發(fā)布時間：2018-04-17 20:16

  本文選題：膿毒血癥 + 腎損傷　； 參考：《浙江大學》2015年博士論文

【摘要】：背景： 嚴重感染(severe sepsis)所繼發(fā)的膿毒血癥和多臟器功能障礙綜合征(multiple organ dysfunction syndrome, MODS)是當前重癥監(jiān)護病房(intensive care unit, ICU)內(nèi)的主要死亡原因,也是當前重癥醫(yī)學面臨的主要焦點及難點。雖然對膿毒癥的病理生理學機制和臨床治療進行了大量研究,然而迄今為止患者的發(fā)病率和病死率仍然居高不下。膿毒血癥發(fā)生后腎臟往往是最容易遭受損傷的器官,根據(jù)醫(yī)學界已基本達成共識的急性腎損傷(Acute kidney injury, AKI)標準(RIFLE標準),有近8%的住院病人及50%以上的ICU患者出現(xiàn)AKI。因此,AKI已成為現(xiàn)代醫(yī)院及ICU的主要臨床難題。此外,有確鑿證據(jù)表明,嚴重感染及膿毒血癥是導致危重癥患者發(fā)生AKI的最主要原因,在ICU中,大約50%以上的AKI由此引發(fā)。雖然目前臨床上臟器的支持治療和液體復蘇治療水平正日益增強,但由膿毒血癥所誘發(fā)的AKI的發(fā)生率及死亡率未有下降,這可能與其確切的發(fā)病機制尚未明確有關(guān)。 新近的研究發(fā)現(xiàn)膿毒血癥的主要病理生理機制——微循環(huán)障礙可能與內(nèi)皮表面糖萼(glycocalyx,多糖-蛋白復合物)的狀態(tài)密切相關(guān)。越來越多的研究已認識到血管內(nèi)皮糖萼對心血管疾病具有重要的意義。糖萼是襯于血管內(nèi)皮細胞管腔膜側(cè),覆蓋著一種富含多糖的絨毛狀細胞結(jié)構(gòu),作為心血管系統(tǒng)的重要屏障,將血液與組織分隔開來。結(jié)構(gòu)完整的糖萼具有維持血管通透性,抑制細胞間粘附等血管保護作用,否則將最終導致組織水腫和臟器功能障礙,并激發(fā)中性粒細胞與內(nèi)皮粘附后所產(chǎn)生的炎癥爆發(fā)。 既往研究已經(jīng)發(fā)現(xiàn)膿毒血癥可導致腎小球毛細血管內(nèi)皮糖萼破壞并伴有蛋白尿,同時該現(xiàn)象的發(fā)生與膿毒血癥后機體產(chǎn)生大量的(tumor necrosis factor-a,TNF-a)可能有關(guān)。然而這其中的確切機制尚不清楚,對于TNF-a的來源,自身的作用,與中性粒細胞貼壁之間的關(guān)系,其產(chǎn)生的調(diào)節(jié)機制以及其損傷糖萼的作用機制仍不清楚。 烏司他丁(urinary trypsin inhibitor,UTI):其以兩種前體分子形式存在于血漿中,inter-a-inhibitor (IαI)和pre-a-inhibitor(Pal),其結(jié)構(gòu)包括兩個Kunitz結(jié)構(gòu)域,O-糖鏈、硫酸軟骨素,N-糖鏈,C端K區(qū)：抑制多種水解酶,N端K區(qū)：抑制組織蛋白酶G和彈性蛋白酶,O-硫酸軟骨素糖鏈：能與細胞、鈣等結(jié)合,與穩(wěn)定溶酶體、抑制炎性細胞釋放有關(guān),N-糖鏈：調(diào)節(jié)內(nèi)皮細胞與單核細胞、粒細胞、淋巴細胞的結(jié)合力。烏司他丁主要是通過兩個Kunitz結(jié)構(gòu)域,分別能抑制多種酶的活性,尤其是減少絲氨酸蛋白酶(包括組織蛋白酶,粒細胞彈性蛋白酶,纖溶酶,激肽原酶,胰蛋白酶或糜蛋白酶,類胰蛋白酶等)活性,通過抑制中性粒細胞,淋巴細胞,巨噬細胞,肥大細胞等過度激活,抑制纖維蛋白降解,抑制緩激肽生成而導致血管擴張,阻止蛋白酶活化受體激活,從而減少炎性細胞活化,將炎性反應局限化,減輕對正常組織的損傷,其直接作用于細胞前體,減少細胞因子的分泌,穩(wěn)定溶酶體膜,減少酶的釋放,達到緩解炎癥反應,改善微循環(huán)的作用。目前尚未發(fā)現(xiàn)烏司他丁對膿毒血癥的腎血管內(nèi)皮糖萼破壞的保護作用及其分子機制的相關(guān)研究。因此,本研究將選用脂多糖內(nèi)毒素(lipopolysaccharide, LPS)誘導膿毒血癥的腎損傷大鼠在體模型,計劃對糖萼破壞與AKI發(fā)生相關(guān)的機制展開研究,并進一步探討腎臟血管內(nèi)皮糖萼在膿毒血癥導致AKI的機制中的作用與地位——僅僅是炎癥作用的“靶子”,抑或是兼具炎癥爆發(fā)的“炸藥桶”。并擬從腎臟血管內(nèi)皮糖萼的病理生理變化及相關(guān)分子水平闡述烏司他丁對膿毒血癥腎損傷的保護作用和具體機制,旨在從藥物治療膿毒血癥后腎血管糖萼破壞提供基礎(chǔ)實驗依據(jù)。 目的： 本研究探討烏司他丁對脂多糖內(nèi)毒素誘導的膿毒血癥大鼠腎臟血管內(nèi)皮的保護作用和分子機制,旨在從新的保護血管內(nèi)皮細胞糖萼破壞的的藥物途徑上提供基礎(chǔ)實驗依據(jù)。 方法： 1)膿毒血癥打擊模型建立及血清學參數(shù)檢測 通過腹部皮下注射水合氯醛對大鼠實施麻醉,對其右側(cè)頸靜脈進行置管。通過尾靜脈注射脂多糖,監(jiān)測平均動脈壓(mean artery pressure, MAP)及在0,180,360分鐘進行血清學(creatinine, Cr),(blood usea nitrogen, BUN)等相關(guān)檢測。 2)腎臟微血流灌注評估-激光多普勒技術(shù) 應用激光多普勒儀(Moor Instruments, Axminister, England)檢測法在活體狀態(tài)下觀察腎臟皮質(zhì)微循環(huán)血流狀態(tài)及灌注并進行實時計算。 3)腎臟血管內(nèi)皮細胞膜側(cè)糖萼的結(jié)構(gòu)分析 通過電鏡直接觀察膿毒血癥大鼠腎小球毛細血管內(nèi)皮糖萼結(jié)構(gòu)及厚度,通過檢測外周血,腎靜脈血(heparan sulfate, HS)和syndecan-1水平來間接評估腎小球毛細血管內(nèi)皮糖萼的破壞。 4)腎臟內(nèi)皮細胞TNF-α合成相關(guān)的分子信號通路研究免疫組化檢測腎小球毛細血管內(nèi)皮細胞漿內(nèi)(p38mitogen-activated protein kinase, p38MAPK)、(nuclear factor of kB,NF-kB)分子表達：通過對外周血和腎靜脈血中TNF-α炎性介質(zhì)(流式細胞儀檢測)的濃度及腎小球毛細血管內(nèi)皮細胞內(nèi)(Mitogenactivatedproteinkinase,MAPK)家族分子及NF-κB分子表達的檢測。 結(jié)果： 1)大鼠尾靜脈注射LPS(10μg/g)后,內(nèi)毒素組大鼠平均動脈壓進行性下降,生理鹽水干預組與烏司他丁組經(jīng)補液復蘇及血管活性藥物支持下,平均動脈壓呈現(xiàn)逐步上升,而腎臟微循環(huán)血流監(jiān)測顯示仍呈下降趨勢。 2)正常標準對照組大鼠腎臟毛細血管糖萼層完整連續(xù),厚約200nnm；膿毒血癥后腎臟毛細血管糖萼不連續(xù),結(jié)構(gòu)松散,變薄。生理鹽水干預組和烏司他丁藥物干預組大鼠腎臟毛細血管糖萼結(jié)構(gòu)同樣存在不連續(xù),較正常組松散,腫脹。膿毒血癥發(fā)生后1小時大鼠腎臟靜脈及外周血中HS、syndecan-1濃度即出現(xiàn)明顯的升高,且其較高濃度維持的時間較長(3小時達到高峰值) 3)膿毒血癥發(fā)生后0.5小時大鼠腎臟靜脈血中TNF-α濃度即出現(xiàn)明顯的升高,且其較高濃度維持的時間較長(3小時后迅速下降),而外周血TNF-a濃度的峰值明顯低于腎靜脈血。相對于標膿毒血癥組及生理鹽水干預組大鼠,烏司他丁能夠降低腎靜脈血和外周血中TNF-a的高峰濃度及其持續(xù)時間(P0.05)。 4)脂多糖內(nèi)毒素誘導的膿毒血癥腎損傷模型組大鼠腎小球毛細血管內(nèi)皮細胞漿內(nèi)NF-kB分子表達比正常對照組顯著升高(P0.01),而烏司他丁藥物治療組較膿毒血癥組也表現(xiàn)為明顯降低(P0.05),其余組間比較無顯著性差異。 5)脂多糖內(nèi)毒素誘導的膿毒血癥腎損傷模型組大鼠腎小球毛細血管內(nèi)皮細胞漿內(nèi)P38MAPK分子表達比正常對照組顯著升高(P0.01),而烏司他丁藥物治療組較膿毒血癥組表現(xiàn)為明顯降低(P0.05),其余組間比較無顯著性差異。 結(jié)論： 烏司他丁能減輕脂多糖內(nèi)毒素誘導的膿毒血癥的腎損傷微循環(huán)屏障破壞,尤其是減輕了腎小球血管內(nèi)皮細胞糖萼的破壞,從而提高腎血流,抑制腎小球血管內(nèi)皮細胞膜側(cè)的炎性反應。糖萼的破壞可能導致了膿毒血癥后腎臟微循環(huán)障礙損傷,腎臟血管內(nèi)皮細胞NF-κB/TNF-α分子機制可能參與了糖萼的破壞。以糖萼作為治療靶點,烏司他丁可能成為修復糖萼損傷而改善微循環(huán)障礙的藥物選擇并具有潛在的治療效果。
[Abstract]:Background :

Severe sepsis and multiple organ dysfunction syndrome ( MODS ) are the main causes of death in intensive care unit ( ICU ) .

Recent studies have found that the main pathological and physiological mechanisms of sepsis are closely related to the state of glycoccodextrin ( polysaccharide - protein complexes ) . More and more studies have recognized that vascular endothelial glycocal is of great importance to cardiovascular diseases . The glycocalycis is a vital barrier to the cardiovascular system . It can maintain vascular permeability , inhibit cell adhesion , etc . Otherwise , it will eventually lead to tissue edema and organ dysfunction , and trigger inflammation of neutrophils and endothelial cells .

Previous studies have found that sepsis can lead to the destruction of glomerular capillary endothelium and associated proteinuria , while the occurrence of this phenomenon may be associated with the occurrence of a large number of tumor necrosis factor - a , TNF - a after sepsis . However , the exact mechanism is unknown . However , the mechanism of regulation and the mechanism of its damage is still unclear for the origin of TNF - a .

Ulinastatin ( UTI ) , which is in the form of two precursor molecules in plasma , inter - a - inhibitor ( I.alpha . I ) and pre - a - inhibitor , the structure of which comprises two Kunitz domains , O - sugar chain , chondroitin sulfate , N - sugar chain , C - terminal K region , and N - sugar chain . In this study , we have not found the protective effect and molecular mechanism of Ulinastatin on the damage of renal vascular endothelium in septic rats , and further study the protective effect and specific mechanism of ullastatin on the renal injury of septic rats .

Purpose :

The purpose of this study was to investigate the protective effect and molecular mechanism of ullastatin on renal vascular endothelium in rats with sepsis induced by lipopolysaccharide ( LPS ) .

Method :

1 ) establishment of sepsis - resistant model and detection of serological parameters

The rats were anesthetized with subcutaneous injection of chloral hydrate , and the right jugular vein was placed . The mean artery pressure ( MAP ) was monitored by intravenous injection of lipopolysaccharide , and serum levels of creatinine ( Cr ) and BUN were monitored at 0,180 , 360 minutes .

2 ) Renal microperfusion assessment - laser Doppler technique

The renal cortex microcirculation blood flow state and perfusion were observed under the condition of living body by using laser Doppler instrument ( Moor Instruments , Axillary , England ) and real - time calculation was performed .

3 ) Structure analysis of vascular endothelial cell membrane side glycocalyon in kidney

The structure and thickness of glomerular capillary endothelium were directly observed by electron microscope . The damage of glomerular capillary endothelium was assessed indirectly by detecting peripheral blood , renal venous blood ( HS ) and syndecan - 1 level .

4 ) The expression of p38MAPK - activated protein kinase ( p38MAPK ) , ( p38MAPK ) , ( nuclear factor of kB , NF - kB ) in glomerular capillary endothelial cells was studied by immunohistochemistry .

Results :

1 ) After intravenous injection of LPS ( 10渭g / g ) in tail vein , the average arterial pressure of endotoxin group decreased gradually , and the average arterial pressure appeared gradually , while renal microcirculation blood flow monitoring showed a downward trend .

2 ) The normal control group rats were intact and continuous with the thickness of 200 nnm .
Compared with normal group , the concentration of HS and syndecan - 1 in the renal vein and peripheral blood of rats with septic shock increased significantly , and the concentration of HS and syndecan - 1 in the renal vein and peripheral blood increased significantly ( reaching the peak value in 3 hours ) .

3 ) The concentration of TNF - 偽 in the renal venous blood of rats after 0.5 hour after sepsis increased significantly , and the peak value of TNF - a in peripheral blood was significantly lower than that in renal venous blood ( P0.05 ) .

4 ) The expression of NF - kB in rat glomerular capillary endothelial cells was significantly higher than that in normal control group ( P0.01 ) .

5 ) The expression of P38MAPK in rat glomerular capillary endothelial cell was significantly higher than that in normal control group ( P0.01 ) .

Conclusion :

Ulinastatin can reduce the renal damage microcirculation barrier damage induced by lipopolysaccharides endotoxin , in particular to reduce the damage of the glycocalycis of the glomerular vascular endothelial cell , thereby improving the renal blood flow and inhibiting the inflammatory response on the membrane side of the glomerular vascular endothelial cell .

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R459.7

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