高迁移率族蛋白box-1对脓毒症时肾小管上皮细胞的影响及机制

发布时间：2018-04-21 04:48

本文选题：HMGB1 + 肾小管上皮细胞　；参考：《武汉大学》2016年博士论文

【摘要】：研究背景：脓毒症是一种常见而凶险的临床综合征,及时和有效的控制脓毒症相关的器官功能障碍是脓毒症救治的重要环节。脓毒症相关性急性肾损伤(sepsis induced acute kidney injury, SAKI)在脓毒症相关的器官功能障碍中发病率最高,且SAKI的发生与脓毒症患者的预后相关性最为密切,然而SAKI的发生机制仍不明确。AKI的发生主要和肾小管上皮细胞功能异常密切相关。研究揭示在许多病理过程中,肾小管上皮细胞可以通过分泌细胞因子等方式参与到疾病的发生发展过程。细胞外高迁移率族蛋白box-1 (high mobility group box-1, HMGB1)是一种哺乳动物中高度保守的重要促炎因子。脓毒症时HMGB1作为一种危险相关分子模式(danger associated molecular patterns, DAMPs)大量释放入血,与Toll样受体4(Toll like receptor 4, TLR4)等受体结合,通过激活细胞内相关信号转导通路,介导细胞炎症反应。循环炎症介质上调是脓毒症肾损伤以及其他并发症发生的关键因素,而分子量小于肾脏滤过屏障截留分子量的循环分子几乎全部进入肾小管与肾小管上皮细胞相互作用。因为HMGB1分子量约为25kD,且脓毒症时肾小球血管通透性增加,本研究假设脓毒症时循环中HMGB1进入肾组织中,与肾小管上皮细胞相互作用,介导炎症反应以及促进SAKI的发生。本研究旨在验证上述假设并对其相关的细胞分子机制进行深入的探讨,以期为脓毒症及SAKI的诊治提供新的依据。方法：第一部分：85只SPF级成年雄性SD大鼠随机分为：正常对照组n=5；盲肠结扎穿刺组(CLP组)、盲肠结扎穿刺+HMGB1输注组(CLP+H组)、假手术+HMGB1输注组(shamOP+H组)、盲肠结扎穿刺+脾切除组(CLP+S组),每组动物20只。采用相应方法制备动物模型,并在不同时点处死动物。分离血清及肾组织中的肾小管上皮细胞,并超滤离心浓缩尿液。采用Western-blotting检测血清及尿液HMGB1水平,采用免疫荧光观察HMGB1在肾组织中的分布,应用外源性带标签的HMGB1输注联合Western-blotting、免疫沉淀及免疫荧光观察循环中HMGB1在肾组织和尿液中的分布,应用实时荧光定量PCR检测肾小管上皮细胞IL-1及IL-6 mRNA表达。第二部分：正常SD大鼠肾组织应用Western-blotting及免疫组化观察TLR4表达及分布。体外培养NRK52E细胞(大鼠肾小管上皮细胞系)应用免疫荧光及Western-blotting观察TLR4在细胞上的表达及定位。第三部分：体外培养NRK52E细胞,随机分为：对照组,HMGB1刺激组,HMGB1+LPS RS刺激组。各组细胞在无血清培养基同步化后分别按照不同分组给予不同时间的相应刺激。流式细胞术观察细胞凋亡,Western-blotting分析MAPK及NF-kB通路相关蛋白磷酸化,实时荧光定量PCR检测细胞IL-1及IL-6mRNA表达,半定量蛋白芯片检测细胞合成IL-1及IL-6。第四部分：1)45只SPF级成年雄性SD大鼠随机分为：正常对照组5只；盲肠结扎穿刺组、盲肠结扎穿刺+脾切除组,每组20只。分离肾小管上皮细胞实时荧光定量PCR检测TIMP2 mRNA水平,超滤浓缩CLP组尿液Western-blotting检测其中TIMP2水平。2)体外培养NRK52E细胞,随机分为：对照组；HMGB1刺激组；HMGB1+LPS RS刺激组,各组细胞使用无血清培养基同步化后分别按照不同分组给予不同时间的相应刺激。流式细胞术观察细胞周期分布,实时荧光定量PCR检测TIMP2 mRNA表达,半定量蛋白芯片检测细胞TIMP2蛋白。结果：第一部分：1)CLP组大鼠血清及尿液HMGB1水平术后较对照显著上升,且显著高于CLP+S组(P0.05)；2)CLP组肾组织内HMGB1不再与细胞核共定位,且分布广泛；3)完整His标记的外源性重组HMGB1在CLP+H组尿中含量高于shamOP+H组,该蛋白在CLP+H组广泛分布于除细胞核外的肾组织中；4)CLP组术后6h肾小管上皮细胞IL-1及IL-6 mRNA表达水平显著增高(P0.05)。第二部分：1)生理状态下肾小管上皮细胞表达TLR4；2)TLR4表达于肾小管上皮细胞细胞膜。第三部分：1)HMGB1刺激后肾小管上皮细胞凋亡率较对照无显著差异(P0.05); 2)HMGB1组肾小管上皮细胞MAPK/NF-kB信号通路相关蛋白磷酸化水平较对照显著上升(P0.05); 3) HMGB1组肾小管上皮细胞IL-1及IL-6mRNA表达水平显著上调,IL-1及IL-6合成显著增加(P0.05)。第四部分：1) HMGB1组肾小管上皮细胞处于G1期细胞比例显著增加(P0.05),而处于细胞周期S/G2期的细胞比例则显著下降(P0.05),HMGB1+LPS RS组各期细胞比例无显著改变(P0.05)；2)体外试验中HMGB1组TIMP2 mRNA转录水平显著上调(P0.05), TIMP2蛋白合成显著增多(P0.05)。HMGB1+LPS RS组TIMP2 mRNA表达水平及蛋白合成无显著改变(P0.05)。3)动物实验中,CLP组及CLP+S组肾小管上皮细胞TIMP2 mRNA表达显著上调(P0.05),但同时间点CLP+S组TIMP2 mRNA表达显著低于CLP组(P0.05),CLP组尿中出现TIMP2。结论：1)脓毒症时循环及肾组织中出现大量HMGB1,循环中HMGB1可以进入肾组织。2)肾小管上皮细胞表达HMGB1的膜受体TLR4。3) HMGB1通过与TLR4相互作用介导肾小管上皮细胞炎症相关信号通路MAPK/NF-kB激活、肾小管上皮细胞释放炎症介质IL-1及IL-6参与炎症反应。4) HMGB1与TLR4相互作用导致肾小管上皮细胞细胞周期G1期阻滞并合成释放TMP2,这些结果说明二者相互作用与脓毒症急性肾损伤的发生密切相关。
[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

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R459.7

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