HDAC6对肺内皮细胞屏障功能的干预机制研究

发布时间：2018-05-20 19:17

本文选题：HDAC6 + 屏障功能　；参考：《吉林大学》2017年博士论文

【摘要】：内皮细胞屏障具有重要的功能。肺内皮细胞屏障是一个半渗透膜,位于血管与周围组织之间,调节体液平衡,维持内环境的稳定[1]。内皮细胞屏障破坏常发生于各种疾病的早期,如急性肺损伤(ALI)等[2],如果纠正不及时甚至可威胁生命。内皮细胞维持其屏障功能完整主要依赖于两方面:内皮细胞骨架结构的完整及紧密的细胞间连接[3,4]。目前已知多种原因可造成内皮细胞屏障功能破坏,如感染诱发炎症因子(TNFα)增加等,有害因素可造成不同程度的细胞收缩,细胞连接缝隙出现,细胞骨架结构重排,进而导致细胞屏障完整性破坏,造成血管内外液体平衡失调。此外,前炎症因子如TNFα可诱导内皮细胞caspase活动增加,激活凋亡信号途径,细胞调亡增加,有报道称caspase活动增加可影响部分细胞连接蛋白,均可导致内皮细胞屏障通透性增强。本文主要针对TNFα、LPS等损伤因素造成的内皮细胞屏障功能障碍进行研究,阐述其主要机制及HDAC6抑制后对上述损害的保护作用分子机制。HDAC6是IIb类去乙酰化酶,主要通过去乙酰化及泛素化作用调节底物发挥功能[5]。已知HDAC6底物主要包括α-tubulin,HSP90,cortactin[6-9]。近年来许多研究表明抑制HDAC6基因表达具有明确的抗肿瘤,免疫抑制,抗炎作用[10-15],但其分子机制仍不明确。体外实验中TNFα刺激肺内皮细胞诱导细胞屏障通透性增加主要通过以下途径[16-18]:1、细胞连接破坏:VE-cadherin与β-catenin复合物形成是维持细胞连接完整性的重要组成,TNFα刺激后可造成上述复合物的不稳定从而导致细胞间缝隙出现;TNFα可诱导肺内皮细胞caspase 3活动增加,也可引起细胞间连接破坏。2、细胞骨架结构重排:细胞微管结构是构成细胞骨架的主要组成部分,主要有α-tubulin及β-tubulin两种单体,二者以聚合体形式存在,维持微管结构完整性,TNFα可导致这种聚合形式破坏,一方面影响细胞微管结构完整性,同时可导致细胞骨架另一结构即细胞微丝结构(Factin,P-MLC)重排。特异性HDAC6抑制剂或HDAC6基因敲除可通过对其底物α-tubulin及β-catenin的乙酰化作用及降低caspase 3活动有效抑制上述改变的发生,从而维持细胞屏障功能的完整性。实验第一部分为体外细胞实验,掌握特异性HDAC6抑制剂或HDAC6基因敲除对细胞屏障功能保护作用及其分子机制。通过TNFα刺激肺动脉血管内皮细胞及肺微血管内皮细胞建立炎症反应模型,我们可观察到实验组细胞屏障通透性明显增加,细胞形态改变,细胞连接破坏(VE-cadherin及β-catenin复合体破坏),微管结构聚合形式降低而α-tubulin单体形式增加,F-actin增加及P-MLC表达增加,同时也观察到cleaved caspase3(c-caspase3)蛋白含量明显升高,提示caspase3活动性增加。而预先应用特异性HDAC6抑制剂干预或者应用si RNA基因敲除HDAC6蛋白后给予TNFα刺激可明显减轻上述改变,同时可检测到α-tubulin及β-catenin乙酰化表达增加,c-caspase3蛋白含量降低,结合以往文献报道,我们认为特异性HDAC6抑制剂或HDAC6基因敲除具有减轻TNFα诱导的内皮细胞通透性增加的作用,主要通过诱导α-tubulin及β-catenin乙酰化表达增加及降低caspase3活动,从而维持细胞连接完整性及细胞微管结构稳定性。实验第二部分为体内动物实验,本部分实验应用两种动物模型即LPS腹腔注射诱导脓毒血症模型及盲肠结扎穿刺术诱导脓毒血症模型。LPS模型组:特异性HDAC6抑制剂(Tub A 9mg/ml及CAY10603 3mg/ml)或DMSO腹腔注射6h后,给予LPS 7.5mg/kg腹腔注,LPS注射24h后收集标本;CLP模型组:CLP手术后或模拟手术后1小时给予特异性HDAC6抑制剂(Tub A 9mg/ml及CAY10603 3mg/ml)腹腔注射,术后24小时处死小鼠,收集样本。实验项目包括测量肺干湿重比,MPO活性,Ace-α-tubulin,Ace-β-catenin,c-caspase3蛋白。我们观察到Tub A或CAY10603干预组较单独应用TNFα组小鼠肺干湿重比及MPO活性明显降低,说明小鼠肺水肿明显改善,炎症减轻,同时检测到TNFα组Ace-α-tubulin及Ace-β-catenin蛋白量较对照组明显降低而Tub A或CAY10603干预组则基本恢复到对照组相同表达水平。进一步说明特异性HDAC6抑制剂可改善炎症介导的肺内皮细胞屏障通透性增加,主要通过诱导底物α-tubulin及β-catenin乙酰化表达增加及抑制caspase3活动性。综上所述,特异性HDAC6抑制剂或HDAC6基因敲除可减轻炎症介导的肺内皮细胞屏障功能障碍,主要通过诱导α-tubulin及β-catenin乙酰化作用及降低capase3活动性,从而增加细胞连接及细胞骨架结构的稳定性,维持细胞屏障功能的完整性。
[Abstract]:The endothelial barrier is an important function. The pulmonary endothelial barrier is 1.5 osmotic membrane, located between the blood vessels and the surrounding tissue, regulating the balance of the body fluid, maintaining the internal environment and maintaining the stability of the [1]. endothelial cell barrier, which often occurs in the early stages of various diseases, such as acute lung injury (ALI) and other [2], if the correction is not timely or even the life can be threatened. The function of the skin cells to maintain its barrier function is mainly dependent on two aspects: the integrity of the endothelial cytoskeleton and the close intercellular connection of [3,4]., which can cause damage to the barrier function of the endothelial cells, such as the increase of the inflammatory factor (TNF alpha) induced by infection, and the harmful factors can cause different degrees of cell contraction and joint seams. In addition, the pro-inflammatory factor such as TNF alpha can induce the increase of caspase activity in endothelial cells, activation of apoptosis signal pathway, and the increase of cell apoptosis. It is reported that the increase of caspase activity may affect some cell connexin. The permeability of endothelial cell barrier is enhanced. This paper focuses on the barrier dysfunction of endothelial cells caused by TNF alpha, LPS and other damage factors, and expounds its main mechanism and the molecular mechanism of the protective effect of HDAC6 inhibition on the above damage,.HDAC6 is the IIb deacetylase, and the main objective is to regulate the substrate through deacetylation and ubiquitination. Function [5]. known HDAC6 substrates include alpha -tubulin, HSP90, and cortactin[6-9]. in recent years, many studies have shown that inhibition of HDAC6 gene expression has clear anti-tumor, immunosuppressive, and anti-inflammatory effects [10-15], but its molecular mechanism is still unclear. In vitro, TNF a stimulates the increased permeability of cell barrier induced by lung endothelial cells to increase the main passage. The following pathways: [16-18]: 1, cell connection destruction: the formation of VE-cadherin and beta -catenin complex is an important component of the maintenance of cell connectivity integrity. TNF a stimulates the instability of the complexes resulting in the emergence of intercellular crevice; TNF alpha induces an increase in caspase 3 activity in the pulmonary endothelial cells and may also cause intercellular connections to destroy.2 The cytoskeleton rearrangement: the cell microtubule structure is the main component of the cytoskeleton, which mainly consists of two monomers of alpha -tubulin and beta -tubulin, and the two is in the form of polymer, maintaining the integrity of microtubule structure. TNF alpha can lead to the destruction of this type of polymerization. On the one hand, it affects the integrity of microtubule structure and can lead to cell bone. The other structure is the rearrangement of Factin (P-MLC). Specific HDAC6 inhibitors or HDAC6 knockout can maintain the integrality of cell barrier power through the acetylation of its substrate, alpha -tubulin and beta -catenin, and the reduction of caspase 3 activity. The first part of the experiment is in vitro cell solid. Test the protective effect of specific HDAC6 inhibitors or HDAC6 knockout on cell barrier function and its molecular mechanism. Through TNF alpha stimulation of pulmonary vascular endothelial cells and pulmonary microvascular endothelial cells to establish an inflammatory response model, we can observe the obvious increase in the permeability of the cell barrier in the experimental group, the change of cell morphology, and the destruction of cell connection. (VE-cadherin and beta -catenin complex), microtubule structure polymerization decreased and the form of alpha -tubulin increased, F-actin increased and P-MLC expression increased. Meanwhile, the content of cleaved Caspase3 (c-caspase3) protein increased significantly, suggesting an increase in Caspase3 activity. NA gene knockout HDAC6 protein to TNF alpha stimulation can significantly reduce the above changes, and can detect the increase in the expression of alpha -tubulin and beta -catenin, and the decrease of c-caspase3 protein content. Combined with previous reports, we believe that specific HDAC6 inhibitors or HDAC6 knockout can reduce the permeability of endothelial cells induced by TNF alpha. The effect is to increase and reduce Caspase3 activity by inducing the acetylation of alpha -tubulin and beta -catenin, thus maintaining the integrity of cell connection and the stability of cell microtubule structure. The second part of the experiment was in vivo animal experiment. This part of the experiment used two animal models, namely, LPS abdominal cavity injection induced sepsis model and cecal ligation wear. .LPS model group: specific HDAC6 inhibitor (Tub A 9mg/ml and CAY10603 3mg/ml) or DMSO intraperitoneally injected 6h, to give LPS 7.5mg/kg peritoneal injection, LPS injection after the collection of specimens. The mice were killed 24 hours after the operation to collect the mice and collect the samples. The experimental items included the measurement of lung dry wet weight ratio, MPO activity, Ace- alpha -tubulin, Ace- beta -catenin, c-caspase3 protein. We observed that the lung dry wet weight ratio and MPO activity in the Tub A or CAY10603 intervention group were significantly lower than those of the mice with TNF a group, which showed that the pulmonary edema was obviously improved and the inflammation was alleviated. At the same time, the amount of Ace- alpha -tubulin and Ace- beta -catenin protein in the TNF alpha group was significantly lower than that in the control group, while the Tub A or CAY10603 intervention group basically recovered to the same expression level in the control group. Further demonstrated that the specific HDAC6 inhibitor could improve the permeability of the inflammatory mediate lung endothelial cell barrier, mainly by inducing the substrate alpha -tubulin and. The expression of beta -catenin acetylation increases and inhibits Caspase3 activity. To sum up, specific HDAC6 inhibitors or HDAC6 knockout can reduce inflammation mediated barrier dysfunction in the pulmonary endothelial cells, mainly by inducing alpha -tubulin and beta -catenin acetylation and reducing capase3 activity, thereby increasing cell connection and cytoskeleton structure Stability, maintaining the integrity of cell barrier function.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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