【作者】 尤青海；

【导师】 孙耕耘；

【作者基本信息】 安徽医科大学 ， 内科学， 2010， 博士

【摘要】 研究背景肺部炎症反应过程中,构成微循环交换血管最内层的肺微血管内皮细胞(pulmonary microvascular endothelial cells, PMVECs)是炎症因子首先攻击的效应细胞,其病理生理改变之一是细胞通透性增加,严重时可导致肺水肿,这是急性肺损伤/急性呼吸窘迫综合征(acute lung injury/acute respiratory distress syndrome, ALI/ARDS)的主要发病环节之一。多种信号转导通路参与PMVECs通透性调控,其中蛋白激酶C(protein kinase C, PKC)信号转导通路被广泛研究。已知活化的PKC磷酸化底物蛋白、促进PMVECs骨架重构以及细胞间黏附连接物解聚,从而增加PMVECs通透性,但细胞内PKC下游信号转导错综复杂,所以PKC底物及其在PMVECs通透性调控中的作用机制值得深入研究。此外,在人体研究PMVECs通透性变化十分困难,目前关于PMVECs通透性的研究结果主要来自体外细胞实验,所以PMVECs单层的融合状态决定实验结果精确性。src抑制的蛋白激酶C底物(src-suppressed C kinase substrate, SSeCKS)是一新发现的PKC底物,可被活化的PKC磷酸化。多种细胞株研究发现SSeCKS蛋白参与肌动蛋白骨架和血脑屏障的动态调控,脂多糖(lipopolysaccharide, LPS)诱导大鼠PMVECs高表达SSeCKS蛋白,这些研究提示SSeCKS可能是PMVECs通透性调控的重要效应分子,但其在炎症因子如白介素(interleukin, IL)-1β和肿瘤坏死因子(tumor necrosin factor, TNF)-α诱导PMVECs通透性增加中的具体作用仍未阐明。除LPS外,其他在肺部炎症反应过程中发挥重要作用的炎症介质是否参与PMVECs中SSeCKS的诱导表达也未见报道。IL-17F是一种新的炎症因子,大量研究表明在支气管上皮细胞和静脉内皮细胞中,IL-17F通过诱导IL-6、IL-8和细胞间黏附分子-1表达触发一系列炎症反应;肺炎克雷伯杆菌、支原体或白色念珠菌感染后,肺部增加的IL-17F诱发中性粒细胞聚集和变态反应,这些研究提示IL-17F是参与肺部炎症反应的重要细胞因子。目前关于肺部IL-17F作用机制的研究主要集中在支气管上皮细胞,并且对其细胞内信号转导通路知之甚少,PMVECs是肺部炎症反应时的主要效应细胞,但关于IL-17F对PMVECs的刺激效应以及细胞内信号转导通路的研究却未见报道。目的观察PMVECs单层融合前后的生物学特征。融合PMVECs单层用于实验,观察IL-17F对PMVECs通透性的影响以及SSeCKS信号通路在IL-1β、TNF-α和IL-17F诱导PMVECs通透性变化中的作用,为认识ALI/ARDS发生机制提供实验依据。方法体外培养大鼠PMVECs;在Transwell和硝酸醋酸混合纤维膜上构建PMVECs单层;倒置显微镜、苏木素-伊红染色、跨内皮细胞电阻(transendothelial electrical resistance, TER)、异硫氰酸荧光素标记葡聚糖法(Pd)以及Hank’s平衡液法(LP)观察细胞单层生物学特征;逆转录聚合酶链反应分析SSeCKS的mRNA变化;免疫印迹技术分析PKC活性以及SSeCKS蛋白变化;应用异硫氰酸荧光素标记的鬼笔环肽观察PMVECs肌动蛋白骨架改变;此外,PMVECs还被特异性的SSeCKS小分子干扰RNA(small interfering RNA, siRNA)转染。结果1)1×105/cm2的PMVECs接种Transwell后,TER随接种时间延长稳定升高,接种后第四天达最高,尔后维持,第十天TER开始下降,而倒置显微镜观察到细胞接种后第三天已融合成单层;2×105/cm2的PMVECs接种Transwell后,TER达高峰时间提前到接种后第二天,第九天TER开始下降;不同密度接种组的TER最高值间比较无显著性差异(P > 0.05)。静息状态下融合PMVECs单层TER为48.07±2.84 ?·cm2、Pd为6.19±0.43×10-6 cm/s、LP为6.80±0.62×10-7 cm/s/cm H2O。2)10mg/L LPS分别刺激融合PMVECs单层0.5h和2h后,与未处理对照组比较,TER显著下降、LP和Pd均显著增加。3)5ng/ml IL-1β和10ng/ml TNF-α分别激活PMVECs的PKC信号通路和诱导SSeCKS的mRNA和蛋白高表达,IL-1β+TNF-α诱导SSeCKS表达的效应更显著;BIM (1μmol/L,PKC抑制剂)显著下调PMVECs的PKC活性和抑制IL-1β+TNF-α诱导SSeCKS的mRNA和蛋白高表达,而蛋白激酶A(protein kinase A, PKA)抑制剂(H89,10μmol/L)和蛋白酪氨酸激酶(protein tyrosine kinase, PTK)抑制剂(金雀异黄素,50μg/ml)不影响IL-1β+TNF-α诱导SSeCKS的mRNA和蛋白高表达。4)与未处理组比较,50nmol/L SSeCKS-siRNA转染PMVECs 12h后,SSeCKS mRNA表达显著下降、24h达最低值、持续到转染后96h,SSeCKS-siRNA抑制SSeCKS蛋白表达的最佳时间在转染48h后,抑制率接近50%,抑制效应持续到转染后120h;SSeCKS-siRNA转染还以浓度依赖方式(10、20、50 nmol/L)沉默SSeCKS mRNA和蛋白表达。无论是脂质体空转染、普通阴性对照siRNA转染还是SSeCKS-siRNA转染均使融合PMVECs的静息状态TER轻微下降、静息状态Pd略升高,但与未处理组比较无显著性差异(P > 0.05)。5 ) IL-1β+TNF-α刺激显著下调PMVECs的TER和增加PMVECs的Pd ,SSeCKS-siRNA转染可显著下调IL-1β+TNF-α诱导细胞单层通透性的增加,但改善效果与BIM的改善效果比较存在差异(P < 0.05)。6)不同浓度IL-17F(0.1、1、10、100ng/ml)刺激PMVECs 3h后,下降的TER和增加的Pd与未处理组比较差异显著;100ng/ml IL-17F刺激细胞0.5h后,Pd与未处理组比较增加了15%、3~6h后Pd达高峰、显著增加的Pd持续到刺激后24h;同Pd变化,IL-17F刺激PMVECs单层时,TER呈时间依赖性下降;1、10μmol/L BIM均能改善IL-17F诱导增加的Pd和下降的TER,但都未能使其恢复到正常值。7)IL-17F刺激3h后,PMVECs的丝状肌动蛋白重组:从细胞周边向细胞中心转移并成平行束状堆积、增厚,同时细胞旁缝隙显著增大;流式细胞技术检测发现IL-17F刺激后,PMVECs中丝状肌动蛋白的相对荧光强度曲线向右位移、其平均荧光强度与未处理组比较显著增加,而1、10μmol/L BIM均能有效改善IL-17F诱导PMVECs肌动蛋白骨架的重构。8)不同浓度(0.1、1、10、100ng/ml)IL-17F刺激PMVECs后,SSeCKS的mRNA和蛋白表达均显著增加;100ng/ml IL-17F刺激PMVECs时,SSeCKS基因表达在刺激后3h达高峰,而蛋白表达在刺激后6h达高峰;IL-17F刺激PMVECs 5min后,SSeCKS蛋白的丝氨酸被磷酸化、30min后显著、3~6h后达高峰、后维持在较高的磷酸化水平至刺激后24h,而1μmol/L BIM预孵育后,IL-17F诱导增加的蛋白磷酸化水平显著下降;此外,IL-17F刺激PMVECs 30min后,不溶于Triton-X-100的SSeCKS蛋白显著增加,3~6h后,不溶于Triton-X-100的SSeCKS蛋白达高峰;最后,静息状态时,SSeCKS蛋白大部分集中在细胞质,IL-17F刺激30min后,细胞膜部SSeCKS蛋白的量与静息状态比较差异显著,刺激90min后,SSeCKS大部分集中在细胞膜。结论1)TER联合倒置显微镜较倒置显微镜单独应用能更准确判定PMVECs单层融合状态;2)TER、Pd、LP均能有效检测PMVECs单层通透性变化,TER联合Pd更多地反映PMVECs通透性变化信息;3)SSeCKS表达增加上调PMVECs通透性在ALI/ARDS复杂机制中占重要地位,IL-1β、TNF-α和IL-17F是PMVECs高表达SSeCKS基因和蛋白的强有力诱导剂,PKC而非PKA、PTK信号通路参与其表达调控;4)IL-17F通过激活PKC-SSeCKS信号转导通路以及诱导PKC依赖的F-actin骨架重构来增加PMVECs通透性,从而促进肺部炎症反应。更多还原

【Abstract】 BackgroundPulmonary microvascular endothelial cells (PMVECs) lined at the critical interface between the blood and microvessel are primary targets of inflammatory cytokines during lung inflammation. A pathophysiologic response of PMVECs to inflammatory cytokines is permeability increase that contributes to high-protein pulmonary edema, which is a characteristic of acute lung injury and acute respiratory distress syndrome (ALI/ARDS). It is well known that the protein kinase C (PKC) signaling pathway significantly contributes to the breakdown of PMVECs barrier though phosphorylation of proteins which can promote cytoskeletal reorganization or dissolution of the adherens junctions. Given the complex interplay between PKC signaling pathway and the possible contribution of other signaling pathways, further studies should be required to delineate downstream targets of PKC and the mechanisms that regulate endothelial barrier function in PMVECs. In addition, it is very difficult in vivo to study changes of PMVECs permeability, and considerable achievements in this research area come from in vitro experiments of cultured cells, therefore, the integrality of study can be determined by the confluent state of PMVECs.Src-suppressed C kinase substrate (SSeCKS) is a PKC substrate that is identified to be phosphorylated by PKC. Of particular interest, evidences that SSeCKS is involved in a dramatic rearrangement of the actin cytoskeleton and the modulation of the blood-brain barrier permeability have been presented. Moreover, in rat PMVECs (RPMVECs), increased levels of SSeCKS have been found after challenge of lipopolysaccharide (LPS). These implicate that SSeCKS is possibly one of the critical proteins in the pathogenesis of endothelial hyperpermeability after exposure of PMVECs to various inflammatory factors, such as interleukin (IL)-1βand tumor necrosin factor (TNF)-α. Nevertheless, the exact role of SSeCKS in the regulation of PMVECs permeability is not well understood. Additionally, besides LPS, other potential inducers of SSeCKS in RPMVECs remain ill-defined.Interleukin-17F is a novel proinflammatory cytokine. Accumulating studies have showed that IL-17F triggers a variety of inflammatory responses such as inducing inflammatory factors, including IL-6, IL-8, and intracellular adhesion molecule-1, in airway epithelial cells and vein endothelial cells. Of note, in lung, increased expression of IL-17F has been found in animal models after Klebsiella pneumoniae, Mycoplasma pneumoniae and Candida albicans infection, and to induce pulmonary neutrophilia and an additive effect on antigen-induced allergic inflammatory responses. These findings have suggested that IL-17F is one of the key cytokines regulating lung inflammation. However, up to date, studies about mechanisms of IL-17F in lung focus on bronchial epithelium, and signaling pathways activated by IL-17F in cells have not been elucidated. It is well known that PMVECs are involved in lung inflammatory disease, yet, little information exists in the role of IL-17F and its signaling pathway in PMVECs.ObjectiveThe aim of this study is firstly to observe some biological characteristics of PMVECs monolayers before and after confluence, and then, we sought to investigate the effects of IL-17F on confluent PMVECs monolayers and assess the role of PKC in this process. The role of SSeCKS in the modulation of PMVECs permeability elicited by IL-1β, TNF-αand IL-17F is also investigated. These may be helpful for elucidating the pathogenesis of ALI/ARDS.MethodsAfter primary RPMVECs were successfully cultured in vitro, the cells were seeded on Transwell polyester membranes or nitric-acid/acetic-acid membranes to construct in vitro models of PMVECs monolayers. The biological characteristics of cells monolayers before and after confluence were observed by the inverted microscope, staining with hematoxylin-eosin, transendothelial electrical resistance (TER) and changes of permeability as measured by means of fluorescein isothiocyanate (FITC)-dextran (Pd) and Hank’s solution (LP) across monolayers. Additional monolayers were stained using FITC-phalloin for observing the filamentous actin (F-actin) cytoskeletal changes. The gene expression of SSeCKS was analyzed by the reverse transcription-polymerase chains. Immunoblotting was used to determine the PKC activity, the levels of SSeCKS protein expression and the biological alterations of SSeCKS protein in PMVECs. In addition, SSeCKS-specific small interfering RNA (siRNA) was transfected into PMVECs.Results1) In group one, after a cell suspension (1×105 cells /cm2) was cultured on Transwell, the normalized TER increased steadily in a time-dependent manner and reached the summit on the fourth day post-seeding, then was sustained. Subsequently, on the tenth day post-seeding, the increased TER began to decrease. However, the monolayer under inversed microscope reached the state of confluence on the third day post-seeding. In group two, a cell suspension (2×105 cells /cm2) was seeded, changes of the normalized TER were similar to that of the group one, reaching the summit on the second day post-seeding and beginning to decrease on the ninth day post-seeding. Of note, no significant difference in the maximum TER was found between both groups. The TER, Pd and LP of confluent PMVECs monolayers under quiescent state were 48.07±2.84 ?·cm2, 6.19±0.43×10-6 cm/s and 6.80±0.62×10-7 cm/s/cm H2O, respectively.2) After confluent PMVECs monolayers were stimulated with 10 mg/L LPS for both 0.5 h and 2 h, there were significant decreases in TER and increases in Pd and LP when respectively compared with the untreated group.3) Interleukin-1β(5 ng/ml) and TNF-α(10 ng/ml) activated PKC signaling pathway in PMVECs, and up-regulated the gene and protein expression of SSeCKS. Meanwhile, we found that the expression of SSeCKS was the greatest after stimulation with the cytokine combination of 5 ng/ml IL-1βand 10 ng/ml TNF-α. PKC inhibitor, Bisindolylmaleimide I (BIM, 1μmol/L) significantly decreased both the increased PKC activity and SSeCKS expression induced by the cytokines in PMVECs. However, both the selective protein kinase A (PKA) inhibitor H89 (10μmol/L) and the broad-spectrum protein tyrosine kinase (PTK) inhibitor genistein (50μg/ml) showed no significant inhibitory effects on cytokine-induced SSeCKS gene expression and protein production in PMVECs.4) Transfection with special SSeCKS-siRNA resulted in a decrease in the expression of SSeCKS gene and protein in a dose-dependent manner (10, 20, 50 nmol/L) and in a time-dependent manner. When compared with the untreated group, significantly inhibitory effects of SSeCKS-siRNA (50 nmol/L) in the expression of SSeCKS gene and protein began at 12 h and 24 h, reached a peak at 24 h and 48 h and were sustained to 96 h and 120 h after PMVECs were transfected, respectively. At 48 h post-transfection, the expression of SSeCKS in PMVECs was inhibited by 50 %. Furthermore, SSeCKS depletion, the mock trasfection and the non-silencing control siRNA transfection induced a slight, but not significant (P > 0.05), decrease in quiescent TER and increase in quiescent Pd across confluent PMVECs monolayers.5) When compared with the untreated group, application of the cytokine combination (IL-1βand TNF-α) to PMVECs monolayers resulted in a significant increase of Pd and a significant decrease of TER, and then, prior transfection with 50 nmol/L SSeCKS-siRNA significantly attenuated the cytokine-induced effects. Notably, it was found that the effect of BIM in blocking cytokine-induced endothelial barrier dysfunction is more significant than that of SSeCKS depletion.6) Interleukin-17F induced, in a dose-dependent manner (0.1, 1, 10, 100ng/ml), significant increases of Pd and decreases of TER after PMVECs monolayers were exposed to stimulations for 3 h. The effects of IL-17F were also time dependent. Pd increased ~15 % as compared with quiescent levels after monolayers were treated by 100ng/ml IL-17F for 0.5 h, peaked at 3h and prolonged lag time of 24 h. Similarly, the progressive decrease in TER produced by IL-17F was time-dependent when compared with untreated monolayer. After PMVECs monolayers were pretreated with BIM (1 or 10μmol/L, respectively), the increased Pd and decreased TER induced by IL-17F can be significantly blocked, but did not return to normal levels.7) In PMVECs, IL-17F stimulation for 3 h produced numerous intercellular gaps and a marked reorganization of F-actin from a web-like peripheral distribution to centrally located parallel stress fibers. The latter became thicker. Meanwhile, after IL-17F stimulation, the flow cytometric profile was shifted to the right and the mean fluorescence intensity of F-actin was conspicuously increased when compared with untreated monolayers. IL-17F-induced cytoskeletal changes indicated above can be significantly amended after PMVECs monolayers were pretreated by 1 and 10μmol/L BIM.8) After PMVECs were treated with increasing concentrations of IL-17F (0.1, 1, 10, 100 ng/ml) and 100 ng/ml IL-17F for various times, the expression of SSeCKS gene and protein were remarkably up-regulated in a dose-dependent manner, occurred maximally at 3 h and 6 h post-stimulation, respectively. The serine phosphorylation of SSeCKS was weakly detected at 5-minutes point, remarkably occurred at 30-minutes point, reached maximal level at 3 ~ 6 h, and stayed at a slightly high level until 24 h after PMVECs were exposed to100 ng/ml IL-17F. BIM (1μmol/L) can significantly abrogate this phosphorylation induced by IL-17F. In addition, our results demonstrated that treatment with IL-17F significantly increased the amount of SSeCKS in the insoluble fraction in a time-dependent manner, beginning at 30 min and peaking at 3 ~ 6 h post-stimulation. Finally, under quiescent conditions, SSeCKS existed for the most part in the cytosolic fraction of PMVECs, after 30 min treatment, IL-17F resulted in significant translocation of SSeCKS from the cytosol to the membrane. At 90-minutes point, SSeCKS predominantly existed in the membrane fraction.Conclusions1) The combination of TER and an inverted microscope is better than the solo application of an inverted microscope to assess the confluent state of PMVECs monolayers.2) Three methods, TER, Pd and LP, can be availably used to determine PMVECs permeability. Combination of Pd and TER maybe greatly reflect the information of changes in permeability across the in vitro PMVECs monolayer model.3) The increased expression of SSeCKS is involved in the regulation of PMVECs hyperpermeability that maybe play an important role in the process of ALI/ARDS. The expression of SSeCKS gene and protein can be potently induced by IL-1β, TNF-αand IL-17F in PMVECs. It is PKC signaling pathway, neither PKA signaling pathway nor PTK signaling pathway, which is involved in the regulation of SSeCKS expression.4) IL-17F promotes the lung inflammatory response though the activated PKC-SSeCKS signaling pathway and the PKC-dependent reorganization of actin cytoskeleton that all contribute to PMVECs hyperpermeabilty induced by IL-17F.更多还原