本文選題：HMGB1 + 腎小管上皮細(xì)胞�。� 參考：《武漢大學(xué)》2016年博士論文

【摘要】：研究背景：膿毒癥是一種常見(jiàn)而兇險(xiǎn)的臨床綜合征,及時(shí)和有效的控制膿毒癥相關(guān)的器官功能障礙是膿毒癥救治的重要環(huán)節(jié)。膿毒癥相關(guān)性急性腎損傷(sepsis induced acute kidney injury, SAKI)在膿毒癥相關(guān)的器官功能障礙中發(fā)病率最高,且SAKI的發(fā)生與膿毒癥患者的預(yù)后相關(guān)性最為密切,然而SAKI的發(fā)生機(jī)制仍不明確。AKI的發(fā)生主要和腎小管上皮細(xì)胞功能異常密切相關(guān)。研究揭示在許多病理過(guò)程中,腎小管上皮細(xì)胞可以通過(guò)分泌細(xì)胞因子等方式參與到疾病的發(fā)生發(fā)展過(guò)程。細(xì)胞外高遷移率族蛋白box-1 (high mobility group box-1, HMGB1)是一種哺乳動(dòng)物中高度保守的重要促炎因子。膿毒癥時(shí)HMGB1作為一種危險(xiǎn)相關(guān)分子模式(danger associated molecular patterns, DAMPs)大量釋放入血,與Toll樣受體4(Toll like receptor 4, TLR4)等受體結(jié)合,通過(guò)激活細(xì)胞內(nèi)相關(guān)信號(hào)轉(zhuǎn)導(dǎo)通路,介導(dǎo)細(xì)胞炎癥反應(yīng)。循環(huán)炎癥介質(zhì)上調(diào)是膿毒癥腎損傷以及其他并發(fā)癥發(fā)生的關(guān)鍵因素,而分子量小于腎臟濾過(guò)屏障截留分子量的循環(huán)分子幾乎全部進(jìn)入腎小管與腎小管上皮細(xì)胞相互作用。因?yàn)镠MGB1分子量約為25kD,且膿毒癥時(shí)腎小球血管通透性增加,本研究假設(shè)膿毒癥時(shí)循環(huán)中HMGB1進(jìn)入腎組織中,與腎小管上皮細(xì)胞相互作用,介導(dǎo)炎癥反應(yīng)以及促進(jìn)SAKI的發(fā)生。本研究旨在驗(yàn)證上述假設(shè)并對(duì)其相關(guān)的細(xì)胞分子機(jī)制進(jìn)行深入的探討,以期為膿毒癥及SAKI的診治提供新的依據(jù)。方法：第一部分：85只SPF級(jí)成年雄性SD大鼠隨機(jī)分為：正常對(duì)照組n=5；盲腸結(jié)扎穿刺組(CLP組)、盲腸結(jié)扎穿刺+HMGB1輸注組(CLP+H組)、假手術(shù)+HMGB1輸注組(shamOP+H組)、盲腸結(jié)扎穿刺+脾切除組(CLP+S組),每組動(dòng)物20只。采用相應(yīng)方法制備動(dòng)物模型,并在不同時(shí)點(diǎn)處死動(dòng)物。分離血清及腎組織中的腎小管上皮細(xì)胞,并超濾離心濃縮尿液。采用Western-blotting檢測(cè)血清及尿液HMGB1水平,采用免疫熒光觀察HMGB1在腎組織中的分布,應(yīng)用外源性帶標(biāo)簽的HMGB1輸注聯(lián)合Western-blotting、免疫沉淀及免疫熒光觀察循環(huán)中HMGB1在腎組織和尿液中的分布,應(yīng)用實(shí)時(shí)熒光定量PCR檢測(cè)腎小管上皮細(xì)胞IL-1及IL-6 mRNA表達(dá)。第二部分：正常SD大鼠腎組織應(yīng)用Western-blotting及免疫組化觀察TLR4表達(dá)及分布。體外培養(yǎng)NRK52E細(xì)胞(大鼠腎小管上皮細(xì)胞系)應(yīng)用免疫熒光及Western-blotting觀察TLR4在細(xì)胞上的表達(dá)及定位。第三部分：體外培養(yǎng)NRK52E細(xì)胞,隨機(jī)分為：對(duì)照組,HMGB1刺激組,HMGB1+LPS RS刺激組。各組細(xì)胞在無(wú)血清培養(yǎng)基同步化后分別按照不同分組給予不同時(shí)間的相應(yīng)刺激。流式細(xì)胞術(shù)觀察細(xì)胞凋亡,Western-blotting分析MAPK及NF-kB通路相關(guān)蛋白磷酸化,實(shí)時(shí)熒光定量PCR檢測(cè)細(xì)胞IL-1及IL-6mRNA表達(dá),半定量蛋白芯片檢測(cè)細(xì)胞合成IL-1及IL-6。第四部分：1)45只SPF級(jí)成年雄性SD大鼠隨機(jī)分為：正常對(duì)照組5只；盲腸結(jié)扎穿刺組、盲腸結(jié)扎穿刺+脾切除組,每組20只。分離腎小管上皮細(xì)胞實(shí)時(shí)熒光定量PCR檢測(cè)TIMP2 mRNA水平,超濾濃縮CLP組尿液Western-blotting檢測(cè)其中TIMP2水平。2)體外培養(yǎng)NRK52E細(xì)胞,隨機(jī)分為：對(duì)照組；HMGB1刺激組；HMGB1+LPS RS刺激組,各組細(xì)胞使用無(wú)血清培養(yǎng)基同步化后分別按照不同分組給予不同時(shí)間的相應(yīng)刺激。流式細(xì)胞術(shù)觀察細(xì)胞周期分布,實(shí)時(shí)熒光定量PCR檢測(cè)TIMP2 mRNA表達(dá),半定量蛋白芯片檢測(cè)細(xì)胞TIMP2蛋白。結(jié)果：第一部分：1)CLP組大鼠血清及尿液HMGB1水平術(shù)后較對(duì)照顯著上升,且顯著高于CLP+S組(P0.05)；2)CLP組腎組織內(nèi)HMGB1不再與細(xì)胞核共定位,且分布廣泛；3)完整His標(biāo)記的外源性重組HMGB1在CLP+H組尿中含量高于shamOP+H組,該蛋白在CLP+H組廣泛分布于除細(xì)胞核外的腎組織中；4)CLP組術(shù)后6h腎小管上皮細(xì)胞IL-1及IL-6 mRNA表達(dá)水平顯著增高(P0.05)。第二部分：1)生理狀態(tài)下腎小管上皮細(xì)胞表達(dá)TLR4；2)TLR4表達(dá)于腎小管上皮細(xì)胞細(xì)胞膜。第三部分：1)HMGB1刺激后腎小管上皮細(xì)胞凋亡率較對(duì)照無(wú)顯著差異(P0.05); 2)HMGB1組腎小管上皮細(xì)胞MAPK/NF-kB信號(hào)通路相關(guān)蛋白磷酸化水平較對(duì)照顯著上升(P0.05); 3) HMGB1組腎小管上皮細(xì)胞IL-1及IL-6mRNA表達(dá)水平顯著上調(diào),IL-1及IL-6合成顯著增加(P0.05)。第四部分：1) HMGB1組腎小管上皮細(xì)胞處于G1期細(xì)胞比例顯著增加(P0.05),而處于細(xì)胞周期S/G2期的細(xì)胞比例則顯著下降(P0.05),HMGB1+LPS RS組各期細(xì)胞比例無(wú)顯著改變(P0.05)；2)體外試驗(yàn)中HMGB1組TIMP2 mRNA轉(zhuǎn)錄水平顯著上調(diào)(P0.05), TIMP2蛋白合成顯著增多(P0.05)。HMGB1+LPS RS組TIMP2 mRNA表達(dá)水平及蛋白合成無(wú)顯著改變(P0.05)。3)動(dòng)物實(shí)驗(yàn)中,CLP組及CLP+S組腎小管上皮細(xì)胞TIMP2 mRNA表達(dá)顯著上調(diào)(P0.05),但同時(shí)間點(diǎn)CLP+S組TIMP2 mRNA表達(dá)顯著低于CLP組(P0.05),CLP組尿中出現(xiàn)TIMP2。結(jié)論：1)膿毒癥時(shí)循環(huán)及腎組織中出現(xiàn)大量HMGB1,循環(huán)中HMGB1可以進(jìn)入腎組織。2)腎小管上皮細(xì)胞表達(dá)HMGB1的膜受體TLR4。3) HMGB1通過(guò)與TLR4相互作用介導(dǎo)腎小管上皮細(xì)胞炎癥相關(guān)信號(hào)通路MAPK/NF-kB激活、腎小管上皮細(xì)胞釋放炎癥介質(zhì)IL-1及IL-6參與炎癥反應(yīng)。4) HMGB1與TLR4相互作用導(dǎo)致腎小管上皮細(xì)胞細(xì)胞周期G1期阻滯并合成釋放TMP2,這些結(jié)果說(shuō)明二者相互作用與膿毒癥急性腎損傷的發(fā)生密切相關(guān)。
[Abstract]:Background: sepsis is a common and dangerous clinical syndrome. Timely and effective organ dysfunction associated with sepsis is an important link in the treatment of sepsis. Acute sepsis associated acute renal injury (sepsis induced acute kidney injury, SAKI) has the highest incidence of organ dysfunction associated with sepsis, and SA The occurrence of KI is most closely related to the prognosis of patients with sepsis. However, the mechanism of the occurrence of SAKI is still not clear that the occurrence of.AKI is closely related to the dysfunction of renal tubular epithelial cells. The high mobility group box-1 (high mobility group box-1, HMGB1) is a highly conserved important pro-inflammatory factor in mammals. In sepsis, HMGB1 is released into blood as a risk related molecular model (danger associated molecular patterns, DAMPs), with receptor like receptor 4 (4) and other receptors. Combined with the activation of intracellular related signal transduction pathway, it mediates the inflammatory reaction of cells. The up regulation of circulating inflammatory mediators is the key factor for the renal injury and other complications of sepsis, and the circulation molecules with less molecular weight than the renal filter barrier intercepting molecular weight are almost all entered into the renal tubules and renal tubular epithelial cells. The molecular weight of HMGB1 is about 25kD, and the glomerular vascular permeability increases in sepsis. This study hypothesized that HMGB1 enters renal tissue during sepsis, interacts with renal tubular epithelial cells, mediates the inflammatory response and promotes the occurrence of SAKI. In order to provide new basis for the diagnosis and treatment of sepsis and SAKI. Method: Part 1: 85 adult male SD rats of grade SPF were randomly divided into normal control group n=5, cecum ligation puncture group (group CLP), caecum ligation puncture +HMGB1 infusion group (group CLP+H), sham operation +HMGB1 infusion group (shamOP+H group), cecum ligation puncture + splenectomy group (CLP+) S group, 20 animals in each group. The animal models were prepared by the corresponding methods and the animals were killed at different points. The renal tubular epithelial cells in the serum and kidney tissues were separated and the urine was centrifuged by ultrafiltration. The serum and urine HMGB1 levels were detected by Western-blotting. The distribution of HMGB1 in the renal tissue was observed by immunofluorescence, and the exogenous band was applied. HMGB1 infusion combined with Western-blotting, the distribution of HMGB1 in renal tissue and urine in immunofluorescence and immunofluorescence observation cycle. The expression of IL-1 and IL-6 mRNA in renal tubular epithelial cells was detected by real-time fluorescence quantitative PCR. The second part: Western-blotting and immunohistochemistry were used to observe the expression and score of TLR4 in normal SD rats. In vitro culture of NRK52E cells (rat renal tubular epithelial cell line) using immunofluorescence and Western-blotting to observe the expression and localization of TLR4 on the cell. The third part: the culture of NRK52E cells in vitro, randomly divided into the control group, the HMGB1 stimulation group and the HMGB1+LPS RS stimulation group. The cells of each group were in the serum-free medium after synchronization, respectively. Different groups were given corresponding stimulation at different time. Flow cytometry was used to observe apoptosis, Western-blotting analysis of MAPK and NF-kB pathway related protein phosphorylation, real-time fluorescence quantitative PCR detection of IL-1 and IL-6mRNA expression, semi quantitative protein chip detection of cell synthesis IL-1 and IL-6. fourth parts: 1) 45 SPF adult male SD rats follow The control group was divided into 5 normal control group, cecum ligation puncture group, cecum ligation puncture and splenectomy group, 20 in each group. The level of TIMP2 mRNA was detected by real-time fluorescence quantitative PCR in renal tubular epithelial cells, and TIMP2 level.2 in the urine Western-blotting of ultrafiltration concentrated CLP group, and NRK52E cells were cultured in vitro. The control group was randomly divided into the control group and HMGB1 thorn. Stimulated group, HMGB1+LPS RS stimulation group, each cell used serum-free media synchronization after the synchronization of different groups to give different time corresponding stimulation. Flow cytometry to observe the cell cycle distribution, real-time quantitative PCR detection of TIMP2 mRNA expression, semi quantitative protein chip to detect TIMP2 protein. Results: the first part: 1) CLP group The serum and urine HMGB1 levels in rats were significantly higher than those in the control group, and significantly higher than that in the CLP+S group (P0.05); 2) HMGB1 in the group CLP was no longer Co located with the nucleus and was widely distributed, and 3) the content of the recombinant HMGB1 in the urine of the complete His marker was higher than that in the shamOP+H group, and the protein in the CLP+H group was widely distributed outside the nucleus except the nucleus. In renal tissue, 4) the expression level of IL-1 and IL-6 mRNA in 6h renal tubular epithelial cells increased significantly after operation in CLP group (P0.05). The second part: 1) renal tubular epithelial cells expressed TLR4; 2) TLR4 was expressed in the renal tubular epithelial cell membrane. Third: 1) there was no significant difference in the apoptosis rate of renal tubular epithelial cells after HMGB1 stimulation (P). 0.05); (0.05) 2) the level of phosphorylation of MAPK/NF-kB signaling pathway related proteins in the renal tubular epithelial cells in HMGB1 group was significantly higher than that of the control (P0.05); 3) the expression level of IL-1 and IL-6mRNA in the renal tubular epithelial cells in the HMGB1 group was significantly up-regulated, and the synthesis of IL-1 and IL-6 increased significantly (P0.05). Fourth parts: 1) the renal tubular epithelial cells in the HMGB1 group were in the G1 phase cell proportion. The proportion of cells in the cell cycle S/G2 phase decreased significantly (P0.05), and there was no significant change in the proportion of cells in the HMGB1+LPS RS group (P0.05). 2) the mRNA transcriptional level of the HMGB1 group in the HMGB1 group was significantly up (P0.05), and the TIMP2 protein synthesis increased (P0.05) and protein synthesis. No significant changes (P0.05).3) in animal experiments, the expression of TIMP2 mRNA in renal tubular epithelial cells in group CLP and CLP+S was significantly up-regulated (P0.05), but the expression of TIMP2 mRNA in group CLP+S was significantly lower than that in CLP group (P0.05) at the same time point. 1) a large number of renal tissue appeared in the urine of sepsis. .2) renal tubular epithelial cells express HMGB1 membrane receptor TLR4.3) HMGB1 by interacting with TLR4 to mediate the activation of inflammation related signaling pathway of renal tubular epithelial cells, MAPK/NF-kB in renal tubular epithelial cells release inflammatory mediators IL-1 and IL-6 involved in the inflammatory response.4) HMGB1 and TLR4 interaction leads to renal tubular epithelial cell cell cycle phase G1 phase resistance The results showed that the interaction between the two was closely related to the occurrence of acute kidney injury in sepsis. TMP2

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R459.7

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6 楊曉;張春;鄧安國(guó);朱忠華;;結(jié)締組織生長(zhǎng)因子在腎小管上皮細(xì)胞細(xì)胞外基質(zhì)積聚中的作用[A];“中華醫(yī)學(xué)會(huì)腎臟病學(xué)分會(huì)2004年年會(huì)”暨“第二屆全國(guó)中青年腎臟病學(xué)術(shù)會(huì)議”論文匯編[C];2004年

7 張瑜;周建華;;肝素對(duì)腎小管上皮細(xì)胞膜結(jié)合補(bǔ)體調(diào)節(jié)蛋白表達(dá)的影響及意義[A];中華醫(yī)學(xué)會(huì)第十四次全國(guó)兒科學(xué)術(shù)會(huì)議論文匯編[C];2006年

8 楊雅麗;程曉霞;;腎小管上皮細(xì)胞凋亡在腎間質(zhì)纖維化中的作用及其防治的研究進(jìn)展[A];2009全國(guó)時(shí)間生物醫(yī)學(xué)學(xué)術(shù)會(huì)議論文集[C];2009年

9 譚玉莉;黃妍;楊亦彬;;魚(yú)膽汁毒素致腎小管上皮細(xì)胞死亡方式及實(shí)施干預(yù)的試驗(yàn)研究[A];貴州省醫(yī)學(xué)會(huì)腎臟病學(xué)分會(huì)2008年學(xué)術(shù)年會(huì)論文匯編[C];2008年

10 喬f^;陳香美;吳鏑;丁瑞;師鎖柱;謝院生;洪權(quán);呂楊;王兆霞;尹忠;;衰老大鼠腎臟缺血/再灌注損傷中腎小管上皮細(xì)胞不同凋亡途徑的研究[A];“中華醫(yī)學(xué)會(huì)腎臟病學(xué)分會(huì)2004年年會(huì)”暨“第二屆全國(guó)中青年腎臟病學(xué)術(shù)會(huì)議”論文匯編[C];2004年

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