同型半胱氨酸對(duì)實(shí)驗(yàn)性結(jié)腸炎大鼠sTM和EPCR表達(dá)的影響
發(fā)布時(shí)間：2019-01-14 07:37
【摘要】：目的:近年來潰瘍性結(jié)腸炎(ulcerative colitis,UC)在我國發(fā)病率明顯上升,UC發(fā)病基礎(chǔ)尚有待于進(jìn)一步的研究。在UC的病理生理過程中,腸粘膜微血管內(nèi)皮細(xì)胞通過蛋白C(protein C,PC)途徑對(duì)腸道炎癥和凝血機(jī)制進(jìn)行調(diào)節(jié)。同型半胱氨酸(homocysteine,Hcy)是一種含硫氨基酸,通過抑制PC途徑中有關(guān)蛋白的表達(dá),降低PC抗凝和抗炎作用,在心腦血管系統(tǒng)疾病的血栓形成過程中具有重要影響。由于IBD患者腸道黏膜中Hcy水平較高,腸道黏膜微血栓形成在UC病理過程中具有重要作用,Hcy是否通過影響腸道黏膜中微血栓形成過程的相關(guān)因素,加重結(jié)腸炎癥反應(yīng)目前尚不明確。因此,本研究通過檢測(cè)Hcy對(duì)實(shí)驗(yàn)性結(jié)腸炎大鼠結(jié)腸黏膜可溶性血栓調(diào)節(jié)蛋白(soluable thrombomodulin,sTM)、PC、游離蛋白S(free protein S,fPS)水平和內(nèi)皮細(xì)胞蛋白C受體(endothelial protein C receptor,EPCR)表達(dá)的影響,探討Hcy在UC發(fā)病機(jī)制中的作用。方法:采用2,4,6-三硝酸苯磺酸(trinitro-benzene-sulfonic acid,TNBS)灌腸制備大鼠結(jié)腸炎模型。對(duì)照組以等體積的生理鹽水(NS)灌腸。SD大鼠分為4組,A組(正常對(duì)照組):生理鹽水灌腸+生理鹽水皮下注射;B組(正常對(duì)照+Hcy注射組):生理鹽水灌腸+Hcy皮下注射;C組(TNBS模型組):TNBS/乙醇溶液灌腸+生理鹽水皮下注射;D組(TNBS模型+Hcy注射):TNBS/乙醇溶液灌腸+Hcy皮下注射。記錄大鼠糞便性狀及體重變化,以肉眼觀察或糞便隱血試紙檢測(cè)便血情況并進(jìn)行疾病活動(dòng)指數(shù)評(píng)分(disease activity index,DAI);ELISA法檢測(cè)結(jié)腸炎大鼠血漿及結(jié)腸勻漿中Hcy和結(jié)腸黏膜中sTM、PC、fPS水平;逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-qPCR)檢測(cè)sTM和EPCRmRNA表達(dá)水平;免疫組化SP法檢測(cè)結(jié)腸組織中TM及EPCR表達(dá)水平。結(jié)果:與正常對(duì)照組比較,TNBS結(jié)腸炎大鼠體重明顯減輕,出現(xiàn)不同程度的肉眼血便,DAI評(píng)分增高(P0.05)。與正常對(duì)照組比較,TNBS模型組大鼠血漿和結(jié)腸組織Hcy水平明顯增高(P0.01)。與正常對(duì)照+Hcy注射組比較,TNBS模型+Hcy注射組大鼠血漿及結(jié)腸組織Hcy水平顯著增加(P0.01)。與正常對(duì)照組比較,TNBS結(jié)腸炎大鼠HI評(píng)分明顯增高(P0.01),結(jié)腸MPO(P0.01)水平明顯增高。與正常對(duì)照組比較,TNBS模型組大鼠結(jié)腸黏膜sTM、PC水平明顯降低,fPS水平降低但差異無統(tǒng)計(jì)學(xué)意義,與TNBS模型組比較,TNBS模型+Hcy注射組大鼠結(jié)腸黏膜sTM、PC、fPS水平顯著下降(P0.05)。免疫組化及RT-qPCR檢測(cè)顯示,與TNBS模型組比較,TNBS模型+Hcy注射組大鼠結(jié)腸黏膜中TM和EPCR表達(dá)顯著降低(P0.05)。結(jié)論:Hcy可能通過降低實(shí)驗(yàn)性結(jié)腸炎大鼠結(jié)腸黏膜中PC通路中相關(guān)蛋白的表達(dá),影響結(jié)腸黏膜微循環(huán)內(nèi)皮細(xì)胞功能,加重大鼠結(jié)腸黏膜炎癥損傷。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R574.62
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