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

【摘要】：背景： 嚴(yán)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(dāng)前重癥監(jiān)護(hù)病房(intensive care unit, ICU)內(nèi)的主要死亡原因,也是當(dāng)前重癥醫(yī)學(xué)面臨的主要焦點(diǎn)及難點(diǎn)。雖然對(duì)膿毒癥的病理生理學(xué)機(jī)制和臨床治療進(jìn)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(xué)界已基本達(dá)成共識(shí)的急性腎損傷(Acute kidney injury, AKI)標(biāo)準(zhǔn)(RIFLE標(biāo)準(zhǔn)),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(yán)重感染及膿毒血癥是導(dǎo)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(fù)蘇治療水平正日益增強(qiáng),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(jī)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(jī)制——微循環(huán)障礙可能與內(nèi)皮表面糖萼(glycocalyx,多糖-蛋白復(fù)合物)的狀態(tài)密切相關(guān)。越來越多的研究已認(rèn)識(shí)到血管內(nèi)皮糖萼對(duì)心血管疾病具有重要的意義。糖萼是襯于血管內(nèi)皮細(xì)胞管腔膜側(cè),覆蓋著一種富含多糖的絨毛狀細(xì)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(xì)胞間粘附等血管保護(hù)作用,否則將最終導(dǎo)致組織水腫和臟器功能障礙,并激發(fā)中性粒細(xì)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(dǎo)致腎小球毛細(xì)血管內(nèi)皮糖萼破壞并伴有蛋白尿,同時(shí)該現(xiàn)象的發(fā)生與膿毒血癥后機(jī)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(jī)制尚不清楚,對(duì)于TNF-a的來源,自身的作用,與中性粒細(xì)胞貼壁之間的關(guān)系,其產(chǎn)生的調(diào)節(jié)機(jī)制以及其損傷糖萼的作用機(jī)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(gè)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(xì)胞、鈣等結(jié)合,與穩(wěn)定溶酶體、抑制炎性細(xì)胞釋放有關(guān),N-糖鏈：調(diào)節(jié)內(nèi)皮細(xì)胞與單核細(xì)胞、粒細(xì)胞、淋巴細(xì)胞的結(jié)合力。烏司他丁主要是通過兩個(gè)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(xì)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(xì)胞,淋巴細(xì)胞,巨噬細(xì)胞,肥大細(xì)胞等過度激活,抑制纖維蛋白降解,抑制緩激肽生成而導(dǎo)致血管擴(kuò)張,阻止蛋白酶活化受體激活,從而減少炎性細(xì)胞活化,將炎性反應(yīng)局限化,減輕對(duì)正常組織的損傷,其直接作用于細(xì)胞前體,減少細(xì)胞因子的分泌,穩(wěn)定溶酶體膜,減少酶的釋放,達(dá)到緩解炎癥反應(yīng),改善微循環(huán)的作用。目前尚未發(fā)現(xiàn)烏司他丁對(duì)膿毒血癥的腎血管內(nèi)皮糖萼破壞的保護(hù)作用及其分子機(jī)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(dǎo)膿毒血癥的腎損傷大鼠在體模型,計(jì)劃對(duì)糖萼破壞與AKI發(fā)生相關(guān)的機(jī)制展開研究,并進(jìn)一步探討腎臟血管內(nèi)皮糖萼在膿毒血癥導(dǎo)致AKI的機(jī)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(duì)膿毒血癥腎損傷的保護(hù)作用和具體機(jī)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(shí)驗(yàn)依據(jù)。 目的： 本研究探討烏司他丁對(duì)脂多糖內(nèi)毒素誘導(dǎo)的膿毒血癥大鼠腎臟血管內(nèi)皮的保護(hù)作用和分子機(jī)制,旨在從新的保護(hù)血管內(nèi)皮細(xì)胞糖萼破壞的的藥物途徑上提供基礎(chǔ)實(shí)驗(yàn)依據(jù)。 方法： 1)膿毒血癥打擊模型建立及血清學(xué)參數(shù)檢測(cè) 通過腹部皮下注射水合氯醛對(duì)大鼠實(shí)施麻醉,對(duì)其右側(cè)頸靜脈進(jìn)行置管。通過尾靜脈注射脂多糖,監(jiān)測(cè)平均動(dòng)脈壓(mean artery pressure, MAP)及在0,180,360分鐘進(jìn)行血清學(xué)(creatinine, Cr),(blood usea nitrogen, BUN)等相關(guān)檢測(cè)。 2)腎臟微血流灌注評(píng)估-激光多普勒技術(shù) 應(yīng)用激光多普勒儀(Moor Instruments, Axminister, England)檢測(cè)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(jìn)行實(shí)時(shí)計(jì)算。 3)腎臟血管內(nèi)皮細(xì)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(xì)血管內(nèi)皮糖萼結(jié)構(gòu)及厚度,通過檢測(cè)外周血,腎靜脈血(heparan sulfate, HS)和syndecan-1水平來間接評(píng)估腎小球毛細(xì)血管內(nèi)皮糖萼的破壞。 4)腎臟內(nèi)皮細(xì)胞TNF-α合成相關(guān)的分子信號(hào)通路研究免疫組化檢測(cè)腎小球毛細(xì)血管內(nèi)皮細(xì)胞漿內(nèi)(p38mitogen-activated protein kinase, p38MAPK)、(nuclear factor of kB,NF-kB)分子表達(dá)：通過對(duì)外周血和腎靜脈血中TNF-α炎性介質(zhì)(流式細(xì)胞儀檢測(cè))的濃度及腎小球毛細(xì)血管內(nèi)皮細(xì)胞內(nèi)(Mitogenactivatedproteinkinase,MAPK)家族分子及NF-κB分子表達(dá)的檢測(cè)。 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(dòng)脈壓進(jìn)行性下降,生理鹽水干預(yù)組與烏司他丁組經(jīng)補(bǔ)液復(fù)蘇及血管活性藥物支持下,平均動(dòng)脈壓呈現(xiàn)逐步上升,而腎臟微循環(huán)血流監(jiān)測(cè)顯示仍呈下降趨勢(shì)。 2)正常標(biāo)準(zhǔn)對(duì)照組大鼠腎臟毛細(xì)血管糖萼層完整連續(xù),厚約200nnm；膿毒血癥后腎臟毛細(xì)血管糖萼不連續(xù),結(jié)構(gòu)松散,變薄。生理鹽水干預(yù)組和烏司他丁藥物干預(yù)組大鼠腎臟毛細(xì)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(shí)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(shí)間較長(zhǎng)(3小時(shí)達(dá)到高峰值) 3)膿毒血癥發(fā)生后0.5小時(shí)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(shí)間較長(zhǎng)(3小時(shí)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(duì)于標(biāo)膿毒血癥組及生理鹽水干預(yù)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(shí)間(P0.05)。 4)脂多糖內(nèi)毒素誘導(dǎo)的膿毒血癥腎損傷模型組大鼠腎小球毛細(xì)血管內(nèi)皮細(xì)胞漿內(nèi)NF-kB分子表達(dá)比正常對(duì)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(dǎo)的膿毒血癥腎損傷模型組大鼠腎小球毛細(xì)血管內(nèi)皮細(xì)胞漿內(nèi)P38MAPK分子表達(dá)比正常對(duì)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(dǎo)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(xì)胞糖萼的破壞,從而提高腎血流,抑制腎小球血管內(nèi)皮細(xì)胞膜側(cè)的炎性反應(yīng)。糖萼的破壞可能導(dǎo)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(xì)胞NF-κB/TNF-α分子機(jī)制可能參與了糖萼的破壞。以糖萼作為治療靶點(diǎn),烏司他丁可能成為修復(fù)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(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R459.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 武曉慧;黃頌敏;樊文星;唐萬欣;邱紅渝;;高糖和MBL途徑的補(bǔ)體激活對(duì)人腎小球內(nèi)皮細(xì)胞表達(dá)IL-6、TNF-α的影響[J];四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2011年01期

2 龍石銀;崔慧輝;張彩平;喬新惠;田英;田汝芳;佟麗;黃良珠;;二苯乙烯苷對(duì)H_2O_2誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞核因子κB、腫瘤壞死因子α表達(dá)的影響[J];中國動(dòng)脈硬化雜志;2010年07期

，

本文編號(hào)：1765111