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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,与对照组相比,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(P<0.01)。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与小鼠结肠黏膜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的新策略。
Hypercoagulable state of blood, thrombosis is the leading cause of ulcerative colitis (UC) deterioration. This article aims to investigate the protein C (PC) system in UC mice and its possible role. (1) In vivo experiments: The model of mouse UC was reproduced by drinking dextran sulfate sodium (DSS) 4%, body weight, length of colon and spleen weight were observed one week after modeling, and the volume, histology The number of macrophages in colonic smooth muscle was observed by immunofluorescence method. The levels of TNF-α and IL-6 in plasma were measured by ELISA. The colonic mucosal microvascular circulation was observed by fluorescence microscope. The protein S (PS) ) Activity, and the expression of endothelial cell protein C receptor (EPCR) and thrombomodulin (TM) was observed by immunohistochemistry. (2) In vitro experiments: Colonic macrophages from mice were isolated and the levels of TNF-α and IL-6 in the supernatant were measured. The colonic mucosal microvascular endothelial cells were isolated and cultured and stimulated with TNF-αand IL-6 The PC, PS, activated protein C (APC) activity and the expression of EPCR and TM were detected. The results of in vivo experiments showed that compared with the control group, the body weight of the mice in the DSS group decreased (P <0.05), the colon shortened (P <0.05), the spleen weight increased (P <0.05) and the colon histological score increased (P <0.01). The results of in vivo microscopy showed that the adhesion of leukocytes to vascular endothelial cells in the colon mucosal microvessels of DSS mice was significantly increased (P <0.01). At the same time, the activity of PC and PS in plasma decreased significantly (P <0.01 or P <0.05), and the expression of EPCR in colon decreased (P <0.01). Correlation analysis showed that the degree of colitis and PC activity was negatively correlated. The results of in vitro experiments showed that the levels of TNF-α and IL-6 secreted by macrophages isolated from the colon of DSS mice were significantly higher than those of the control group (P <0.05), but TNF-α or IL- After co-cultured with microvascular endothelial cells, the activity of APC in endothelial cells was significantly decreased (P <0.05 or P <0.01), and its ability to express EPCR was decreased (P <0.05). The above results suggest that the PC system is inhibited when UC, the possible mechanism is that macrophages through the secretion of proinflammatory cytokines further affect the function of vascular endothelial cells, thereby inhibiting the PC system. Improving PC system level may be a new strategy for the treatment of UC.