活化二价锌离子对碱烧伤后角膜融解病理机制中基质金属蛋白酶抑制作用的研宄

发布时间：2019-04-10 19:46

【摘要】：目的：探讨在碱烧伤后角膜融解损伤病理作用中不同时间内起主要组织损伤作用的基质金属蛋白酶的类型；探讨活化Zn2+离子对碱烧伤后角膜融解损伤不同时期异常高表达的基质金属蛋白酶的抑制作用。方法：建立中度角膜碱烧伤小鼠实验动物模型,应用明胶酶谱、蛋白质印迹法以及免疫组织化学技术检测碱烧伤后3日、1周以及1个月时角膜组织内基质金属蛋白酶-1,2及9的表达情况。应用不同浓度Zn2+离子溶液(0.1mmol/L及1mmol/L)对碱烧伤后角膜组织进行局部干预。应用明胶酶谱以及蛋白质印迹法检测角膜碱烧伤后3日及1周时不同浓度Zn2+离子溶液实验亚组以及对照组角膜组织内中-1,2及9的表达情况。结果：应用明胶酶谱方法观察到碱烧伤后3日角膜组织内基质金属蛋白酶-9表达显著的升高,呈现高水平表达,碱烧伤后1周其表达量显著下降,而碱烧伤后1个月时角膜组织内观察不到基质金属蛋白酶-9的表达。基质金属蛋白酶-2与之相反,在碱烧伤后3日角膜组织内仅呈现低水平表达,碱烧伤后1周其角膜组织内表达量显著升高,呈现高水平表达,碱烧伤后1个月时其表达量再次降低,呈现中度水平表达。应用蛋白质印迹法以及免疫组织化学方法观察的碱烧伤后3日、1周及1个月组织内基质金属蛋白酶-9及2的表达变化情况与应用明胶酶谱方法观察的情况相符,即：基质金属蛋白酶-9在碱烧伤后急性期高表达,且其高表达主要位于靠近角膜上皮层区域,而后表达量逐渐降低；基质金属蛋白酶-2在碱烧伤后急性期表达水平较低,而在碱烧伤后1周呈现高水平表达,不同于基质金属蛋白酶-9,其高表达分布于角膜基质内,其后表达量再次降低,呈现中度水平表达。应用蛋白质印迹法以及免疫组织化学方法观察到碱烧伤后组织内不同时期基质金属蛋白酶-1在角膜碱烧伤后组织内的表达变化趋势与基质金属蛋白酶-2相似,在碱烧伤后1周时表达水平最高,其后表达量下降,但其各时期表达量普遍较低。另一方面,研究通过明胶酶谱以及蛋白质印迹法观察到应用活化Zn2+离子干预的实验组较对照组角膜组织在碱烧伤后3日对高水平表达的基质金属蛋白酶-9以及碱烧伤后1周对呈现最高峰表达的基质金属蛋白酶-2及1的表达量均有明显的抑制作用,其抑制作用呈现剂量正相关性。结论：基质金属蛋白酶-9在碱烧伤后主要通过降解角膜上皮基底膜打通组织屏障使更多免疫损伤介质浸润角膜基质的方式,参与碱烧伤后角膜组织的病理性损伤过程。而基质金属蛋白酶-2则承担了更为重要的实质性组织损伤作用。研究亦从活体体内观察层面证实了活化Zn2+离子的局部应用对角膜碱烧伤后组织内基质金属蛋白酶的表达存在有效的抑制作用,从而为其应用于临床碱烧伤后角膜组织进行保护性治疗提供了理论依据。
[Abstract]:Objective: to investigate the types of matrix metalloproteinases (MMP) which play a major role in tissue injury of corneal fusion injury after alkali burn. To investigate the inhibitory effect of activated Zn2 ion on abnormal high expression of matrix metalloproteinases (MMP) in different stages of corneal fusion injury after alkali burn. Methods: an experimental animal model of moderate corneal alkali burn in mice was established. Gelatin zymogram, protein blot and immunohistochemical technique were used to detect the effects of alkali burn on the 3rd day after alkali burn. Expression of matrix metalloproteinase-1, 2 and 9 in corneal tissue at 1 week and 1 month. Different concentrations of Zn2 ion solutions (0.1mmol/L and 1mmol/L) were used to treat corneal tissue after alkali burn. Gelatin zymogram and Western blot were used to detect the expression of-1, 2 and 9 in corneal tissue of the experimental subgroup of different concentration of Zn2 ion solution at 3 days and 1 week after alkali burn as well as the control group. Results: the expression of matrix metalloproteinase-9 (MMP-9) in corneal tissue was significantly increased at 3 days after alkali burn by gelatin zymogram method, and decreased significantly one week after alkali burn, while the expression of MMP-9 in corneal tissue was significantly higher than that in control group (P < 0.05). The expression of matrix metalloproteinase-9 (MMP-9) was not observed in corneal tissue 1 month after alkali burn. On the contrary, the expression of matrix metalloproteinase-2 (MMP-2) in corneal tissue was only low on the 3rd day after alkali burn, and the expression of MMP-2 in corneal tissue increased significantly one week after alkali burn, showing a high level of expression. One month after alkali burn, the expression level decreased again, showing a moderate level of expression. The expression of matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-2 (MMP-2) were observed by protein blot and immunohistochemical method at 3, 1 and 1 month after alkali burn, which were consistent with those observed by gelatin zymogram. That is to say, matrix metalloproteinase-9 was highly expressed in acute stage after alkali burn, and it was mainly located near the corneal epithelium, and then decreased gradually. The expression level of MMP-2 in acute stage of alkali burn was lower than that in 1 week after alkali burn, which was different from that of matrix metalloproteinase-9. The expression level of MMP-2 was lower in corneal stroma after alkali burn than that of MMP-9, and the expression of MMP-2 was decreased again after alkali burn. There was a moderate level of expression. Protein blot and immunohistochemistry were used to observe the expression trend of matrix metalloproteinase-1 (MMP-1) in corneal tissue after alkali burn, which was similar to that of matrix metalloproteinase-2 (MMP-2). At 1 week after alkali burn, the expression level was the highest, and then decreased, but the expression level was generally low at each stage of alkali burn. On the other hand, The expression of matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-9 (MMP-9) were observed by gelatin zymogram and protein blotting in the corneal tissue of the experimental group treated with activated Zn2 ion on the 3rd day after alkali burn and after alkali burn. Zhou inhibited the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-1 (MMP-1), which was the highest expression of MMP-2 and MMP-1. The inhibitory effect showed a positive dose correlation. Conclusion: Matrix metalloproteinases-9 (MMP-9) is involved in the pathological process of corneal tissue injury after alkali burn by breaking down the epithelial basement membrane to open the tissue barrier and allowing more immune injury mediators to infiltrate the corneal stroma after alkali burn. [WT5 "HZ] conclusion: [WT5" BZ] [WT5 "BZ] Matrix metalloproteinase-2 (MMP-2) plays a more important role in tissue damage. The study also confirmed that the local application of activated Zn2 ion can effectively inhibit the expression of matrix metalloproteinases (MMP) in corneal tissue after alkali burn in vivo. It provides a theoretical basis for the protective treatment of corneal tissue after clinical alkali burn.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R772.2

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