【摘要】：目的：探討在堿燒傷后角膜融解損傷病理作用中不同時(shí)間內(nèi)起主要組織損傷作用的基質(zhì)金屬蛋白酶的類型；探討活化Zn2+離子對(duì)堿燒傷后角膜融解損傷不同時(shí)期異常高表達(dá)的基質(zhì)金屬蛋白酶的抑制作用。 方法：建立中度角膜堿燒傷小鼠實(shí)驗(yàn)動(dòng)物模型,應(yīng)用明膠酶譜、蛋白質(zhì)印跡法以及免疫組織化學(xué)技術(shù)檢測(cè)堿燒傷后3日、1周以及1個(gè)月時(shí)角膜組織內(nèi)基質(zhì)金屬蛋白酶-1,2及9的表達(dá)情況。應(yīng)用不同濃度Zn2+離子溶液(0.1mmol/L及1mmol/L)對(duì)堿燒傷后角膜組織進(jìn)行局部干預(yù)。應(yīng)用明膠酶譜以及蛋白質(zhì)印跡法檢測(cè)角膜堿燒傷后3日及1周時(shí)不同濃度Zn2+離子溶液實(shí)驗(yàn)亞組以及對(duì)照組角膜組織內(nèi)中-1,2及9的表達(dá)情況。 結(jié)果：應(yīng)用明膠酶譜方法觀察到堿燒傷后3日角膜組織內(nèi)基質(zhì)金屬蛋白酶-9表達(dá)顯著的升高,呈現(xiàn)高水平表達(dá),堿燒傷后1周其表達(dá)量顯著下降,而堿燒傷后1個(gè)月時(shí)角膜組織內(nèi)觀察不到基質(zhì)金屬蛋白酶-9的表達(dá)。基質(zhì)金屬蛋白酶-2與之相反,在堿燒傷后3日角膜組織內(nèi)僅呈現(xiàn)低水平表達(dá),堿燒傷后1周其角膜組織內(nèi)表達(dá)量顯著升高,呈現(xiàn)高水平表達(dá),堿燒傷后1個(gè)月時(shí)其表達(dá)量再次降低,呈現(xiàn)中度水平表達(dá)。應(yīng)用蛋白質(zhì)印跡法以及免疫組織化學(xué)方法觀察的堿燒傷后3日、1周及1個(gè)月組織內(nèi)基質(zhì)金屬蛋白酶-9及2的表達(dá)變化情況與應(yīng)用明膠酶譜方法觀察的情況相符,即：基質(zhì)金屬蛋白酶-9在堿燒傷后急性期高表達(dá),且其高表達(dá)主要位于靠近角膜上皮層區(qū)域,而后表達(dá)量逐漸降低；基質(zhì)金屬蛋白酶-2在堿燒傷后急性期表達(dá)水平較低,而在堿燒傷后1周呈現(xiàn)高水平表達(dá),不同于基質(zhì)金屬蛋白酶-9,其高表達(dá)分布于角膜基質(zhì)內(nèi),其后表達(dá)量再次降低,呈現(xiàn)中度水平表達(dá)。應(yīng)用蛋白質(zhì)印跡法以及免疫組織化學(xué)方法觀察到堿燒傷后組織內(nèi)不同時(shí)期基質(zhì)金屬蛋白酶-1在角膜堿燒傷后組織內(nèi)的表達(dá)變化趨勢(shì)與基質(zhì)金屬蛋白酶-2相似,在堿燒傷后1周時(shí)表達(dá)水平最高,其后表達(dá)量下降,但其各時(shí)期表達(dá)量普遍較低。另一方面,研究通過明膠酶譜以及蛋白質(zhì)印跡法觀察到應(yīng)用活化Zn2+離子干預(yù)的實(shí)驗(yàn)組較對(duì)照組角膜組織在堿燒傷后3日對(duì)高水平表達(dá)的基質(zhì)金屬蛋白酶-9以及堿燒傷后1周對(duì)呈現(xiàn)最高峰表達(dá)的基質(zhì)金屬蛋白酶-2及1的表達(dá)量均有明顯的抑制作用,其抑制作用呈現(xiàn)劑量正相關(guān)性。 結(jié)論：基質(zhì)金屬蛋白酶-9在堿燒傷后主要通過降解角膜上皮基底膜打通組織屏障使更多免疫損傷介質(zhì)浸潤(rùn)角膜基質(zhì)的方式,參與堿燒傷后角膜組織的病理性損傷過程。而基質(zhì)金屬蛋白酶-2則承擔(dān)了更為重要的實(shí)質(zhì)性組織損傷作用。研究亦從活體體內(nèi)觀察層面證實(shí)了活化Zn2+離子的局部應(yīng)用對(duì)角膜堿燒傷后組織內(nèi)基質(zhì)金屬蛋白酶的表達(dá)存在有效的抑制作用,從而為其應(yīng)用于臨床堿燒傷后角膜組織進(jìn)行保護(hù)性治療提供了理論依據(jù)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R772.2

【共引文獻(xiàn)】

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本文編號(hào)：2456074