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蛋白C系统在溃疡性结肠炎中的变化及可能的作用

林旭红, 王慧超, 魏丹丹, 王斌, 葛全兴, 白春洋, 王亚强, 任学群

河南大学淮河医院检验科；第一附属医院肾内科；淮河医院消化内科；淮河医院普外科，开封 475000

摘要

血液高凝状态、血栓形成是导致溃疡性结肠炎(ulcerate colitis, UC)恶化的主要原因。本文旨在探讨蛋白C (protein C, PC)系统在UC小鼠中的变化及其可能的作用。(1)体内实验：采用饮用4%硫酸葡聚糖钠(dextran sulfate sodium, DSS)复制小鼠UC模型，造模后1周观察体重、结肠长度、脾重变化，并进行大体积分、组织学积分，免疫荧光法观察结肠平滑肌组织巨噬细胞数量，ELISA法测定血浆TNF-α、IL-6水平；活体荧光显微镜观察结肠黏膜微血管循环，免疫比浊法观察PC、蛋白S (protein S, PS)活性，免疫组化观察内皮细胞蛋白C受体(endothelial cell protein C receptor, EPCR)、血栓调节蛋白(thrombomodulin, TM)表达。(2)体外实验：分离小鼠结肠组织巨噬细胞，测定上清液中TNF-α、IL-6水平；分离、培养小鼠结肠黏膜微血管内皮细胞，分别以TNF-α、IL-6刺激后，检测其PC、PS、激活的蛋白C (activated protein C, APC)活性及EPCR、TM的表达。体内实验结果显示，DSS组小鼠体重减轻(P < 0.05)，结肠缩短(P < 0.05)，伴脾重增加(P < 0.05)，结肠组织学积分升高(P < 0.05)，结肠组织大量巨噬细胞浸润，血浆TNF-α、IL-6水平显著升高(P < 0.01)；活体显微镜结果显示，DSS组小鼠结肠黏膜微血管中白细胞与血管内皮细胞的粘附力大大升高(P < 0.01)，同时，血浆PC、PS活性明显降低(P < 0.01或P < 0.05)，结肠组织EPCR表达下调。相关性分析表明，结肠炎症程度与PC活性呈负相关。体外实验结果显示，DSS小鼠结肠组织中分离的巨噬细胞分泌TNF-α、IL-6水平均比对照组升高(P < 0.05)，而TNF-α或IL-6与小鼠结肠微血管内皮细胞共孵育后，内皮细胞APC活性明显降低(P < 0.05或P < 0.01)，其表达EPCR的能力均有所降低(P < 0.05)。以上结果提示，UC时PC系统被抑制，其可能的机制是巨噬细胞通过分泌促炎细胞因子进一步影响血管内皮细胞功能，从而抑制PC系统。提高PC系统水平可能是治疗UC的新靶点。

关键词： 蛋白C系统; 溃疡性结肠炎; 血栓形成

分类号：R363.2

[Study of the change and role of protein C system in ulcerate colitis.] [Article in Chinese]

LIN Xu-Hong, WANG Hui-Chao, WEI Dan-Dan, WANG Bin, GE Quan-Xing, BAI Chun-Yang, WANG Ya-Qiang, REN Xue-Qun

Department of Clinical Laboratory, Huaihe Hospital Affiliated to Henan University, Kaifeng 475000, China； Department of Nephrology, the First Hospital Affiliated to Henan University, Kaifeng 475000, China； Department of Digestive Medicine, Huaihe Hospital Affiliated to Henan University, Kaifeng 475000, China； Department of General Surgery, Huaihe Hospital Affiliated to Henan University, Kaifeng 475000, China

Abstract

Hypercoagulable state and thrombosis are major lethal causes of ulcerate colitis (UC). The aim of the present study is to explore the change and role of protein C (PC) system in UC thrombosis. 4% dextran sulfate sodium (DSS) was used to induce the UC model, and the body weight, the length of colon, and the weight of spleen were measured after intake of DSS as drinking water for 1 week. The macroscore and microscore were examined. The quantity of macrophage in colon smooth muscle was observed by immunofluorescence, and TNF-α and IL-6 levels in plasma were evaluated by ELISA. Intravital microscopy was applied to observe colonic mucosal microvascular circulation, activities of PC and protein S (PS) were determined by immunoturbidimetry, endothelial cell protein C receptor (EPCR) and thrombomodulin (TM) expressions were detected by immunohistochemistry. In vitro, TNF-α and IL-6 levels were tested in supernatant of macrophage separated from colonic tissue. After stimulation of mouse colonic mucosa mocrovascular endothelial cells by TNF-α and IL-6 respectively, the activities of PC, PS, activated protein C (APC) were evaluated, and the expressions of EPCR and TM were detected by Western blotting. The results revealed that the DSS mouse showed weight loss (P < 0.05), a shorten colon (P < 0.05), and swelled spleen (P < 0.05), accompanied by higher histological score (P < 0.05), as well as infiltration of macrophages, elevated TNF-α and IL-6 levels in plasma (P < 0.01). The intravital microscopy results revealed that compared with control, DSS mice showed significantly enhanced adhesion of leukocytes and colonic mucosal microvascular endothelial cells (P < 0.01), meanwhile, activity of PC and PS decreased obviously in plasma (P < 0.01 or P < 0.05), and expression of EPCR was down regulated. The degree of inflammation was negatively correlated with the PC activity. In vitro, TNF-α and IL-6 level were increased in the supernatant of macrophages from DSS mice colonic tissue (P < 0.05), and after incubation of TNF-α or IL-6 with colonic mucosal microvascular endothelial cells, the APC activity was decreased (P < 0.05 or P < 0.01), and expression of EPCR was down regulated (P < 0.05). These results suggest that PC system is inhibited in UC mouse. Presumably, the mechanism may be due to the secretion of cytokines from macrophages and subsequential influence on the function of endothelia cells. Furthermore, enhancement of PC system activity may serve as a new target for the treatment of UC.

Key words: protein C system; ulcerate colitis; thrombosis

收稿日期：2014-10-09　　录用日期：2014-12-24

通讯作者：任学群　　E-mail: hhyyrxq@126.com

引用本文：

林旭红, 王慧超, 魏丹丹, 王斌, 葛全兴, 白春洋, 王亚强, 任学群. 蛋白C系统在溃疡性结肠炎中的变化及可能的作用[J]. 生理学报 2015; 67 (2): 201-206.

LIN Xu-Hong, WANG Hui-Chao, WEI Dan-Dan, WANG Bin, GE Quan-Xing, BAI Chun-Yang, WANG Ya-Qiang, REN Xue-Qun. [Study of the change and role of protein C system in ulcerate colitis.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (2): 201-206 (in Chinese with English abstract).