乙酰肝素酶在脓毒症急性肾损伤中的作用及机制

发布时间：2018-05-23 19:18

  本文选题：乙酰肝素酶 + 脓毒症　； 参考：《第二军医大学》2014年博士论文

【摘要】：研究目的 脓毒症是一种细菌、病毒或真菌等感染后引发的严重全身性炎症反应，可导致休克、甚至死亡。在脓毒症初期24小时内急性肾损伤（acute kidney injury）发生率约为64%；一旦并发急性肾损伤，脓毒症患者死亡率增加一倍，因此脓毒症急性肾损伤的发生率和死亡率很高。目前脓毒症急性肾损伤发病机制尚未完全阐明，除抗感染治疗和支持疗之外，没有针对性治疗。所以，深入探讨脓毒症急性肾损伤发病机制，寻找新的治疗靶点具有重要的意义。 乙酰肝素酶（heparanase，HPSE）是哺乳动物体内唯一能够降解细胞糖萼中硫酸乙酰肝素成分的β-葡萄糖醛酸内切酶。因此，乙酰肝素酶与对糖萼的结构和功能起重要的调节作用。糖萼是被覆于内皮细胞表面的一层多糖蛋白复合物，与炎症的关系密切。研究发现血管内皮细胞糖萼在炎症反应中发生降解和脱落，并与白细胞的黏附和血管渗透性改变相关。近期的一项实验研究证实：小鼠注射脂多糖后，肺内皮细胞的乙酰肝素酶活化增加，引起糖萼的降解，暴露黏附分子，从而引起中性粒细胞对血管内皮细胞的黏附，发生炎症性肺损伤；硫酸乙酰肝素类似物抑制乙酰肝素酶后能使脓毒症急性肺损伤减轻。这提示，乙酰肝素酶在脓毒症急性肾损伤发病中也可能起到的作用，但目前尚无研究证据。 因此，我们应用脓毒症急性肾损伤动物和细胞模型，研究脓毒症中乙酰肝素酶对肾脏血管内皮细胞糖萼的作用，观察干预乙酰肝素酶后对肾脏的炎细胞黏附和血管内皮渗透性的影响，阐明乙酰肝素酶是否为肾脏炎症性损伤发生的关键因素；通过药物干预乙酰肝素酶，观察对脓毒症急性肾损伤的影响，阐明乙酰肝素酶抑制剂是否具有肾脏保护作用。该项研究为脓毒症急性肾损伤发病机制的研究开辟新思路，为治疗提供新靶点。 研究方法 首先，观察乙酰肝素酶是否参与脓毒症急性肾损伤的发病过程。脂多糖腹腔注射建立脓毒症小鼠模型，利用Western Blot和免疫组织化学方法，观察脓毒症对肾脏乙酰肝素酶的表达和分布的影响，探讨乙酰肝素酶是否参与脓毒症急性肾损伤的发生以及可能的影响部位。 其次，明确乙酰肝素酶对脓毒症肾间质微血管糖萼及炎细胞浸润的影响。WesternBlot观察脓毒症不同时间点肾脏乙酰肝素酶的蛋白表达与黏附分子表达之间的关系和TNF-α作用下人脐静脉内皮细胞乙酰肝素酶的活化情况；镧示踪法透射电镜观察脓毒症中乙酰肝素酶对肾间质微血管糖萼影响以及TNF-α所起的作用；HE染色观察干预乙酰肝素酶后肾间质炎细胞浸润的变化。以探讨乙酰肝素酶对脓毒症肾间质炎症发生的作用及其机制。 第三，明确乙酰肝素酶对肾小球内皮细胞糖萼和蛋白尿的影响。Western Blot观察TNF-α作用肾小球内皮细胞后，乙酰肝素酶的蛋白表达情况；镧示踪法透射电镜观察乙酰肝素酶对肾小球内皮细胞糖萼的影响以及TNF-α所起的作用；观察干预乙酰肝素酶后尿白蛋白-肌酐比值的变化。以探讨乙酰肝素酶对脓毒症急性肾损伤蛋白尿发生的影响及其机制。 第四，明确硫酸乙酰肝素类似物是否对脓毒症急性肾损伤起保护作用。不同剂量脂多糖分别建立脓毒症急性肾损伤和脓毒症生存率小鼠模型；观察硫酸乙酰肝素类似物对脓毒症小鼠的肾功能、24小时尿量和生存率的影响。 结果 （1）脓毒症小鼠肾脏乙酰肝素酶的蛋白表达量较正常明显增加，主要分布在肾间质血管内皮细胞和肾小球内皮细胞和系膜区。 （2）脓毒症小鼠肾脏的乙酰肝素酶表达量增加的时间点明显早于黏附分子（ICAM-1和VCAM-1）；人脐静脉内皮细胞在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，肾间质微血管内皮细胞糖萼减少至消失；加用乙酰肝素酶抑制剂后，血管内皮糖萼恢复；脓毒症小鼠肾间质可见炎细胞浸润；给予乙酰肝素酶抑制剂后肾间质则无炎细胞浸润；加用肝素酶Ⅲ促进糖萼降解，肾间质可见炎细胞浸润。 （3）TNF-α刺激肾小球内皮细胞后，乙酰肝素酶的活化蛋白表达显著增高；小鼠注射脂多糖或TNF-α后，肾小球内皮细胞糖萼层减少至消失；加用乙酰肝素酶抑制剂后糖萼层恢复；脓毒症小鼠的尿白蛋白-肌酐比值较正常明显增高，加用乙酰肝素酶抑制剂后，尿白蛋白-肌酐比值显著降低；加用肝素酶Ⅲ促进糖萼降解，脓毒症小鼠尿白蛋白-肌酐比值无下降。 （4）小鼠给予脂多糖10mg/kg，腹腔注射，24小时可作为脓毒症急性肾损伤模型的方案，观察肾功能的变化；小鼠给予脂多糖20mg/kg，腹腔注射可用来观察生存率的变化；脓毒症小鼠给予硫酸乙酰肝素类似物后，血清肌酐和尿素氮显著下降，24小时尿量增多，生存率显著上升。 结论 乙酰肝素酶是脓毒症急性肾损伤发病的重要因素，其机制可能为：脓毒症发生后，TNF-α使肾间质微血管内皮细胞的乙酰肝素酶活化增加，糖萼降解，引起肾间质白细胞浸润；同时，TNF-α使肾小球内皮细胞的乙酰肝素酶活化增加，糖萼降解，肾小球滤过屏障破坏，，引起蛋白尿。硫酸乙酰肝素类似物能够通过抑制乙酰肝素酶的活性，改善脓毒症急性肾损伤的肾功能损伤，提高脓毒症生存率。
[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 - 伪 .
The effect of heparanase in sepsis and the role of TNF - 伪 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 - 伪 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 - 伪 in human umbilical vein endothelial cells .
After the mice were injected with lipopolysaccharide or TNF - 伪 , 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 - 伪 , the expression of heparanase - activated protein was significantly increased .
After injection of lipopolysaccharide or TNF - 伪 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

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