophages in the cornea of the mice on the 2nd and 4th day after early intervention by IP-10. There was no significant difference between the experimental group and the control group. There was no significant difference between the experimental group and the control group. Conclusion 1. The early intervention of exogenous IP-10 protein after alkali burn could inhibit the formation of CNV, but the late intervention had no obvious effect on CNV induced by alkali burn of cornea. 2. The early intervention of exogenous IP-10 protein after alkali burn could inhibit the formation of CNV by up-regulating CXCR3 and down-regulating the expression of VEGF and TGF- 尾 1, but did not change the infiltration of macrophages.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R772.2

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