免疫组织化学在心肌梗死中一氧化氮合酶同工酶鉴定中的应用

Immunohistochemistry in the identification of nitric oxide synthase isoenzymes in myocardial infarction.

作者信息

Wildhirt S M, Dudek R R, Suzuki H, Pinto V, Narayan K S, Bing R J

机构信息

Department of Experimental Cardiology, Huntington Medical Research Institutes, Pasadena, CA 91101, USA.

出版信息

Cardiovasc Res. 1995 Apr;29(4):526-31.

PMID:7540956

Abstract

OBJECTIVE

Inducible nitric oxide synthase (iNOS) activity, as measured by conversion of L-14C-arginine to L-14C-citrulline is significantly increased in infarcted rabbit myocardium as compared to healthy myocardium 48 h after coronary occlusion. The aim of this study was to localise the nitric oxide synthase (NOS) isoforms in normal myocardium and compare these findings with NOS activity in cells of the infarcted region.

METHODS

Activities of NOS isoforms were enzymatically assayed in normal and infarcted myocardium. Localisation of NOS was performed on identical sections using specific monoclonal IgG antibodies against endothelial constitutive (cNOS) and macrophage inducible (iNOS) nitric oxide synthase. In addition, macrophages were identified using fluorescein conjugated ChromPure rabbit IgG, Fc fragment.

RESULTS

iNOS activity increased significantly in infarcted as compared to normal myocardium [0.42(SEM 0.03) v 0.85(0.08) fmol.microgram-1.min-1, P = 0.02, respectively]. However, cNOS did not increase significantly in infarcted regions [0.18(0.04) v 0.24(0.06) fmol.microgram-1.min-1; P = 0.16, respectively]. cNOS was immunohistochemically localised in endothelial and endocardial cells in normal and infarcted tissues. The presence of iNOS activity in macrophages in infarcted myocardium was identified immunohistochemically. Cardiomyocytes and neutrophils did not label with the antibodies to cNOS and iNOS.

CONCLUSIONS

(1) Infiltrating macrophages are the main site of increased iNOS activity in infarcted rabbit myocardium. (2) cNOS activity is not significantly increased in infarcted tissues as compared to normal myocardium. (3) Neutrophils and cardiomyocytes do not express NOS immunoreactivity in infarcted and normal rabbit myocardium.

摘要

目的

通过检测 L-14C-精氨酸转化为 L-14C-瓜氨酸来测定诱导型一氧化氮合酶（iNOS）活性，结果显示，与冠状动脉闭塞 48 小时后的健康心肌相比，梗死兔心肌中的该活性显著增加。本研究的目的是确定正常心肌中一氧化氮合酶（NOS）同工型的定位，并将这些结果与梗死区域细胞中的 NOS 活性进行比较。

方法

采用酶法检测正常和梗死心肌中 NOS 同工型的活性。使用针对内皮组成型（cNOS）和巨噬细胞诱导型（iNOS）一氧化氮合酶的特异性单克隆 IgG 抗体，在相同切片上进行 NOS 的定位。此外，使用异硫氰酸荧光素偶联的 ChromPure 兔 IgG Fc 片段鉴定巨噬细胞。

结果

与正常心肌相比，梗死心肌中的 iNOS 活性显著增加[分别为 0.42（标准误 0.03）对 0.85（0.08）fmol·μg-1·min-1，P = 0.02]。然而，梗死区域的 cNOS 没有显著增加[分别为 0.18（0.04）对 0.24（0.06）fmol·μg-1·min-1；P = 0.16]。cNOS 通过免疫组织化学法定位在正常和梗死组织的内皮细胞和心内膜细胞中。通过免疫组织化学法鉴定出梗死心肌中巨噬细胞存在 iNOS 活性。心肌细胞和中性粒细胞未被 cNOS 和 iNOS 的抗体标记。