血管缺氧再灌注损伤时超氧化物自由基过氧化氢分布的电镜细胞化学研究

CYTOCHEMICAL LOCALIZATION OF SUPEROXIDE FREE RADICAL HYDROGEN PEROXIDE IN HYPOXIA-REPERFUSED VESSELS

用电镜细胞化学方法，从形态上研究超氧化物自由基的分布。经５、１０、２０ｍｉｎ缺氧灌注和含ＣｅＣｌ３的富氧液再灌注１０ｍｉｎ的颈总动脉为样品。结果发现，缺氧时间越长，内皮细胞超微结构改变逐渐明显，甚至脱落。内皮的腔面有电子致密物出现，缺氧时间越长，致密层越厚。用Ｑ５２０图像分析仪测量内皮单位长度沉积物的面积，三个时间组分别为１５０．３±１１．４、１６５．６±９．８和２０３．５±６．８ｎｍ２／ｎｍ（ｎ＝５）。经统计分析，５、１０ｍｉｎ组与２０ｍｉｎ组间差异很显著（Ｐ＜０．０１）；５、１０ｍｉｎ组间差异显著（Ｐ＜０．０５）。富氧灌注液中无ＣｅＣｌ３时，则致密沉淀不出现。当加入过氧化氢酶３００ｕ／ｍｌ进行灌注时，沉淀明显减少至２２．７±４．４ｎｍ２／ｎｍ（Ｐ＜０．０１）；６００ｕ／ｍｌ时，沉淀完全清除。对血管内皮沉淀物进行Ｘ射线微区分析，发现在４．８４、５．２６、５．８１ｋｅＶ处有三个Ｃｅ特征峰（ＣｅＬα、ＣｅＬβ、ＣｅＬ３ａｂｓ），其中４．８４ｋｅＶ处的ＣｅＬα峰最高，证明沉淀物中有Ｃｅ存在，又由于过氧化氢酶消除沉淀的作用明显，说明沉淀物为Ｎ２Ｏ２和Ｃｅ所形成。这一方法为研究超氧自由基在超微结构水平上的分布提供了?

The electron microscopic cytochemical method, using CeCl3 as a capture agent to from electron - dense products of Ce - H2O2 complex, was adapted to localize ultrastructurally H2O2 produced by endotheelial cells of vessels afters hypoxia - reperfusion. Rats were perfused via their left ventricle by hypoxia buffer for 5, 10, and 20min respectively followed by 10min reperfusion wiith oxygenated buffer containing CeCl3. The ultrastructure alteration in endothelial cells of a common carotid artery was found to be more serious when the hypoxia duration was getting longer. After 20min hypoxia perfusion, the endothelial cells even dropped away from the endothelium. At the same time, the eelectron dense precipitate appeared on the luminal face of endotheial cells. The longer hypoxia lasted, the thicker precipitate formed. The area/length ratio (nm2/nm) of the precipitate was assessed by imaging analyzer. The precipitate were 150. 3±11. 4, 165. 6±9. 8 and 203. 5±6. 8nm2/nm(n = 5) respectively in 5, 10 and 20min group. The differences betweeen 20min and 5, 10min groups were very significant (P <0. 01), and the signficant difference between 5min and 10min group was also showed (P<0. 05). No precipitate appeared when CeCl3 did not be added in the reperfusion buffer. When catalase was added into perfusion buffers, the precipitate was decreased apparently or almost disappeared. Electron microscopic x- ray microanalysis of the endothelial electron dense precipitate revealed three characteristic peaks of Ce, indicating the presence of Ce. In connection with that catalase eliminated significantly the precipitate, it indicated that the precipitate was formed by H2O2 and Ce .This method may provide a new way to localize peroxide free radicals at the ultrastructural level.