本文選題：HDAC6 + 屏障功能��； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：內(nèi)皮細(xì)胞屏障具有重要的功能。肺內(nèi)皮細(xì)胞屏障是一個(gè)半滲透膜,位于血管與周圍組織之間,調(diào)節(jié)體液平衡,維持內(nèi)環(huán)境的穩(wěn)定[1]。內(nèi)皮細(xì)胞屏障破壞常發(fā)生于各種疾病的早期,如急性肺損傷(ALI)等[2],如果糾正不及時(shí)甚至可威脅生命。內(nèi)皮細(xì)胞維持其屏障功能完整主要依賴于兩方面:內(nèi)皮細(xì)胞骨架結(jié)構(gòu)的完整及緊密的細(xì)胞間連接[3,4]。目前已知多種原因可造成內(nèi)皮細(xì)胞屏障功能破壞,如感染誘發(fā)炎癥因子(TNFα)增加等,有害因素可造成不同程度的細(xì)胞收縮,細(xì)胞連接縫隙出現(xiàn),細(xì)胞骨架結(jié)構(gòu)重排,進(jìn)而導(dǎo)致細(xì)胞屏障完整性破壞,造成血管內(nèi)外液體平衡失調(diào)。此外,前炎癥因子如TNFα可誘導(dǎo)內(nèi)皮細(xì)胞caspase活動(dòng)增加,激活凋亡信號(hào)途徑,細(xì)胞調(diào)亡增加,有報(bào)道稱caspase活動(dòng)增加可影響部分細(xì)胞連接蛋白,均可導(dǎo)致內(nèi)皮細(xì)胞屏障通透性增強(qiáng)。本文主要針對(duì)TNFα、LPS等損傷因素造成的內(nèi)皮細(xì)胞屏障功能障礙進(jìn)行研究,闡述其主要機(jī)制及HDAC6抑制后對(duì)上述損害的保護(hù)作用分子機(jī)制。HDAC6是IIb類去乙�；�,主要通過去乙酰化及泛素化作用調(diào)節(jié)底物發(fā)揮功能[5]。已知HDAC6底物主要包括α-tubulin,HSP90,cortactin[6-9]。近年來許多研究表明抑制HDAC6基因表達(dá)具有明確的抗腫瘤,免疫抑制,抗炎作用[10-15],但其分子機(jī)制仍不明確。體外實(shí)驗(yàn)中TNFα刺激肺內(nèi)皮細(xì)胞誘導(dǎo)細(xì)胞屏障通透性增加主要通過以下途徑[16-18]:1、細(xì)胞連接破壞:VE-cadherin與β-catenin復(fù)合物形成是維持細(xì)胞連接完整性的重要組成,TNFα刺激后可造成上述復(fù)合物的不穩(wěn)定從而導(dǎo)致細(xì)胞間縫隙出現(xiàn);TNFα可誘導(dǎo)肺內(nèi)皮細(xì)胞caspase 3活動(dòng)增加,也可引起細(xì)胞間連接破壞。2、細(xì)胞骨架結(jié)構(gòu)重排:細(xì)胞微管結(jié)構(gòu)是構(gòu)成細(xì)胞骨架的主要組成部分,主要有α-tubulin及β-tubulin兩種單體,二者以聚合體形式存在,維持微管結(jié)構(gòu)完整性,TNFα可導(dǎo)致這種聚合形式破壞,一方面影響細(xì)胞微管結(jié)構(gòu)完整性,同時(shí)可導(dǎo)致細(xì)胞骨架另一結(jié)構(gòu)即細(xì)胞微絲結(jié)構(gòu)(Factin,P-MLC)重排。特異性HDAC6抑制劑或HDAC6基因敲除可通過對(duì)其底物α-tubulin及β-catenin的乙�；饔眉敖档蚦aspase 3活動(dòng)有效抑制上述改變的發(fā)生,從而維持細(xì)胞屏障功能的完整性。實(shí)驗(yàn)第一部分為體外細(xì)胞實(shí)驗(yàn),掌握特異性HDAC6抑制劑或HDAC6基因敲除對(duì)細(xì)胞屏障功能保護(hù)作用及其分子機(jī)制。通過TNFα刺激肺動(dòng)脈血管內(nèi)皮細(xì)胞及肺微血管內(nèi)皮細(xì)胞建立炎癥反應(yīng)模型,我們可觀察到實(shí)驗(yàn)組細(xì)胞屏障通透性明顯增加,細(xì)胞形態(tài)改變,細(xì)胞連接破壞(VE-cadherin及β-catenin復(fù)合體破壞),微管結(jié)構(gòu)聚合形式降低而α-tubulin單體形式增加,F-actin增加及P-MLC表達(dá)增加,同時(shí)也觀察到cleaved caspase3(c-caspase3)蛋白含量明顯升高,提示caspase3活動(dòng)性增加。而預(yù)先應(yīng)用特異性HDAC6抑制劑干預(yù)或者應(yīng)用si RNA基因敲除HDAC6蛋白后給予TNFα刺激可明顯減輕上述改變,同時(shí)可檢測(cè)到α-tubulin及β-catenin乙�；磉_(dá)增加,c-caspase3蛋白含量降低,結(jié)合以往文獻(xiàn)報(bào)道,我們認(rèn)為特異性HDAC6抑制劑或HDAC6基因敲除具有減輕TNFα誘導(dǎo)的內(nèi)皮細(xì)胞通透性增加的作用,主要通過誘導(dǎo)α-tubulin及β-catenin乙酰化表達(dá)增加及降低caspase3活動(dòng),從而維持細(xì)胞連接完整性及細(xì)胞微管結(jié)構(gòu)穩(wěn)定性。實(shí)驗(yàn)第二部分為體內(nèi)動(dòng)物實(shí)驗(yàn),本部分實(shí)驗(yàn)應(yīng)用兩種動(dòng)物模型即LPS腹腔注射誘導(dǎo)膿毒血癥模型及盲腸結(jié)扎穿刺術(shù)誘導(dǎo)膿毒血癥模型。LPS模型組:特異性HDAC6抑制劑(Tub A 9mg/ml及CAY10603 3mg/ml)或DMSO腹腔注射6h后,給予LPS 7.5mg/kg腹腔注,LPS注射24h后收集標(biāo)本;CLP模型組:CLP手術(shù)后或模擬手術(shù)后1小時(shí)給予特異性HDAC6抑制劑(Tub A 9mg/ml及CAY10603 3mg/ml)腹腔注射,術(shù)后24小時(shí)處死小鼠,收集樣本。實(shí)驗(yàn)項(xiàng)目包括測(cè)量肺干濕重比,MPO活性,Ace-α-tubulin,Ace-β-catenin,c-caspase3蛋白。我們觀察到Tub A或CAY10603干預(yù)組較單獨(dú)應(yīng)用TNFα組小鼠肺干濕重比及MPO活性明顯降低,說明小鼠肺水腫明顯改善,炎癥減輕,同時(shí)檢測(cè)到TNFα組Ace-α-tubulin及Ace-β-catenin蛋白量較對(duì)照組明顯降低而Tub A或CAY10603干預(yù)組則基本恢復(fù)到對(duì)照組相同表達(dá)水平。進(jìn)一步說明特異性HDAC6抑制劑可改善炎癥介導(dǎo)的肺內(nèi)皮細(xì)胞屏障通透性增加,主要通過誘導(dǎo)底物α-tubulin及β-catenin乙酰化表達(dá)增加及抑制caspase3活動(dòng)性。綜上所述,特異性HDAC6抑制劑或HDAC6基因敲除可減輕炎癥介導(dǎo)的肺內(nèi)皮細(xì)胞屏障功能障礙,主要通過誘導(dǎo)α-tubulin及β-catenin乙酰化作用及降低capase3活動(dòng)性,從而增加細(xì)胞連接及細(xì)胞骨架結(jié)構(gòu)的穩(wěn)定性,維持細(xì)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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8 苑t，

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