MAP4在炎症导致肺微血管通透性增高中的作用及机制研究

发布时间：2018-06-25 14:12

本文选题：炎症 + 人肺微血管内皮细胞　；参考：《第三军医大学》2015年硕士论文

【摘要】：研究背景严重烧伤后,由于应激刺激,神经、体液和内分泌紊乱,体内常有致炎因子释放,容易引起局部或全身的炎症反应(inflammation)。炎症反应发生后,炎性细胞又被进一步激活,细胞和体液均可释放大量的炎症介质,通过直接损伤血管内皮细胞或间接影响其它化学因子而最终引起血管通透性的增高。作为人体与外界沟通的重要门户,肺在严重烧伤后,极易受到多种因素影响而引起肺微血管通透性增高,导致肺微血管内皮细胞屏障破坏,进而引起急性肺损伤(acute lung injury,ALI)的发生。截至目前,临床尚无有效降低血管通透性的方法,对于炎症导致肺微血管通透性增高的机制我们也依然不清楚。近年来,大量研究结果提示,微管(microtubule,MT)作为细胞骨架系统的重要成分,在内皮细胞通透性的调节中具有举足轻重的地位,而微管的稳定受到其它多种因素的影响,例如GTP、温度、药物、微管相关蛋白等。相关研究表明,微管解聚可以引起细胞形态、细胞间连接结构等一系列改变,造成细胞不可逆的损伤。而微管稳定剂则可稳定微管,抑制这些破坏作用的发生。但是在炎症条件下,微管是如何参与并调节人肺微血管内皮细胞(human pulmonary microvascular endothelial cells,HPMECs)通透性的增高,我们尚不清楚。微管相关蛋白4(microtubule associated protein 4,MAP4)是一种广泛表达于全身组织的微管动力学调节蛋白,它能促进微管聚合,维持细胞骨架和连接结构的完整。MAP4通过磷酸化的方式来调节自身活性,当其磷酸化增高时,MAP4的活性下降,并且从微管上脱落下来,导致微管动力学的失稳。我们前期研究发现,大鼠乳鼠心肌细胞缺氧后,p38/MAPK(p38 mitogen-activated protein kinase)激活,MAP4磷酸化增高,活性下降,同时伴随微管的解聚;此外,我们还发现,烧伤血清处理后,人的脐静脉内皮细胞(HUVECs)中的p38/MAPK通路激活,并且导致HUVECs通透性的增高。但是,炎症条件下,HPMECs中MAP4的活性变化情况我们还不清楚,而且MAP4是否参与炎症导致的HPMECs通透性增高以及可能的调节机制也有待我们进一步发现和证实。因此,本研究将检测map4在炎症时的活性变化情况,并且通过构建map4突变体map4(ala),模拟map4的去磷酸化状态,通过瞬时转染重组腺病毒,进一步明确map4磷酸化是否介导炎症引起的hpmecs通透性增高。在此基础之上,我们还将初步探索map4参与调节炎症导致hpmecs通透性增高的相关机制。旨在为临床相关疾病的治疗提供新的靶点和研究方向。材料和方法:1、我们以hpmecs为研究对象,利用致炎因子脂多糖(lipopolysaccharide,lps)和肿瘤坏死因子-α(tumournecrosisfactor-α)处理细胞,模拟体外炎症刺激。观察致炎因子lps(200、500、1000ng/ml)和tnf-α(200、500、1000ng/ml)处理前后,hpmecs通透性及微管的变化情况,并采用以下方法进行相应检测和观察:1)检测fitc-dextran(40kda)荧光物质漏出情况和测量跨内皮细胞电阻值(ter),以此观察肺微血管内皮细胞屏障功能的改变状况;2)对α-微管蛋白进行免疫荧光染色(immunofluorescence,if),以此观察微管的形态、结构和分布状况;3)将游离态与聚合态的微管蛋白分离、提取并定量,采用westernblot(wb)方法进行检测,观察游离态与聚合态微管蛋白含量的变化情况。2、使用微管稳定剂紫杉醇(taxol,1um)预处理细胞,观察紫杉醇预处理前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激对hpmecs通透性和微管的影响。通过检测fitc-dextran和ter,来观察肺微血管内皮细胞单层通透性的改变情况;采用if法观察微管的形态、结构和分布的改变;分离并提取游离态和聚合态的微管蛋白,通过wb检测其含量的变化。3、通过wb进行检测,观察致炎因子lps(500ng/ml)和tnf-α(500ng/ml)处理hpmecs1h、3h、6h、12h后,map4活性的变化情况。构建模拟map4去磷酸化状态的突变体map4(ala),瞬时转染hpmecs72h,通过测量fitc-dextran荧光漏出和ter,观察转染前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激对hpmecs通透性的影响,并采用if法检测微管的形态、结构和分布状况,同时分离提取聚合态与游离态的微管蛋白,通过wb法检测其含量的改变,另外,使用免疫共沉淀(immunoprecipitation,ip)检测map4与微管间的相互作用情况,以此明确map4在调节hpmecs通透性和微管改变中发挥的作用。4、以wb法检测致炎因子lps(500ng/ml)和tnf-α(500ng/ml)处理hpmecs1h、3h、6h、12h后,p38/mapk的激活情况。构建p38/mapk的上游激酶mkk6(glu),同时应用p38/mapk抑制剂sb203580(5um),观察mkk6(glu)转染细胞和sb203580预处理细胞前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激对map4(s696、s787、s768)和p-p38活性的变化情况,同时采用ip法检测map4和微管间的相互作用,以此明确p38/mapk是否为map4的上游激酶。5、应用p38/mapk上游激酶mkk6(glu)和p38/mapk抑制剂sb203580(5um),通过检测fitc-dextran和ter,观察sb203580预处理细胞前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激或mkk6(glu)转染后对hpmecs通透性的影响,采用if法检测微管的形态结构和分布状况,通过分离提取聚合/游离态的微管蛋白,使用wb法检测其含量改变情况。同时,转染或共转染cmv-null、map4(ala)和mkk6(glu),并采用上述方法检测hpmecs通透性、微管结构以及游离/聚合态微管蛋白含量的变化情况,以明确p38/mapk激酶通路是否通过调节map4活性参与并调控lps和tnf-α介导的hpmecs通透性与微管的改变。6、利用caspase-3抑制剂(z-dqmd-fmk,10um)、p38/mapk抑制剂sb203580(5um)和map4(ala)预处理或转染细胞,观察预处理细胞前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激对hpmecs凋亡和hpmecs通透性的影响,通过tunel法检测细胞凋亡,测量fitc-dextran和ter来评判hpmecs通透性的改变,以此明确map4介导的炎症引起的hpmecs通透性增高不依赖细胞凋亡。结果1、致炎因子lps(200、500、1000ng/ml)或tnf-α(200、500、1000ng/ml)处理hpmecs6h后,hpmecs单层对fitc-dextran荧光物质漏出增多,跨内皮细胞电阻值降低,并且呈剂量依赖性改变;致炎因子lps或tnf-α刺激浓度为200ng/ml时,微管结构不规则,少量微管断裂;lps或tnf-α刺激浓度为500ng/ml时,细胞膜周微管解聚明显,细胞核周微管皱缩;当lps或tnf-α刺激浓度增至1000ng/ml时,只残留极少的微管结构和片段;游离/聚合态微管蛋白定量检测提示,致炎因子lps(200、500、1000ng/ml)或tnf-α(200、500、1000ng/ml)处理后,游离态微管蛋白增多而聚合态微管蛋白减少,并呈剂量依赖性改变。2、根据上述实验结果,选择lps(500ng/ml)或tnf-α(500ng/ml)两个典型的致炎因子用于后续实验。在微管稳定剂紫杉醇(1um)预处理细胞1h后,正常细胞通透性无明显改变,微管聚合增加,解聚减少;同时,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激6h导致的fitc-dextran荧光物质漏出明显减少,跨内皮细胞电阻值明显增加;并且微管网状结构破坏减少,细胞膜周微管密度增加;聚合态微管蛋白含量增加,游离态微管蛋白含量减少。3、致炎因子lps(500ng/ml)或tnf-α(500ng/ml)处理hpmecs6h后,map4(s696与s787)磷酸化增高,而map4(ser768)磷酸化无改变。转染map4(ala)干预map4磷酸化后,正常hpmecs通透性无明显改变,微管聚合增加,解聚减少;同时,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激6h后的hpmecs单层对fitc-dextran荧光物质漏出明显减少,跨内皮细胞电阻值明显升高;而且微管的动力学更加稳定,微管的结构得到改善,游离态的微管含量减少而聚合态微管蛋白增多;此外,map4与微管之间的相互结合也增多。4、致炎因子lps(500ng/ml)或tnf-α(500ng/ml)处理hpmecs1h、3h、6h、12h后,p38/mapk激活,并且呈时间依赖性改变。同时,转染mkk6(glu)持续激活p38/mapk后,map4(s696与s787)和p38磷酸化水平显著增加,map4与微管相互结合减少;反之,p38/mapk阻断剂sb203580(5um)预处理细胞后,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激6h导致的p38/mapk激活显著受抑,且map4(s696与s787)磷酸化水平明显下降,此外,map4与微管的相互结合也显著增多。5、sb203580(5um)预处理1h后,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激的hpmecs单层对fitc-dextran荧光物质漏出明显减少,跨内皮细胞电阻值明显增高;且微管结构破坏受抑,游离态的微管蛋白含量减少而聚合态的微管蛋白含量增多;相反,mkk6(glu)转染细胞后,hpmecs单层对fitc-dextran荧光物质漏出增多,跨内皮细胞电阻值降低,微管解聚增加而聚合减少。此外,我们转染mkk6(glu)+cmv-null后发现,hpmecs单层对fitc-dextran荧光漏出增多,跨内皮细胞电阻值降低;微管结构破坏,游离态微管蛋白增加而聚合态微管蛋白减少;而共转染mkk6(glu)和map4(ala)后,上述变化得到明显抑制。6、caspase-3抑制剂(z-dqmd-fmk,10um)预处理细胞1h后,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)诱导的细胞凋亡明显受抑;同时,辅以sb203580(5um)预处理和map4(ala)转染后,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激的hpmecs单层对fitc-dextran荧光物质漏出仍然减少,跨内皮细胞电阻值仍然增加,提示map4磷酸化介导的致炎因子所致hpmecs通透性增加不依赖细胞凋亡。结论致炎因子lps或tnf-α处理hpmecs后,细胞中p38/mapk激酶通路激活,通过map4(s696与s787)磷酸化使map4失活,并引起微管解聚,导致hpmecs通透性增高;同时,map4磷酸化介导的致炎因子所致的hpmecs通透性增高不依赖细胞凋亡。以上结果提示map4介导的微管结构变化可能参与了炎症导致的hpmecs通透性增高,有助于我们进一步深入认识烧伤休克、炎症时急性肺损伤的发病机理,为临床相关疾病的干预和治疗提供新靶点和方向。
[Abstract]:After severe burns, due to stress stimulation, nerve, body fluid and endocrine disorder, the release of inflammatory factors is often caused in the body, and it is easy to cause local or systemic inflammatory response (inflammation). After the inflammation, inflammatory cells are further activated, and cells and body fluid can release a large number of inflammatory mediators and directly damage the blood vessels. Skin cells or indirect effects of other chemical factors lead to increased vascular permeability. As an important portal for communication between the human body and the outside world, lung microvascular permeability increases greatly after severe burns, causing lung microvascular endothelial cell barrier damage and causing acute lung injury (acute lung inj). Ury, ALI). Up to now, there is no effective method for reducing vascular permeability, and we are still not clear about the mechanism of inflammation leading to increased pulmonary microvascular permeability. In recent years, a large number of research results suggest that microtubule (microtubule, MT), as an important component of the cytoskeleton system, can be used in the regulation of endothelial cell permeability. The stability of the microtubule is affected by many other factors, such as GTP, temperature, drug, microtubule related protein and so on. Related studies have shown that microtubule depolymerization can cause a series of changes in cell morphology, intercellular connection structure and other changes, causing cell irreversible damage. Microtubule stabilizers can stabilize microtubules and inhibit these breaks. It is not clear how microtubules participate in and regulate the permeability of human pulmonary microvascular endothelial cells (HPMECs) in human lung microvascular endothelial cells under inflammatory conditions. Microtubule related protein 4 (microtubule associated protein 4, MAP4) is a microtubule that is widely expressed in whole body tissue. The kinetic regulation protein, which promotes microtubule polymerization, maintains a complete.MAP4 of the cytoskeleton and connection structure to regulate its own activity by phosphorylation. When its phosphorylation is increased, the activity of MAP4 is reduced and the microtubule dynamics is unstable. Our previous study found that the rat myocardial cells were deficient in the rat milk. After oxygen, p38/MAPK (p38 mitogen-activated protein kinase) activated, MAP4 phosphorylation increased, activity decreased and microtubule disaggregation. In addition, we also found that after burn serum treatment, the p38/MAPK pathway in human umbilical vein endothelial cells (HUVECs) was activated and led to the increase of HUVECs permeability. But, under inflammatory conditions, MA in HPMECs We are not clear about the changes in the activity of P4, and the increase in HPMECs permeability and the possible regulatory mechanism of MAP4 in inflammation need further discovery and confirmation. Therefore, this study will detect the changes in the activity of Map4 in inflammation, and simulate dephosphorylation of Map4 by constructing a Map4 mutant Map4 (ALA). State, through transient transfection of recombinant adenovirus, further clarify whether Map4 phosphorylation mediated inflammation induced hpmecs permeability increase. On the basis of this, we will also initially explore the relevant mechanisms of Map4 to regulate the increase of hpmecs permeability caused by inflammation. The aim is to provide new targets and research directions for the treatment of clinical related diseases. And methods: 1, we use hpmecs as the research object, using lipopolysaccharide (LPS) and tumor necrosis factor - alpha (tumournecrosisfactor- alpha) to treat the cells in vitro, and simulate the inflammatory stimulation in vitro. The changes of hpmecs permeability and microtubule changes are observed before and after the treatment of LPS (2005001000ng/ml) and tnf- alpha (2005001000ng/ml). The following methods were used to detect and observe the following methods: 1) detect the leakage of FITC-dextran (40kDa) fluorescent substance and measure the resistance value of the cross endothelial cell (TER) to observe the changes in the barrier function of the pulmonary microvascular endothelial cells, and 2) to observe the microtubule by immunofluorescence staining (immunofluorescence, if) on the alpha microtubulin. State, structure and distribution status; 3) separation of free and polymerized microtubules, extracted and quantified, detected by Westernblot (WB), observed the changes in free and polymerized microtubule protein content.2, using microtubule stabilizer paclitaxel (Taxol, 1um) pretreated cells, before and after paclitaxel pretreatment, to observe the inflammatory factor LPS (50). 0ng/ml) and the effect of tnf- alpha (500ng/ml) stimulation on the permeability and microtubule of hpmecs. By detecting FITC-dextran and ter, the changes in the permeability of the pulmonary microvascular endothelial cells were observed. The morphology, structure and distribution of microtubules were observed by if, and the microtubules in the isolated and polymerized states were isolated and extracted, and the content of the microtubules was detected by WB. .3, detected by WB, and observed the changes in the activity of hpmecs1h, 3h, 6h, 12h after hpmecs1h, 3h, 6h, 12h, and tnf- alpha (500ng/ml) in treating hpmecs1h, 3h, 6h, 12h. The effect of ML) and tnf- alpha (500ng/ml) stimulation on the permeability of hpmecs, and using if to detect the morphology, structure and distribution of microtubules, and the separation and extraction of microtubules of the polymeric and free microtubules, and the changes in the content of the microtubules by WB method. In addition, the interaction between Map4 and microtubules was detected by the immunoprecipitation (immunoprecipitation, IP). In order to clarify the role of Map4 in regulating hpmecs permeability and microtubule change,.4 was used to detect the activation of hpmecs1h, 3h, 6h, 12h, and tnf- alpha (500ng/ml) were detected by WB method. The changes in the activity of Map4 (s696, s787, s768) and p-p38 were induced by LPS (500ng/ml) and tnf- alpha (500ng/ml) before and after the pretreatment of SB203580 cells, and the interaction between Map4 and microtubules was detected by IP method. 203580 (5um), by detecting FITC-dextran and ter, the effects of LPS (500ng/ml) and tnf- alpha (500ng/ml) stimulation or MKK6 (Glu) on the hpmecs permeability were observed before and after SB203580 pretreatment cells. The morphology and distribution of microtubules were detected by if method, and microtubules were extracted by separation and dissociation. At the same time, cmv-null, Map4 (ALA) and MKK6 (Glu) were transfected or co transfected, and the changes of hpmecs permeability, microtubule structure and free / polymerized microtubule protein content were detected by the above methods to determine whether the p38/mapk kinase pathway was involved in the regulation of Map4 activity and regulated the hpmecs permeability mediated by LPS and tnf- alpha. .6, caspase-3 inhibitor (z-dqmd-fmk, 10um), p38/mapk inhibitor SB203580 (5um) and Map4 (ALA) were pretreated or transfected to cells. The effects of inflammatory factors LPS (500ng/ml) and tnf- alpha stimulation on apoptosis and permeability were observed before and after the pretreated cells. Ran and ter were used to judge the changes in the permeability of hpmecs so that the increase of hpmecs permeability caused by Map4 mediated inflammation was not dependent on apoptosis. Results 1, after LPS (2005001000ng/ml) or tnf- alpha (2005001000ng/ml) treated hpmecs6h, hpmecs monolayer increased the leakage of FITC-dextran fluorescein, and the resistance of trans endothelial cells decreased. When the concentration of LPS or tnf- alpha was 200ng/ml, the microtubule structure was irregular and a small number of microtubules were broken. When the concentration of LPS or tnf- alpha was 500ng/ml, the cell membrane microtubules were depolymerization obviously and the peripheral microtubules were crinkled. When the concentration of LPS or tnf- alpha was increased to 1000ng/ml, only a few microtubule structures remained. Fragments; free / polymerized microtubule protein quantitative detection suggested that after the treatment of inflammatory factor LPS (2005001000ng/ml) or tnf- alpha (2005001000ng/ml), free microtubule protein was increased and the polymerized microtubule decreased, and.2 was changed in a dose-dependent manner. According to the experimental results, two typical inflammation of LPS (500ng/ml) or tnf- alpha (500ng/ml) was selected. After the pretreated cell 1H by microtubule stabilizer paclitaxel (1um), the permeability of normal cells was not obviously changed, microtubule polymerization increased and the depolymerization decreased; meanwhile, the leakage of FITC-dextran fluorescent substance caused by LPS (500ng/ml) or tnf- alpha (500ng/ml) stimulated 6h significantly decreased, and the resistance value of trans endothelial cells increased significantly. The microtubule microtubule density of microtubule was decreased and the density of microtubule microtubules increased, the content of microtubule protein in polymerized microtubule increased, the content of free microtubule protein decreased by.3, and the phosphorylation of Map4 (s696 and s787) increased, while Map4 (ser768) phosphorylation was not changed after hpmecs6h of LPS (500ng/ml) or tnf- alpha (500ng/ml). After that, there was no obvious change in the permeability of the normal hpmecs, the increase of microtubule polymerization and the decrease of the depolymerization. At the same time, the leakage of the fluorescent substance in the hpmecs monolayer after the stimulation of the inflammatory factor LPS (500ng/ml) or tnf- a (500ng/ml) was significantly reduced, the resistance value of the trans endothelial cells increased obviously, and the microtubule dynamics was more stable and the microstructure of microtubules was improved. The free microtubule content was reduced and the aggregate microtubule increased; in addition, the combination of Map4 and microtubule increased by.4, and inflammatory factor LPS (500ng/ml) or tnf- alpha (500ng/ml) treated hpmecs1h, 3h, 6h, 12h after hpmecs1h, 3h, 6h, 12h, and showed a time dependent change. The level of 38 phosphorylation increased significantly, and the combination of Map4 and microtubule decreased. On the contrary, p38/mapk blocker SB203580 (5um) pretreated the cells, and the activation of p38/mapk activation caused by LPS (500ng/ml) or tnf- alpha (500ng/ml) stimulated 6h, and Map4 (s696 and 500ng/ml) phosphorylation level decreased significantly. Furthermore, the combination of microtubules and microtubules was also significant. After the increase of.5, SB203580 (5um) pretreated 1H, the leakage of hpmecs monolayer stimulated by LPS (500ng/ml) or tnf- alpha (500ng/ml) significantly decreased the leakage of FITC-dextran fluorescent substance and increased the resistance value of the trans endothelial cells, and the microtubule structure destruction was suppressed, the free microtubule content was reduced and the microtubule protein content in the polymerized state increased; on the contrary, MKK After transfection of 6 (Glu) cells, the leakage of FITC-dextran fluorescent substance increased in hpmecs monolayer, the resistance value of cross endothelial cells decreased, microtubule depolymerization increased and polymerization decreased. Furthermore, after transfecting MKK6 (Glu) +cmv-null, we found that the hpmecs monolayer increased the fluorescent leakage of FITC-dextran, reduced the resistance value of the endothelial cells, microtubule structure destruction and free microtubules. The protein increased and the polymerized microtubule decreased, and after CO transfection of MKK6 (Glu) and Map4 (ALA), the above changes were obviously inhibited by.6. After the caspase-3 inhibitor (z-dqmd-fmk, 10um) pretreated the cell 1H, the apoptotic cell apoptosis induced by LPS (500ng/ml) or tnf- alpha was obviously suppressed. After staining, the leakage of hpmecs monolayer stimulated by inflammatory factor LPS (500ng/ml) or tnf- alpha (500ng/ml) still decreases, and the resistance value of trans endothelial cells still increases. It suggests that the increase of hpmecs permeability induced by Map4 phosphorylation mediated inflammation factor is not dependent on cell withering. Conclusion the inflammation factor LPS or tnf- alpha treatment hpmecs, cells P38/mapk kinase pathway is activated by phosphorylation of Map4 (s696 and s787) to deactivate Map4 and cause microtubule depolymerization and lead to higher hpmecs permeability. Meanwhile, the increase of hpmecs permeability induced by Map4 phosphorylation mediated inflammation factor is not dependent on apoptosis. The above results suggest that Map4 mediated microtubule structure changes may be involved in inflammation LED HPME. The increase of CS permeability will help us to further understand the pathogenesis of acute lung injury in burn shock and inflammation, and provide new targets and directions for the intervention and treatment of clinical related diseases.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R54

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