同型半胱氨酸对实验性结肠炎大鼠sTM和EPCR表达的影响
发布时间：2019-01-14 07:37
【摘要】：目的:近年来溃疡性结肠炎(ulcerative colitis,UC)在我国发病率明显上升,UC发病基础尚有待于进一步的研究。在UC的病理生理过程中,肠粘膜微血管内皮细胞通过蛋白C(protein C,PC)途径对肠道炎症和凝血机制进行调节。同型半胱氨酸(homocysteine,Hcy)是一种含硫氨基酸,通过抑制PC途径中有关蛋白的表达,降低PC抗凝和抗炎作用,在心脑血管系统疾病的血栓形成过程中具有重要影响。由于IBD患者肠道黏膜中Hcy水平较高,肠道黏膜微血栓形成在UC病理过程中具有重要作用,Hcy是否通过影响肠道黏膜中微血栓形成过程的相关因素,加重结肠炎症反应目前尚不明确。因此,本研究通过检测Hcy对实验性结肠炎大鼠结肠黏膜可溶性血栓调节蛋白(soluable thrombomodulin,sTM)、PC、游离蛋白S(free protein S,fPS)水平和内皮细胞蛋白C受体(endothelial protein C receptor,EPCR)表达的影响,探讨Hcy在UC发病机制中的作用。方法:采用2,4,6-三硝酸苯磺酸(trinitro-benzene-sulfonic acid,TNBS)灌肠制备大鼠结肠炎模型。对照组以等体积的生理盐水(NS)灌肠。SD大鼠分为4组,A组(正常对照组):生理盐水灌肠+生理盐水皮下注射;B组(正常对照+Hcy注射组):生理盐水灌肠+Hcy皮下注射;C组(TNBS模型组):TNBS/乙醇溶液灌肠+生理盐水皮下注射;D组(TNBS模型+Hcy注射):TNBS/乙醇溶液灌肠+Hcy皮下注射。记录大鼠粪便性状及体重变化,以肉眼观察或粪便隐血试纸检测便血情况并进行疾病活动指数评分(disease activity index,DAI);ELISA法检测结肠炎大鼠血浆及结肠匀浆中Hcy和结肠黏膜中sTM、PC、fPS水平;逆转录聚合酶链反应(RT-qPCR)检测sTM和EPCRmRNA表达水平;免疫组化SP法检测结肠组织中TM及EPCR表达水平。结果:与正常对照组比较,TNBS结肠炎大鼠体重明显减轻,出现不同程度的肉眼血便,DAI评分增高(P0.05)。与正常对照组比较,TNBS模型组大鼠血浆和结肠组织Hcy水平明显增高(P0.01)。与正常对照+Hcy注射组比较,TNBS模型+Hcy注射组大鼠血浆及结肠组织Hcy水平显著增加(P0.01)。与正常对照组比较,TNBS结肠炎大鼠HI评分明显增高(P0.01),结肠MPO(P0.01)水平明显增高。与正常对照组比较,TNBS模型组大鼠结肠黏膜sTM、PC水平明显降低,fPS水平降低但差异无统计学意义,与TNBS模型组比较,TNBS模型+Hcy注射组大鼠结肠黏膜sTM、PC、fPS水平显著下降(P0.05)。免疫组化及RT-qPCR检测显示,与TNBS模型组比较,TNBS模型+Hcy注射组大鼠结肠黏膜中TM和EPCR表达显著降低(P0.05)。结论:Hcy可能通过降低实验性结肠炎大鼠结肠黏膜中PC通路中相关蛋白的表达,影响结肠黏膜微循环内皮细胞功能,加重大鼠结肠黏膜炎症损伤。
[Abstract]:Objective: in recent years, the incidence of ulcerative colitis (ulcerative colitis,UC) has increased significantly in China, and the pathogenesis of UC remains to be further studied. In the pathophysiological process of UC, intestinal microvascular endothelial cells regulate the mechanism of intestinal inflammation and coagulation through the protein C (protein Cpc pathway. Homocysteine (homocysteine,Hcy) is a kind of sulfur-containing amino acid. By inhibiting the expression of related proteins in the PC pathway and reducing the anticoagulant and anti-inflammatory effects of PC, homocysteine (homocysteine,Hcy) plays an important role in the thrombosis of cardiovascular and cerebrovascular diseases. Because of the high level of Hcy in the intestinal mucosa of IBD patients, intestinal mucosal microthrombosis plays an important role in the pathological process of UC. The reaction to aggravated colitis is unclear. Therefore, the effect of Hcy on the levels of soluble thrombomodulin (soluable thrombomodulin,sTM), PC, free protein S (free protein SfPS) and endothelial cell protein C receptor (endothelial protein C receptor, (endothelial protein C receptor,) in colonic mucosa of experimental colitis rats was determined. To explore the role of Hcy in the pathogenesis of UC. Methods: the colitis model of rats was established by enema with 2'4'4'6'- trinitrobenzene sulfonic acid (trinitro-benzene-sulfonic acid,TNBS). SD rats were divided into four groups: group A (normal control group), group B (normal control group): normal saline enema Hcy subcutaneous injection; Group C (TNBS model group): TNBS/ ethanol solution enema normal saline subcutaneous injection, group D (TNBS model Hcy injection): TNBS/ ethanol solution enema Hcy subcutaneous injection. The changes of fecal traits and body weight of rats were recorded. The hematochezia was detected by naked eye observation or fecal occult blood test paper and the disease activity index score (disease activity index,DAI) was carried out. The expression of sTM and EPCRmRNA was detected by reverse transcriptase polymerase chain reaction (RT-qPCR), and the expression of TM and EPCR in colonic tissue was detected by immunohistochemical SP method, and the levels of Hcy in plasma and colonic homogenate of colitis rats were detected by ELISA method, and the expression of sTM and EPCRmRNA in colon mucosa by reverse transcriptase polymerase chain reaction (RT-qPCR). Results: compared with the normal control group, the weight of TNBS colitis rats was significantly reduced, the naked blood stool appeared in varying degrees, and the DAI score was increased (P0.05). Compared with the normal control group, the level of Hcy in plasma and colon tissue in TNBS group was significantly higher (P0.01). Compared with the normal control Hcy injection group, the plasma and colon Hcy levels in the TNBS model Hcy injection group were significantly increased (P0.01). Compared with the normal control group, the HI score of TNBS colitis rats was significantly higher (P0.01), and the colon MPO (P0.01) level was significantly higher. Compared with the normal control group, the colonic mucosal sTM,PC level and the fPS level in the TNBS model group were significantly decreased, but the difference was not statistically significant. Compared with the TNBS model group, the TNBS model Hcy injection group showed the colon mucosal sTM,PC, of the rats. The level of fPS decreased significantly (P0.05). Compared with TNBS model group, the expression of TM and EPCR in colonic mucosa of TNBS model Hcy injection group was significantly lower than that of TNBS model group (P0.05). Conclusion: Hcy may decrease the expression of PC related protein in colonic mucosa of experimental colitis rats, and affect the function of endothelial cells of microcirculation of colonic mucosa and aggravate the inflammatory injury of colonic mucosa in rats.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62
[Abstract]:Objective: in recent years, the incidence of ulcerative colitis (ulcerative colitis,UC) has increased significantly in China, and the pathogenesis of UC remains to be further studied. In the pathophysiological process of UC, intestinal microvascular endothelial cells regulate the mechanism of intestinal inflammation and coagulation through the protein C (protein Cpc pathway. Homocysteine (homocysteine,Hcy) is a kind of sulfur-containing amino acid. By inhibiting the expression of related proteins in the PC pathway and reducing the anticoagulant and anti-inflammatory effects of PC, homocysteine (homocysteine,Hcy) plays an important role in the thrombosis of cardiovascular and cerebrovascular diseases. Because of the high level of Hcy in the intestinal mucosa of IBD patients, intestinal mucosal microthrombosis plays an important role in the pathological process of UC. The reaction to aggravated colitis is unclear. Therefore, the effect of Hcy on the levels of soluble thrombomodulin (soluable thrombomodulin,sTM), PC, free protein S (free protein SfPS) and endothelial cell protein C receptor (endothelial protein C receptor, (endothelial protein C receptor,) in colonic mucosa of experimental colitis rats was determined. To explore the role of Hcy in the pathogenesis of UC. Methods: the colitis model of rats was established by enema with 2'4'4'6'- trinitrobenzene sulfonic acid (trinitro-benzene-sulfonic acid,TNBS). SD rats were divided into four groups: group A (normal control group), group B (normal control group): normal saline enema Hcy subcutaneous injection; Group C (TNBS model group): TNBS/ ethanol solution enema normal saline subcutaneous injection, group D (TNBS model Hcy injection): TNBS/ ethanol solution enema Hcy subcutaneous injection. The changes of fecal traits and body weight of rats were recorded. The hematochezia was detected by naked eye observation or fecal occult blood test paper and the disease activity index score (disease activity index,DAI) was carried out. The expression of sTM and EPCRmRNA was detected by reverse transcriptase polymerase chain reaction (RT-qPCR), and the expression of TM and EPCR in colonic tissue was detected by immunohistochemical SP method, and the levels of Hcy in plasma and colonic homogenate of colitis rats were detected by ELISA method, and the expression of sTM and EPCRmRNA in colon mucosa by reverse transcriptase polymerase chain reaction (RT-qPCR). Results: compared with the normal control group, the weight of TNBS colitis rats was significantly reduced, the naked blood stool appeared in varying degrees, and the DAI score was increased (P0.05). Compared with the normal control group, the level of Hcy in plasma and colon tissue in TNBS group was significantly higher (P0.01). Compared with the normal control Hcy injection group, the plasma and colon Hcy levels in the TNBS model Hcy injection group were significantly increased (P0.01). Compared with the normal control group, the HI score of TNBS colitis rats was significantly higher (P0.01), and the colon MPO (P0.01) level was significantly higher. Compared with the normal control group, the colonic mucosal sTM,PC level and the fPS level in the TNBS model group were significantly decreased, but the difference was not statistically significant. Compared with the TNBS model group, the TNBS model Hcy injection group showed the colon mucosal sTM,PC, of the rats. The level of fPS decreased significantly (P0.05). Compared with TNBS model group, the expression of TM and EPCR in colonic mucosa of TNBS model Hcy injection group was significantly lower than that of TNBS model group (P0.05). Conclusion: Hcy may decrease the expression of PC related protein in colonic mucosa of experimental colitis rats, and affect the function of endothelial cells of microcirculation of colonic mucosa and aggravate the inflammatory injury of colonic mucosa in rats.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62
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