本文選題：乙酰肝素酶 + 膿毒癥��； 參考：《第二軍醫(yī)大學》2014年博士論文

【摘要】：研究目的 膿毒癥是一種細菌、病毒或真菌等感染后引發(fā)的嚴重全身性炎癥反應，可導致休克、甚至死亡。在膿毒癥初期24小時內(nèi)急性腎損傷（acute kidney injury）發(fā)生率約為64%；一旦并發(fā)急性腎損傷，膿毒癥患者死亡率增加一倍，因此膿毒癥急性腎損傷的發(fā)生率和死亡率很高。目前膿毒癥急性腎損傷發(fā)病機制尚未完全闡明，除抗感染治療和支持療之外，沒有針對性治療。所以，深入探討膿毒癥急性腎損傷發(fā)病機制，尋找新的治療靶點具有重要的意義。 乙酰肝素酶（heparanase，HPSE）是哺乳動物體內(nèi)唯一能夠降解細胞糖萼中硫酸乙酰肝素成分的β-葡萄糖醛酸內(nèi)切酶。因此，乙酰肝素酶與對糖萼的結(jié)構(gòu)和功能起重要的調(diào)節(jié)作用。糖萼是被覆于內(nèi)皮細胞表面的一層多糖蛋白復合物，與炎癥的關(guān)系密切。研究發(fā)現(xiàn)血管內(nèi)皮細胞糖萼在炎癥反應中發(fā)生降解和脫落，并與白細胞的黏附和血管滲透性改變相關(guān)。近期的一項實驗研究證實：小鼠注射脂多糖后，肺內(nèi)皮細胞的乙酰肝素酶活化增加，引起糖萼的降解，暴露黏附分子，從而引起中性粒細胞對血管內(nèi)皮細胞的黏附，發(fā)生炎癥性肺損傷；硫酸乙酰肝素類似物抑制乙酰肝素酶后能使膿毒癥急性肺損傷減輕。這提示，乙酰肝素酶在膿毒癥急性腎損傷發(fā)病中也可能起到的作用，但目前尚無研究證據(jù)。 因此，我們應用膿毒癥急性腎損傷動物和細胞模型，研究膿毒癥中乙酰肝素酶對腎臟血管內(nèi)皮細胞糖萼的作用，觀察干預乙酰肝素酶后對腎臟的炎細胞黏附和血管內(nèi)皮滲透性的影響，闡明乙酰肝素酶是否為腎臟炎癥性損傷發(fā)生的關(guān)鍵因素；通過藥物干預乙酰肝素酶，觀察對膿毒癥急性腎損傷的影響，闡明乙酰肝素酶抑制劑是否具有腎臟保護作用。該項研究為膿毒癥急性腎損傷發(fā)病機制的研究開辟新思路，為治療提供新靶點。 研究方法 首先，觀察乙酰肝素酶是否參與膿毒癥急性腎損傷的發(fā)病過程。脂多糖腹腔注射建立膿毒癥小鼠模型，利用Western Blot和免疫組織化學方法，觀察膿毒癥對腎臟乙酰肝素酶的表達和分布的影響，探討乙酰肝素酶是否參與膿毒癥急性腎損傷的發(fā)生以及可能的影響部位。 其次，明確乙酰肝素酶對膿毒癥腎間質(zhì)微血管糖萼及炎細胞浸潤的影響。WesternBlot觀察膿毒癥不同時間點腎臟乙酰肝素酶的蛋白表達與黏附分子表達之間的關(guān)系和TNF-α作用下人臍靜脈內(nèi)皮細胞乙酰肝素酶的活化情況；鑭示蹤法透射電鏡觀察膿毒癥中乙酰肝素酶對腎間質(zhì)微血管糖萼影響以及TNF-α所起的作用；HE染色觀察干預乙酰肝素酶后腎間質(zhì)炎細胞浸潤的變化。以探討乙酰肝素酶對膿毒癥腎間質(zhì)炎癥發(fā)生的作用及其機制。 第三，明確乙酰肝素酶對腎小球內(nèi)皮細胞糖萼和蛋白尿的影響。Western Blot觀察TNF-α作用腎小球內(nèi)皮細胞后，乙酰肝素酶的蛋白表達情況；鑭示蹤法透射電鏡觀察乙酰肝素酶對腎小球內(nèi)皮細胞糖萼的影響以及TNF-α所起的作用；觀察干預乙酰肝素酶后尿白蛋白-肌酐比值的變化。以探討乙酰肝素酶對膿毒癥急性腎損傷蛋白尿發(fā)生的影響及其機制。 第四，明確硫酸乙酰肝素類似物是否對膿毒癥急性腎損傷起保護作用。不同劑量脂多糖分別建立膿毒癥急性腎損傷和膿毒癥生存率小鼠模型；觀察硫酸乙酰肝素類似物對膿毒癥小鼠的腎功能、24小時尿量和生存率的影響。 結(jié)果 （1）膿毒癥小鼠腎臟乙酰肝素酶的蛋白表達量較正常明顯增加，主要分布在腎間質(zhì)血管內(nèi)皮細胞和腎小球內(nèi)皮細胞和系膜區(qū)。 （2）膿毒癥小鼠腎臟的乙酰肝素酶表達量增加的時間點明顯早于黏附分子（ICAM-1和VCAM-1）；人臍靜脈內(nèi)皮細胞在TNF-α作用后，乙酰肝素酶活化蛋白增加；小鼠注射脂多糖或TNF-α后，腎間質(zhì)微血管內(nèi)皮細胞糖萼減少至消失；加用乙酰肝素酶抑制劑后，血管內(nèi)皮糖萼恢復；膿毒癥小鼠腎間質(zhì)可見炎細胞浸潤；給予乙酰肝素酶抑制劑后腎間質(zhì)則無炎細胞浸潤；加用肝素酶Ⅲ促進糖萼降解，腎間質(zhì)可見炎細胞浸潤。 （3）TNF-α刺激腎小球內(nèi)皮細胞后，乙酰肝素酶的活化蛋白表達顯著增高；小鼠注射脂多糖或TNF-α后，腎小球內(nèi)皮細胞糖萼層減少至消失；加用乙酰肝素酶抑制劑后糖萼層恢復；膿毒癥小鼠的尿白蛋白-肌酐比值較正常明顯增高，加用乙酰肝素酶抑制劑后，尿白蛋白-肌酐比值顯著降低；加用肝素酶Ⅲ促進糖萼降解，膿毒癥小鼠尿白蛋白-肌酐比值無下降。 （4）小鼠給予脂多糖10mg/kg，腹腔注射，24小時可作為膿毒癥急性腎損傷模型的方案，觀察腎功能的變化；小鼠給予脂多糖20mg/kg，腹腔注射可用來觀察生存率的變化；膿毒癥小鼠給予硫酸乙酰肝素類似物后，血清肌酐和尿素氮顯著下降，24小時尿量增多，生存率顯著上升。 結(jié)論 乙酰肝素酶是膿毒癥急性腎損傷發(fā)病的重要因素，其機制可能為：膿毒癥發(fā)生后，TNF-α使腎間質(zhì)微血管內(nèi)皮細胞的乙酰肝素酶活化增加，糖萼降解，引起腎間質(zhì)白細胞浸潤；同時，TNF-α使腎小球內(nèi)皮細胞的乙酰肝素酶活化增加，糖萼降解，腎小球濾過屏障破壞，，引起蛋白尿。硫酸乙酰肝素類似物能夠通過抑制乙酰肝素酶的活性，改善膿毒癥急性腎損傷的腎功能損傷，提高膿毒癥生存率。
[Abstract]:Purpose of study

Septicemia is a serious systemic inflammatory response induced after infection , such as bacteria , viruses , or fungi , leading to shock or even death . Acute kidney injury ( acute kidney injury ) occurs at a rate of about 64 % in the first 24 hours of sepsis ;
At present , the pathogenesis of acute renal injury in sepsis has not been fully elucidated , and the pathogenesis of acute renal injury in sepsis has not been fully elucidated . Therefore , it is of great significance to study the pathogenesis of acute renal injury in sepsis and find new therapeutic target .

heparanase ( HPSE ) is the only 尾 - glucuronase which can degrade heparan sulfate component in the cell glycocalycis . Therefore , heparanase and the structure and function of glycocalyse play an important role in regulating the structure and function of glycocalycis .
heparan sulfate analogs inhibit heparanase and reduce acute lung injury . This suggests that heparanase may play a role in the pathogenesis of sepsis acute renal injury , but there is no evidence available at present .

Therefore , we used the animal and cellular model of sepsis acute kidney injury to study the effect of heparanase on the vascular endothelial cells of renal vascular endothelial cells , observe the effect of heparanase on the adhesion of inflammatory cells and vascular endothelial cell permeability of the kidney , and clarify whether heparanase is a key factor in the pathogenesis of renal inflammatory injury .
The effect of heparanase inhibitor on acute renal injury of sepsis was observed by the intervention of heparanase and the effect of heparanase inhibitor on acute renal injury . The study opened a new way for the study of pathogenesis of sepsis acute renal injury , which provided a new target for treatment .

Research Methods

First , whether heparanase was involved in the pathogenesis of septic acute renal injury was observed . The effect of sepsis on the expression and distribution of heparanase was observed by Western Blot and immunohistochemistry .

Western Blot was used to observe the relationship between the expression of heparanase and adhesion molecule and the activation of heparanase in human umbilical vein endothelial cells under the action of TNF - 偽 .
The effect of heparanase in sepsis and the role of TNF - 偽 in sepsis were observed by lanthanum tracer transmission electron microscope .
To investigate the effect of heparanase on renal interstitial inflammation in sepsis and its mechanism .

Thirdly , the effects of heparanase on the glycocal and proteinuria in glomerular endothelial cells were determined . Western Blot was used to observe the expression of heparanase in glomerular endothelial cells .
The effect of heparanase on the glycocal of glomerular endothelial cells and the function of TNF - 偽 were observed by lanthanum tracer transmission electron microscope ( TEM ) .
To investigate the effect of heparanase on proteinuria and its mechanism in acute renal injury caused by sepsis .

Fourthly , it is clear whether heparan sulfate analogues protect the acute renal injury of sepsis .
To observe the effect of heparan sulfate analogues on renal function , 24 hour urine volume and survival rate in septic mice .

Results

( 1 ) The expression of heparanase was significantly increased in septic mice , mainly in renal interstitial vascular endothelial cells and glomerular endothelial cells and mesangial area .

( 2 ) The time point of the increase of heparanase expression in the kidney of septic mice was significantly earlier than that of adhesion molecules ( ICAM - 1 and VCAM - 1 ) ;
The activity of heparanase activated protein increased after TNF - 偽 in human umbilical vein endothelial cells .
After the mice were injected with lipopolysaccharide or TNF - 偽 , the vascular endothelial cells of renal interstitial microvessels decreased to disappear .
after adding heparanase inhibitor , the vascular endothelial glycocalyse is recovered ;
The infiltration of inflammatory cells was seen in the renal interstitial cells of septic mice .
There was no inflammatory cell infiltration in the renal stroma after the heparanase inhibitor was given .
In addition , heparanase III is added to promote the degradation of glycocalycis , and the infiltration of inflammatory cells can be seen in the renal stroma .

( 3 ) After stimulation of glomerular endothelial cells with TNF - 偽 , the expression of heparanase - activated protein was significantly increased .
After injection of lipopolysaccharide or TNF - 偽 into mice , the content of glycocalyses of glomerulus endothelial cells decreased to disappear .
adding heparanase inhibitor , and recovering the glycocalyse layer ;
The urinary albumin - creatinine ratio of septic mice was significantly higher than that of normal mice . After adding heparanase inhibitor , the ratio of urinary albumin to creatinine was significantly decreased .
The ratio of urinary albumin and creatinine in septic mice was not decreased with heparinase 鈪

本文編號：1926009