脂多糖致脑微血管内皮细胞通透性升高的机制研究
摘要
细菌性脑膜炎被认为是全球前十大致死性感染性疾病之一,致死、致残率高,目前仍约30-50%的患者留有不可逆转的神经系统后遗症。血脑屏障(Blood-brain barrier, BBB)通透性增加致血管源性脑水肿在其发病中起关键作用,发病机制未明,治疗较困难。因此阐明细菌性脑膜炎时BBB通透性改变及调控机制有非常重要的实用意义。
研究表明脑微血管内皮细胞(brain micro vascular endothelial cells, BMECs)及紧密连接(tight junction, TJ)是BBB结构和功能的主要基础。LPS为革兰氏阴性细菌细胞壁的组成成分,释放入血后被称为内毒素,对BBB屏障功能破坏有重要作用。我们团队的前期实验证实中枢神经系统感染性时LPS表达显著升高,且可引起BEMC紧密连接蛋白Occludin和ZO-1表达下调,但其具体的调控方式不明。目前研究显示PKC、Rho、PI3K和酪氨酸激酶以及核转录因子NF-κB均可能参与调控紧密连接的组装和分解,维持内皮细胞低通透性。但对于上述信号分子是否参与细菌性脑膜炎或感染性脑损伤时LPS致BBB通透性升高的调控过程,他们之间的调控关系如何等方面的研究均少见报道。对上述问题的深入研究,有助于进一步阐明感染性脑损伤时BBB通透性增高的病理过程和发病机制,为其临床防治提供新的思路。
本研究分为四部分：
第一章永生化Bend.3细胞株具有原代鼠脑微血管内皮细胞屏障特性
目的：评价永生化小鼠脑微血管内皮细胞株Bend.3是否具有原代培养的鼠脑微血管内皮细胞的屏障及生理特性。
方法：将小鼠脑微血管内皮细胞株Bend.3和原代培养的鼠脑微血管内皮细胞接种于细胞培养插内,跨内皮细胞电阻抗(transendothelial electrical resistance,TEER)和辣根过氧化物酶(horseradish peroxidase,HRP)通透性实验检测其屏障功能。Westernblot法和直接荧光染色法观察其紧密连接相关蛋白Occludin、ZO-1的表达及细胞骨架蛋白F-actin的分布。
结果：Bend.3细胞的TEER随培养时间延长逐渐升高,10d达82.33±6.03Ω·cm2,与培养3d时相比,差异有统计学意义(P<0.05),与同期原代培养的鼠脑微血管内皮细胞的TEER相比,无明显差异(P>0.05)。Bend.3细胞培养10d和3d的平均HRP通透率在120min分别为(2.±20.05)%和(4.3±0.20)%,差异有统计学意义(P<0.05),与同期原代培养的鼠脑微血管内皮细胞的TEER相比,无明显差异(P>0.05)。培养10d时Bend.3细胞和原代培养的鼠脑微血管内皮细胞均表达高浓度的紧密连接蛋白Occludin、ZO-1,且F-actin主要分布在细胞周边,线条完整连续,未见明显缝隙形成。
结论：小鼠脑微血管内皮细胞株Bend.3具有原代培养的脑微血管内皮细胞的屏障特性,且其屏障功能在接种10d后可达到最理想的状态。
第二章初步筛选脂多糖致脑微血管内皮细胞通透性改变的信号分子
目的：证实脂多糖通过引起Actin重组、紧密连接表达和分布变化导致脑微血管内皮细胞通透性增高,初步探讨PKC、Rho、PI3K和酪氨酸激酶等信号是否参与脂多糖致脑微血管内皮细胞通透性改变的调控。
方法：运用TEER测定法、F-actin染色法、western blot和免疫荧光法分别检测了LPS作用不同时间下Bend.3细胞的通透性,F-actin分布以及紧密连接蛋白cluaudin-5,Occludin和ZO-1的状态,以动态观察LPS是否通过破坏紧密连接蛋白增加脑血管内皮细胞通透性,然后分别利用calphostin C (PKC抑制剂)、C3transferase (Rho抑制剂)、wortmannin (PI3K抑制剂)、PP2(酪氨酸激酶抑制剂)预处理Bend.3细胞,再通过TEER检测LPS对各组细胞屏障功能的影响,了解PKC、酪氨酸激酶、PI3K、Rho信号是否参与了LPS致血脑屏障通透性升高的调控过程。
结果：LPS可致Bend.3细胞TEER以时间依赖性的方式下降,同时伴有紧密连接蛋白ZO-1、Occludin和Claudin-5表达下调,Claudin-5蛋白分布改变,以及细胞骨架F-actin重组。PKC、Rho抑制剂可改善LPS引起的Bend.3细胞TEER下降；而PI3K、酪氨酸激酶抑制剂不能阻断LPS对Bend.3屏障功能的破坏。
结论：脂多糖引起脑微血管内皮细胞Actin重组、紧密连接表达和分布变化而导致其通透性增高；PKC和Rho,而非PI3K和酪氨酸激酶,参与了此过程调控。
第三章PKC和RhoA信号相互作用调控脂多糖致脑微血管内皮细胞通透性升高过程
目的：探讨PKC各亚型(α、β和ζ)如何参与脂多糖致脑微血管内皮细胞通透性升高调控过程,他们与RhoA之间的调控关系如何。
方法：1.pull-down法和免疫共沉淀结合体外酶学实验法分别检测LPS作用不同时间Bend.3细胞的RhoA和PKC各亚基(α、β和ζ)活化状态。2.分别利用脂质体2000将PcDN A3.1hygro-n19RhoA、 PcDNA3.1hygro-vector(空载对照质粒)导入Bend.3细胞,利用潮霉素B筛选出稳定表达株。Pull Down法鉴定RhoA活性的抑制情况。并将PLKO.1-puro-PKCα-shRNA、PLKO.1-puro-PKCβ-shRNA PLKO.1-puro-PKCζ-shRNA和empty PLKO.1-puro vector导入Bend.3细胞,利用嘌呤霉素B筛选出稳定表达株。Western blot分别鉴定PKC-α PKC-β和PKCζ蛋白的表达抑制情况。并根据导入质粒不同分5组,运用F-actin染色法、western blot和免疫荧光法分别检测了LPS作用不同时间下各组Bend.3细胞F-actin分布以及紧密连接蛋白cluaudin-5,Occludin和ZO-1的状态,以了解抑制RhoA及PKC亚基(α、β和ζ)后LPS致紧密连接破坏作用的改变。3.利用N19RhoA和C3转移酶抑制RhoA活性后,体外酶学法检测LPS对PKC各亚基活化作用；利用shRNA分别抑制PKC-α、PKC-β和PKC-ζ活性后Pull Down法分别检测各组LPS对RhoA活化作用,以初步探讨PKC各亚型与RhoA调控关系；为进一步确认在LPS致BBB屏障功能破坏过程中PKC-α是否为RhoA上游分子信号,抑制PKC-a活性后,比较稳定表达N19RhoA和vector-1质粒的两组Bend.3细胞在LPS作用前后的TEER值变化；为明确PKC-ζ和RhoA在LPS致BBB屏障功能破坏过程中的调控关系,抑制稳定表达PKCζ-ShRNA和vector-2的Bend.3细胞的RhoA活性,比较其在LPS作用前后的TEER值的改变。
结果：1.LPS作用5min RhoA及PKC各亚型(α、β和ζ)均开始活化。2.成功建立稳定表达PKC-ακ、PKC-β、PKC-ζ及N19RhoA的Bend.3细胞株。分别抑制RhoA及PKC各亚型(α、β和ζ)均可改善LPS对Bend.3细胞TJ的破坏作用。3.PKC-ζ活性受RhoA调控,RhoA活性受PKC-α调控,且在LPS致Ben.3细胞通透性上升调控过程中,PKC-ζ PKC-α分别是RhoA的下游及上游调控信号。
结论：PKC各亚基(α,β,ζ)和RhoA活化均促使BBB-TJ开放而导致BBB通透性上升,其中PKC-a、PKC-ζ分别为RhoA的上游调控分子和下游调控事件。
第四章RhoA/NF-κB/MLCK信号参与调控脂多糖致脑微血管内皮细胞通透性升高过程
目的：探讨RhoA/NF-κB/MLCK信号是否参与LPS致BBB通透性升高的调控。
方法：利用脂质体2000将DNMu-IκBa质粒导入Bend.3细胞,潮霉素B筛选出稳定表达株。报告基因法鉴定NF-κB活性的抑制情况。首先为了解RhoA和NF-κB是否参与LPS致BBB通透性升高的调控：利用pull down法和荧光素酶报告基因法分别测定LPS对Bend.3细胞的RhoA和NF-κB的活化作用。同时比较LPS对稳定表达N19RhoA和DNMu-IκBa质粒的Bend.3细胞的通透性、F-actin分布以及紧密连接蛋白表达分布等指标的影响。为明确上述过程中NF-κB是否由RhoA活化而激活,进一步比较了LPS作用不同时间,Bend.3细胞、稳定表达Vector-1和N19RhoA质粒的Bend.3细胞的NF-κB活性变化,同时比较了LPS对Bend.3细胞、稳定表达Vector-1和DNMu-IκBα质粒的Bend.3细胞的RhoA活性的影响。最后,为阐明上述过程中NF-κB是否通过增加MLCK转录,导致肌球蛋白轻链(Myosin light Chain, MLC)磷酸化,本研究利用western blot及RT-PCR分别检测了MLC磷酸化和MLCK转录水平。
结果：稳定表达DNMu-IκBα和N19RhoA质粒均可改善LPS致Bend.3细胞紧密连接破坏、通透性升高的作用。LPS作用5min RhoA活化,作用30min NF-κB活化；抑制RhoA活化,LPS致NF-κB活化的作用也被明显抑制,但抑制NF-κB活化对RhoA活性水平无影响。LPS作用0.5h, MLCK转录水平上升,3h MLC磷酸化水平明显增高,阻断NF-κB活化后上述表现被抑制。
结论：RhoA/NF-KB/MLCK信号通过磷酸化MLC,参与了LPS致BBB-TJ破坏、通透性升高过程的调控。
Despite the availability of effective antibacterial therapies, bacterial meningitis in infants and children is associated with significant morbidity and mortality. Even survivors,30-50%of them, sustain neurological sequelae. The mechanisms of CNS damage during meningitis have not been conclusively identified, but several lines of evidence indicate that the inflammatory response to bacterial lipopolysaccharides (LPS) promotes increased blood brain barrier (BBB) permeability; leading to vasogenic brain edema, could play a key role in its pathogeneses.
The BMECs, which are linked by tight junctions, were important structural and functional roles in maintaining the integrity of the BBB. LPS, the major component of the outer membrane of gram-negative bacteria, could ruin the BMEC barrier by promoting TJ disassembly, leading to vasogenic brain edema formation. We previously demonstrated that elevated LPS is highly correlated with the occurrence of decrease of Occluding and ZO-1as well as BBB dysfunction. However, little is known about the complex signaling events in the above process. In vivo and in vitro studies indicate that a complex network of signaling pathways is involved in regulating TJ structure and function, such as protein kinase C (PKC), tyrosine kinase, Ras or Rho GTP-binding proteins and phosphatidylinositol3-kinase (PI3Ks). Recently, it has been demonstrated that, in response to stimulus, NF-κB also can participate in regulating tight junction proteins. All the above signal molecular could participate regulating LPS induced BBB hyperpermeability, but it remains unclearly that how they regulate the TJ protein and interact with each other. Therefore, to make cleary these question will contribute to clarify the mechanism of infectious brain edema, and will be possible to design new methods to ameliorating brain edema formation.
Section1The immortalized mouse brain endothelial cell line Bend3displayed the comparative barrier characteristics as the primary brain microvascular endothelial cells
Objective The purpose of this study is to assesse weather the immortalized mouse brain endothelial cell line Bend3displayed the comparative barrier characteristics as the primary brain microvascular endothelial cells(BEMC). Methods Bend.3cells and primary BEMC, whose restrictive characteristics were assessed by transendothelial electrical resistance (TEER) assay and HRP permeability assay. Meanwhile, Western Blot and F-actin Stain were used to detect the tight junction protein and cytoskeleton. Results The TEER over the10days Bend.3samples was82.33±6.03Ω-cm2, which was significantly increased compared to the3days samples(P<0.05). Meanwhile, the permeability rates for HRP were (2.2±0.05)%and (4.3±0.20)%in3days samples and10days samples respectively, the difference was obvious in the2groups(P<0.05). Compared to the primary BEMC, there were no difference in TEER in Bend.3cells at the same time(P<0.05). Futhermore, in Bend.3and primary BEMC, Western blot indicated high level expression of multiple tight junction proteins Occludin, Claudin-5and ZO-1. Furthermore, F-actin Stain showed that the presence of F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence. Conclusions Bend.3cell has the similar barrier characteristics with primary BEMC, whose barrier function may reach to the most advantageous state after10days culture.
Section2the preparatory mechanism for LPS induced BEMC hyperpermeability
Objective To address the impair ment of TJ are close to LPS induced-Bend.3cell barrier dysfunction and to examine the signaling networks involved in the hyperpermeability of the brain endothelial barrier caused by LPS. Methods To confirm that LPS induces endothelial barrier hyperpermeability by disrupting tight junction, Bend.3cells were exposed to LPS, and changes in endothelial permeability (TEER assay), F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein expression (western blot or immunofluorescence) were monitored. To make clearly that wether PKC, RhoA, PI3K and tyrosine kinase were involve in the hyperpermeability of the brain endothelial barrier caused by LPS, calphostin C (PKC inhibitor), C3transferase (Rho inhibitor), wortmannin (PI3K inhibitor), PP2(tyrosine kinase inhibitor)were pretreated with Bend.3cell, then TEER assay were used to assay the barrier function of each group cells. Result LPS caused a significant decrease in TEER in a time-dependent manner, accompanied by decrease of TJ protein ZO-1, Occludin and Claudin-5, distribution of Claudin-5, rearrangement of F-actin. Inhititors of PKC and Rho could improve LPS-induced TEER decrease, while inhibitors of PI3K and tyrosine kinase could have no effect on suppressing LPS-induced BBB hyperpermeability. Conclusions These data indicate that LPS ruined TJ protein and induced rearrangement of F-actin, which lead to BEMC hyperpermeability, Moreover, PKC and Rho but not PI3K and tyrosine kinase mediates LPS disruptive effect on BEMC barrier.
Section3PKC and RhoA signals cross-talk in LPS induced alterations in brain endothelial permeability
Objective to explore how PKC a, β, ζ, to mediated the LPS disruptive signal to BEMC permeability and to detected how they interact with RhoA in the process. Methods1. pull-down assay and enzyme assay were used to detected the LPS activated effect on RhoA and PKC (α、β和ζ).2. PcDNA3.1hygro-N19RhoA (the dominant negative mutant of RhoA), and PcDNA3.1hygro vector plasmids (vector-1) were transfected into Bend.3cells by Lipofectamine2000. Stably transfected cells were obtained using the Hygromycin B (400ug/ml) selection method after transfection. The remarkable inhibitory effect of N19RhoA was confirmed by pull-down assay. Meanwhile, he Bend.3cells transfected with PLKO.1-puro-PKCa-shRNA, PLKO.1-puro-PKCp-shRNA, PLKO.1-puro-PKCζ-shRNA and empty PLKO.1-puro vector respectively. Stably transfected cells were used for experiments after selection by Puromycin (300ug/ml). The inhibitory levels of PKC-a, β, ζ were confirmed by Western blot. To detected whether PKC-a, β, ζ, and RhoA were involved in regulating LPS induced TJ dysfunction in BEMC, Cells were divided to5groups relate to their different transfected plasmids, and each group cells were exposed to LPS, and changes in F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein (western blot or immunofluorescence) were monitored.3. The interactions between the PKC and Rho pathways were therefore examined. Experiments were performed in which RhoA activation was inhibited (by N19RhoA plasmid or C3transferase) and the activity of PKC-a, PKC-β and PKC-ζ was observed by enzyme assay in Bend.3cells. Then opposite experiments were performed where ShRNA was used to deplete PKC (PKC-a, PKC-β and PKC-ζ,respectively), and RhoA activity was observed. To further detect the relationship between PKC-a and RhoA in regulating BMEC TJ dysfunction induced by LPS, Bend.3/N19RhoA cells and Bend.3/vector-1cells were pretreated with inhibitor of PKC-a, and then their TEER was detected. to confirm whether PKC-ζ acts as downstream molecular target of RhoA signals in the process, Bend.3/Sh-PKCζ and Bend.3/vector-2cells were pretreated with inhibitor of Rho, then TEER detection was performed as mentioned before. Result1. LPS active RhoA, PKC-a, PKC-β and PKC-ζ at5min.2. Established stably transfected cells for N19RhoA and PKCa, β,ζ-ShRNA. Inhibition of RhoA, PKC-a, PKC-p and PKC-ζ could improve LPS-induced TJ destruction.3. PKC-a and PKC-ζ, but not PKC-β, interact with RhoA in Bend.3cells stimulated by LPS; moreover, PKC-a and PKC-ζ act as the upstream and downstream targets for Rho, respectively. Conclusions These data indicate that LPS induced activation of PKC (a, β, ζ and RhoA mediated TJ destruction and BEMC barrier dysfunction, PKC-a and PKC-ζ, respectively, act as the upstream and downstream regulators for RhoA in the process.
Section4RhoA/NF-KB/MLCK pathway involved in LPS-induced BEMC hyperpermeability
Objective to explore whether RhoA/NF-κB/MLCK pathway were involved in LPS-induced BEMC hyperpermeability. Methods DNMu-IκBαplasmids were transfected into Bend.3cells by Lipofectamine2000. Stably transfected cells were obtained using the Hygromycin B (400ug/ml) selection method after transfection. The inhibitory effect was confirmed by Reporter gene analysis. To ensure that RhoA and NF-κB participated in the regulatory mechanisms of LPS mediated BBB dysfunction, pull down assay and Reporter gene analysis were used to detected LPS active effect on RhoA and NF-κB. Men while, Cells were divided to4groups:Bend.3, Bend.3/vector-1, Bend.3/N19RhoA and Bend.3/DNMu-IκBα, and each group cells were exposed to LPS, and changes in permeability (TEER assay), F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein (western blot or immunofluorescence) were monitored. To clarify the relationship between RhoA and NF-κB in the process, the activities of NF-κB (via luciferase reporter assays) and RhoA (via pull-down assays) were detected in the above4group cells. Lastly, to investigate whether NF-Kb/MLCK signal regulate LPS induced MLC phosphorylation, we measured changes in myosin light chain (MLC) phosphorylation and MLCK transcription by western blot and RT-PCR respectively. Result LPS caused RhoA active earlier than NF-κB. And LPS induced F-actin rearrangement, tight junction disruption and barrier dysfunction were suppressed by inhibitors of RhoA or NF-κB, in which inhibiting RhoA was more efficient. Inactivating RhoA prohibited LPS-induced NF-KB activation, but the inverse was not true. LPS increase MLCK transcription and MLC phosphorylation at30min and3h respectively, and inhibition of NF-KB could suppressed these incensement. Conclusions PhoA/NF-κB/MLCK pathway were involved in LPS-induced BEMC TJ disruption and hyperpermeability by phosphorylated MLC.
研究表明脑微血管内皮细胞(brain micro vascular endothelial cells, BMECs)及紧密连接(tight junction, TJ)是BBB结构和功能的主要基础。LPS为革兰氏阴性细菌细胞壁的组成成分,释放入血后被称为内毒素,对BBB屏障功能破坏有重要作用。我们团队的前期实验证实中枢神经系统感染性时LPS表达显著升高,且可引起BEMC紧密连接蛋白Occludin和ZO-1表达下调,但其具体的调控方式不明。目前研究显示PKC、Rho、PI3K和酪氨酸激酶以及核转录因子NF-κB均可能参与调控紧密连接的组装和分解,维持内皮细胞低通透性。但对于上述信号分子是否参与细菌性脑膜炎或感染性脑损伤时LPS致BBB通透性升高的调控过程,他们之间的调控关系如何等方面的研究均少见报道。对上述问题的深入研究,有助于进一步阐明感染性脑损伤时BBB通透性增高的病理过程和发病机制,为其临床防治提供新的思路。
本研究分为四部分：
第一章永生化Bend.3细胞株具有原代鼠脑微血管内皮细胞屏障特性
目的：评价永生化小鼠脑微血管内皮细胞株Bend.3是否具有原代培养的鼠脑微血管内皮细胞的屏障及生理特性。
方法：将小鼠脑微血管内皮细胞株Bend.3和原代培养的鼠脑微血管内皮细胞接种于细胞培养插内,跨内皮细胞电阻抗(transendothelial electrical resistance,TEER)和辣根过氧化物酶(horseradish peroxidase,HRP)通透性实验检测其屏障功能。Westernblot法和直接荧光染色法观察其紧密连接相关蛋白Occludin、ZO-1的表达及细胞骨架蛋白F-actin的分布。
结果：Bend.3细胞的TEER随培养时间延长逐渐升高,10d达82.33±6.03Ω·cm2,与培养3d时相比,差异有统计学意义(P<0.05),与同期原代培养的鼠脑微血管内皮细胞的TEER相比,无明显差异(P>0.05)。Bend.3细胞培养10d和3d的平均HRP通透率在120min分别为(2.±20.05)%和(4.3±0.20)%,差异有统计学意义(P<0.05),与同期原代培养的鼠脑微血管内皮细胞的TEER相比,无明显差异(P>0.05)。培养10d时Bend.3细胞和原代培养的鼠脑微血管内皮细胞均表达高浓度的紧密连接蛋白Occludin、ZO-1,且F-actin主要分布在细胞周边,线条完整连续,未见明显缝隙形成。
结论：小鼠脑微血管内皮细胞株Bend.3具有原代培养的脑微血管内皮细胞的屏障特性,且其屏障功能在接种10d后可达到最理想的状态。
第二章初步筛选脂多糖致脑微血管内皮细胞通透性改变的信号分子
目的：证实脂多糖通过引起Actin重组、紧密连接表达和分布变化导致脑微血管内皮细胞通透性增高,初步探讨PKC、Rho、PI3K和酪氨酸激酶等信号是否参与脂多糖致脑微血管内皮细胞通透性改变的调控。
方法：运用TEER测定法、F-actin染色法、western blot和免疫荧光法分别检测了LPS作用不同时间下Bend.3细胞的通透性,F-actin分布以及紧密连接蛋白cluaudin-5,Occludin和ZO-1的状态,以动态观察LPS是否通过破坏紧密连接蛋白增加脑血管内皮细胞通透性,然后分别利用calphostin C (PKC抑制剂)、C3transferase (Rho抑制剂)、wortmannin (PI3K抑制剂)、PP2(酪氨酸激酶抑制剂)预处理Bend.3细胞,再通过TEER检测LPS对各组细胞屏障功能的影响,了解PKC、酪氨酸激酶、PI3K、Rho信号是否参与了LPS致血脑屏障通透性升高的调控过程。
结果：LPS可致Bend.3细胞TEER以时间依赖性的方式下降,同时伴有紧密连接蛋白ZO-1、Occludin和Claudin-5表达下调,Claudin-5蛋白分布改变,以及细胞骨架F-actin重组。PKC、Rho抑制剂可改善LPS引起的Bend.3细胞TEER下降；而PI3K、酪氨酸激酶抑制剂不能阻断LPS对Bend.3屏障功能的破坏。
结论：脂多糖引起脑微血管内皮细胞Actin重组、紧密连接表达和分布变化而导致其通透性增高；PKC和Rho,而非PI3K和酪氨酸激酶,参与了此过程调控。
第三章PKC和RhoA信号相互作用调控脂多糖致脑微血管内皮细胞通透性升高过程
目的：探讨PKC各亚型(α、β和ζ)如何参与脂多糖致脑微血管内皮细胞通透性升高调控过程,他们与RhoA之间的调控关系如何。
方法：1.pull-down法和免疫共沉淀结合体外酶学实验法分别检测LPS作用不同时间Bend.3细胞的RhoA和PKC各亚基(α、β和ζ)活化状态。2.分别利用脂质体2000将PcDN A3.1hygro-n19RhoA、 PcDNA3.1hygro-vector(空载对照质粒)导入Bend.3细胞,利用潮霉素B筛选出稳定表达株。Pull Down法鉴定RhoA活性的抑制情况。并将PLKO.1-puro-PKCα-shRNA、PLKO.1-puro-PKCβ-shRNA PLKO.1-puro-PKCζ-shRNA和empty PLKO.1-puro vector导入Bend.3细胞,利用嘌呤霉素B筛选出稳定表达株。Western blot分别鉴定PKC-α PKC-β和PKCζ蛋白的表达抑制情况。并根据导入质粒不同分5组,运用F-actin染色法、western blot和免疫荧光法分别检测了LPS作用不同时间下各组Bend.3细胞F-actin分布以及紧密连接蛋白cluaudin-5,Occludin和ZO-1的状态,以了解抑制RhoA及PKC亚基(α、β和ζ)后LPS致紧密连接破坏作用的改变。3.利用N19RhoA和C3转移酶抑制RhoA活性后,体外酶学法检测LPS对PKC各亚基活化作用；利用shRNA分别抑制PKC-α、PKC-β和PKC-ζ活性后Pull Down法分别检测各组LPS对RhoA活化作用,以初步探讨PKC各亚型与RhoA调控关系；为进一步确认在LPS致BBB屏障功能破坏过程中PKC-α是否为RhoA上游分子信号,抑制PKC-a活性后,比较稳定表达N19RhoA和vector-1质粒的两组Bend.3细胞在LPS作用前后的TEER值变化；为明确PKC-ζ和RhoA在LPS致BBB屏障功能破坏过程中的调控关系,抑制稳定表达PKCζ-ShRNA和vector-2的Bend.3细胞的RhoA活性,比较其在LPS作用前后的TEER值的改变。
结果：1.LPS作用5min RhoA及PKC各亚型(α、β和ζ)均开始活化。2.成功建立稳定表达PKC-ακ、PKC-β、PKC-ζ及N19RhoA的Bend.3细胞株。分别抑制RhoA及PKC各亚型(α、β和ζ)均可改善LPS对Bend.3细胞TJ的破坏作用。3.PKC-ζ活性受RhoA调控,RhoA活性受PKC-α调控,且在LPS致Ben.3细胞通透性上升调控过程中,PKC-ζ PKC-α分别是RhoA的下游及上游调控信号。
结论：PKC各亚基(α,β,ζ)和RhoA活化均促使BBB-TJ开放而导致BBB通透性上升,其中PKC-a、PKC-ζ分别为RhoA的上游调控分子和下游调控事件。
第四章RhoA/NF-κB/MLCK信号参与调控脂多糖致脑微血管内皮细胞通透性升高过程
目的：探讨RhoA/NF-κB/MLCK信号是否参与LPS致BBB通透性升高的调控。
方法：利用脂质体2000将DNMu-IκBa质粒导入Bend.3细胞,潮霉素B筛选出稳定表达株。报告基因法鉴定NF-κB活性的抑制情况。首先为了解RhoA和NF-κB是否参与LPS致BBB通透性升高的调控：利用pull down法和荧光素酶报告基因法分别测定LPS对Bend.3细胞的RhoA和NF-κB的活化作用。同时比较LPS对稳定表达N19RhoA和DNMu-IκBa质粒的Bend.3细胞的通透性、F-actin分布以及紧密连接蛋白表达分布等指标的影响。为明确上述过程中NF-κB是否由RhoA活化而激活,进一步比较了LPS作用不同时间,Bend.3细胞、稳定表达Vector-1和N19RhoA质粒的Bend.3细胞的NF-κB活性变化,同时比较了LPS对Bend.3细胞、稳定表达Vector-1和DNMu-IκBα质粒的Bend.3细胞的RhoA活性的影响。最后,为阐明上述过程中NF-κB是否通过增加MLCK转录,导致肌球蛋白轻链(Myosin light Chain, MLC)磷酸化,本研究利用western blot及RT-PCR分别检测了MLC磷酸化和MLCK转录水平。
结果：稳定表达DNMu-IκBα和N19RhoA质粒均可改善LPS致Bend.3细胞紧密连接破坏、通透性升高的作用。LPS作用5min RhoA活化,作用30min NF-κB活化；抑制RhoA活化,LPS致NF-κB活化的作用也被明显抑制,但抑制NF-κB活化对RhoA活性水平无影响。LPS作用0.5h, MLCK转录水平上升,3h MLC磷酸化水平明显增高,阻断NF-κB活化后上述表现被抑制。
结论：RhoA/NF-KB/MLCK信号通过磷酸化MLC,参与了LPS致BBB-TJ破坏、通透性升高过程的调控。
Despite the availability of effective antibacterial therapies, bacterial meningitis in infants and children is associated with significant morbidity and mortality. Even survivors,30-50%of them, sustain neurological sequelae. The mechanisms of CNS damage during meningitis have not been conclusively identified, but several lines of evidence indicate that the inflammatory response to bacterial lipopolysaccharides (LPS) promotes increased blood brain barrier (BBB) permeability; leading to vasogenic brain edema, could play a key role in its pathogeneses.
The BMECs, which are linked by tight junctions, were important structural and functional roles in maintaining the integrity of the BBB. LPS, the major component of the outer membrane of gram-negative bacteria, could ruin the BMEC barrier by promoting TJ disassembly, leading to vasogenic brain edema formation. We previously demonstrated that elevated LPS is highly correlated with the occurrence of decrease of Occluding and ZO-1as well as BBB dysfunction. However, little is known about the complex signaling events in the above process. In vivo and in vitro studies indicate that a complex network of signaling pathways is involved in regulating TJ structure and function, such as protein kinase C (PKC), tyrosine kinase, Ras or Rho GTP-binding proteins and phosphatidylinositol3-kinase (PI3Ks). Recently, it has been demonstrated that, in response to stimulus, NF-κB also can participate in regulating tight junction proteins. All the above signal molecular could participate regulating LPS induced BBB hyperpermeability, but it remains unclearly that how they regulate the TJ protein and interact with each other. Therefore, to make cleary these question will contribute to clarify the mechanism of infectious brain edema, and will be possible to design new methods to ameliorating brain edema formation.
Section1The immortalized mouse brain endothelial cell line Bend3displayed the comparative barrier characteristics as the primary brain microvascular endothelial cells
Objective The purpose of this study is to assesse weather the immortalized mouse brain endothelial cell line Bend3displayed the comparative barrier characteristics as the primary brain microvascular endothelial cells(BEMC). Methods Bend.3cells and primary BEMC, whose restrictive characteristics were assessed by transendothelial electrical resistance (TEER) assay and HRP permeability assay. Meanwhile, Western Blot and F-actin Stain were used to detect the tight junction protein and cytoskeleton. Results The TEER over the10days Bend.3samples was82.33±6.03Ω-cm2, which was significantly increased compared to the3days samples(P<0.05). Meanwhile, the permeability rates for HRP were (2.2±0.05)%and (4.3±0.20)%in3days samples and10days samples respectively, the difference was obvious in the2groups(P<0.05). Compared to the primary BEMC, there were no difference in TEER in Bend.3cells at the same time(P<0.05). Futhermore, in Bend.3and primary BEMC, Western blot indicated high level expression of multiple tight junction proteins Occludin, Claudin-5and ZO-1. Furthermore, F-actin Stain showed that the presence of F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence. Conclusions Bend.3cell has the similar barrier characteristics with primary BEMC, whose barrier function may reach to the most advantageous state after10days culture.
Section2the preparatory mechanism for LPS induced BEMC hyperpermeability
Objective To address the impair ment of TJ are close to LPS induced-Bend.3cell barrier dysfunction and to examine the signaling networks involved in the hyperpermeability of the brain endothelial barrier caused by LPS. Methods To confirm that LPS induces endothelial barrier hyperpermeability by disrupting tight junction, Bend.3cells were exposed to LPS, and changes in endothelial permeability (TEER assay), F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein expression (western blot or immunofluorescence) were monitored. To make clearly that wether PKC, RhoA, PI3K and tyrosine kinase were involve in the hyperpermeability of the brain endothelial barrier caused by LPS, calphostin C (PKC inhibitor), C3transferase (Rho inhibitor), wortmannin (PI3K inhibitor), PP2(tyrosine kinase inhibitor)were pretreated with Bend.3cell, then TEER assay were used to assay the barrier function of each group cells. Result LPS caused a significant decrease in TEER in a time-dependent manner, accompanied by decrease of TJ protein ZO-1, Occludin and Claudin-5, distribution of Claudin-5, rearrangement of F-actin. Inhititors of PKC and Rho could improve LPS-induced TEER decrease, while inhibitors of PI3K and tyrosine kinase could have no effect on suppressing LPS-induced BBB hyperpermeability. Conclusions These data indicate that LPS ruined TJ protein and induced rearrangement of F-actin, which lead to BEMC hyperpermeability, Moreover, PKC and Rho but not PI3K and tyrosine kinase mediates LPS disruptive effect on BEMC barrier.
Section3PKC and RhoA signals cross-talk in LPS induced alterations in brain endothelial permeability
Objective to explore how PKC a, β, ζ, to mediated the LPS disruptive signal to BEMC permeability and to detected how they interact with RhoA in the process. Methods1. pull-down assay and enzyme assay were used to detected the LPS activated effect on RhoA and PKC (α、β和ζ).2. PcDNA3.1hygro-N19RhoA (the dominant negative mutant of RhoA), and PcDNA3.1hygro vector plasmids (vector-1) were transfected into Bend.3cells by Lipofectamine2000. Stably transfected cells were obtained using the Hygromycin B (400ug/ml) selection method after transfection. The remarkable inhibitory effect of N19RhoA was confirmed by pull-down assay. Meanwhile, he Bend.3cells transfected with PLKO.1-puro-PKCa-shRNA, PLKO.1-puro-PKCp-shRNA, PLKO.1-puro-PKCζ-shRNA and empty PLKO.1-puro vector respectively. Stably transfected cells were used for experiments after selection by Puromycin (300ug/ml). The inhibitory levels of PKC-a, β, ζ were confirmed by Western blot. To detected whether PKC-a, β, ζ, and RhoA were involved in regulating LPS induced TJ dysfunction in BEMC, Cells were divided to5groups relate to their different transfected plasmids, and each group cells were exposed to LPS, and changes in F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein (western blot or immunofluorescence) were monitored.3. The interactions between the PKC and Rho pathways were therefore examined. Experiments were performed in which RhoA activation was inhibited (by N19RhoA plasmid or C3transferase) and the activity of PKC-a, PKC-β and PKC-ζ was observed by enzyme assay in Bend.3cells. Then opposite experiments were performed where ShRNA was used to deplete PKC (PKC-a, PKC-β and PKC-ζ,respectively), and RhoA activity was observed. To further detect the relationship between PKC-a and RhoA in regulating BMEC TJ dysfunction induced by LPS, Bend.3/N19RhoA cells and Bend.3/vector-1cells were pretreated with inhibitor of PKC-a, and then their TEER was detected. to confirm whether PKC-ζ acts as downstream molecular target of RhoA signals in the process, Bend.3/Sh-PKCζ and Bend.3/vector-2cells were pretreated with inhibitor of Rho, then TEER detection was performed as mentioned before. Result1. LPS active RhoA, PKC-a, PKC-β and PKC-ζ at5min.2. Established stably transfected cells for N19RhoA and PKCa, β,ζ-ShRNA. Inhibition of RhoA, PKC-a, PKC-p and PKC-ζ could improve LPS-induced TJ destruction.3. PKC-a and PKC-ζ, but not PKC-β, interact with RhoA in Bend.3cells stimulated by LPS; moreover, PKC-a and PKC-ζ act as the upstream and downstream targets for Rho, respectively. Conclusions These data indicate that LPS induced activation of PKC (a, β, ζ and RhoA mediated TJ destruction and BEMC barrier dysfunction, PKC-a and PKC-ζ, respectively, act as the upstream and downstream regulators for RhoA in the process.
Section4RhoA/NF-KB/MLCK pathway involved in LPS-induced BEMC hyperpermeability
Objective to explore whether RhoA/NF-κB/MLCK pathway were involved in LPS-induced BEMC hyperpermeability. Methods DNMu-IκBαplasmids were transfected into Bend.3cells by Lipofectamine2000. Stably transfected cells were obtained using the Hygromycin B (400ug/ml) selection method after transfection. The inhibitory effect was confirmed by Reporter gene analysis. To ensure that RhoA and NF-κB participated in the regulatory mechanisms of LPS mediated BBB dysfunction, pull down assay and Reporter gene analysis were used to detected LPS active effect on RhoA and NF-κB. Men while, Cells were divided to4groups:Bend.3, Bend.3/vector-1, Bend.3/N19RhoA and Bend.3/DNMu-IκBα, and each group cells were exposed to LPS, and changes in permeability (TEER assay), F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein (western blot or immunofluorescence) were monitored. To clarify the relationship between RhoA and NF-κB in the process, the activities of NF-κB (via luciferase reporter assays) and RhoA (via pull-down assays) were detected in the above4group cells. Lastly, to investigate whether NF-Kb/MLCK signal regulate LPS induced MLC phosphorylation, we measured changes in myosin light chain (MLC) phosphorylation and MLCK transcription by western blot and RT-PCR respectively. Result LPS caused RhoA active earlier than NF-κB. And LPS induced F-actin rearrangement, tight junction disruption and barrier dysfunction were suppressed by inhibitors of RhoA or NF-κB, in which inhibiting RhoA was more efficient. Inactivating RhoA prohibited LPS-induced NF-KB activation, but the inverse was not true. LPS increase MLCK transcription and MLC phosphorylation at30min and3h respectively, and inhibition of NF-KB could suppressed these incensement. Conclusions PhoA/NF-κB/MLCK pathway were involved in LPS-induced BEMC TJ disruption and hyperpermeability by phosphorylated MLC.
引文
[1]Afonso PV, Ozden S, Prevost MC, Schmitt C, Seilhean D, Weksler B, Couraud PO, Gessain A, Romero IA, Ceccaldi PE (2007) Human blood-brain barrier disruption by retroviral-infected lymphocytes:role of myosin light chain kinase in endothelial tight-junction disorganization. J Immunol 179:2576-83.
[2]Alkon DL, Sun MK, Nelson TJ (2007) PKC signaling deficits:a mechanistic hypothesis for the origins of Alzheimer's disease. Trends Pharmacol Sci 28:51-60.
[3]Anderson JM, Van Itallie CM (1995) Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol 269:G467-75.
[4]Andreeva AY, Krause E, Muller EC, Blasig IE, Utepbergenov DI (2001) Protein kinase C regulates the phosphorylation and cellular localization of Occludin. J Biol Chem 276:38480-6.
[5]Andreeva AY, Piontek J, Blasig IE, Utepbergenov DI (2006) Assembly of tight junction is regulated by the antagonism of conventional and novel protein kinase C isoforms. Int J Biochem Cell Biol 38:222-33.
[6]Asanuma K, Yanagida-Asanuma E, Faul C, Tomino Y, Kim K, Mundel P (2006) Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling. Nat Cell Biol 8:485-91.
[7]Avila-Flores A, Rendon-Huerta E, Moreno J, Islas S, Betanzos A, Robles-Flores M, Gonzalez-Mariscal L (2001) Tight-junction protein zonula occludens 2 is a target of phosphorylation by protein kinase C. Biochem J 360:295-304.
[8]Banan A, Farhadi A, Fields JZ, Mutlu E, Zhang L, Keshavarzian A (2003) Evidence that nuclear factor-kappa B activation is critical in oxidant-induced disruption of the microtubule cytoskeleton and barrier integrity and that its inactivation is essential in epidermal growth factor-mediated protection of the monolayers of intestinal epithelia. J Pharmacol Exp Ther 306:13-28.
[9]Banks WA, Erickson MA (2010) The blood-brain barrier and immune function and dysfunction. Neurobiol Dis 37:26-32.
[10]Birukova AA, Smurova K, Birukov KG, Kaibuchi K, Garcia JG, Verin AD (2004) Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. Microvasc Res 67:64-77.
[11]Bogatcheva NV, Zemskova MA, Kovalenkov Y, Poirier C, Verin AD (2009) Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability. J Cell Physiol 221:750-9.
[12]Brown RC, Morris AP, OT'Neil RG (2007) Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells. Brain Res 1130:17-30.
[13]Bruewer M, Hopkins AM, Hobert ME, Nusrat A, Madara JL (2004) RhoA, Racl, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin. Am J Physiol Cell Physiol 287:C327-35.
[14]Chen YH, Lu Q, Goodenough DA, Jeansonne B (2002) Nonreceptor tyrosine kinase c-Yes interacts with Occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell 13:1227-37.
[15]Clayburgh DR, Barrett TA, Tang Y, Meddings JB, Van Eldik LJ, Watterson DM, Clarke LL, Mrsny RJ, Turner JR (2005) Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J Clin Invest 115:2702-15.
[16]Clayburgh DR, Rosen S, Witkowski ED, Wang F, Blair S, Dudek S, Garcia JG, Alverdy JC, Turner JR (2004) A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability. J Biol Chem 279:55506-13.
[17]Clements RT, Minnear FL, Singer HA, Keller RS, Vincent PA (2005) RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability. Am J Physiol Lung Cell Mol Physiol 288:L294-306.
[18]Cohen G, Rubinstein S, Gur Y, Breitbart H (2004) Crosstalk between protein kinase A and C regulates phospholipase D and F-actin formation during sperm capacitation. Dev Biol 267:230-41.
[19]Colgan OC, Collins NT, Ferguson G, Murphy RP, Birney YA, Cahill PA, Cummins PM (2008) Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function. Brain Res 1193:84-92.
[20]Condliffe AM, Cadwallader KA, Walker TR, Rintoul RC, Cowburn AS, Chilvers ER (2000) Phosphoinositide 3-kinase:a critical signalling event in pulmonary cells. Respir Res 1:24-9.
[21]Cucullo L, Couraud PO, Weksler B, Romero IA, Hossain M, Rapp E, Janigro D (2008a) Immortalized human brain endothelial cells and flow-based vascular modeling:a marriage of convenience for rational neurovascular studies. J Cereb Blood Flow Metab 28:312-28.
[22]Destaing O, Sanjay A, Itzstein C, Home WC, Toomre D, De Camilli P, Baron R (2008) The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts. Mol Biol Cell 19:394-404.
[23]Dohgu S, Banks WA (2008) Lipopolysaccharide-enhanced transcellular transport of HIV-1 across the blood-brain barrier is mediated by the p38 mitogen-activated protein kinase pathway. Exp Neurol 210:740-9.
[24]Drexler HG, Quentmeier H, Dirks WG, MacLeod RA (2002) Bladder carcinoma cell line ECV304 is not a model system for endothelial cells. In Vitro Cell Dev Biol Anim 38:185-6,187.
[25]Fleegal MA, Horn S, Borg LK, Davis TP (2005) Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation. Am J Physiol Heart Circ Physiol 289:H2012-9.
[26]Georgiadis A, Tschernutter M, Bainbridge JW, Balaggan KS, Mowat F, West EL, Munro PM, Thrasher AJ, Matter K, Balda MS, Ali RR (2010) The tight junction associated signalling proteins ZO-1 and ZONAB regulate retinal pigment epithelium homeostasis in mice. PLoS One 5:e 15730.
[27]Ghosh S, May MJ, Kopp EB (1998) NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16:225-60.
[28]Gumbleton M, Audus KL (2001) Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier. J Pharm Sci 90:1681-98.
[29]Harhaj NS, Antonetti DA (2004) Regulation of tight junctions and loss of barrier function in pathophysiology. Int J Biochem Cell Biol 36:1206-37.
[30]Hasegawa H, Fujita H, Katoh H, Aoki J, Nakamura K, Ichikawa A, Negishi M (1999) Opposite regulation of transepithelial electrical resistance and paracellular permeability by Rho in Madin-Darby canine kidney cells. J Biol Chem 274:20982-8.
[31]Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, Tsukita S, Yokoyama M, Staddon JM (2001) Regulation of tight junction permeability and Occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. J Biol Chem 276:10423-31.
[32]Holinstat, M., et al.,2003. Protein kinase Calpha-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement. J Biol Chem. 278,28793-8.
[33]Izumi Y, Hirose T, Tamai Y, Hirai S, Nagashima Y, Fujimoto T, Tabuse Y, Kemphues KJ, Ohno S (1998) An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol 143:95-106.
[34]Jin R, Song Z, Yu S, Piazza A, Nanda A, Penninger JM, Granger DN, Li G (2011) Phosphatidylinositol-3-kinase gamma plays a central role in blood-brain barrier dysfunction in acute experimental stroke. Stroke 42:2033-44.
[35]Jou, T. S., et al.,1998. Structural and functional regulation of tight junctions by Rho A and Racl small GTPases. J Cell Biol.142,101-15.
[36]Kaya, M., et al.,2004. Effects of lipopolysaccharide on the radiation-induced changes in the blood-brain barrier and the astrocytes. Brain Res.1019,105-12.
[37]Kim, K. S.,2003. Pathogenesis of bacterial meningitis:from bacteraemia to neuronal injury. Nat Rev Neurosci.4,376-85.
[38]Konopatskaya O, Poole AW (2010) Protein kinase Calpha:disease regulator and therapeutic target. Trends Pharmacol Sci 31:8-14.
[39]Lapointe TK, Buret AG (2012) Interleukin-18 facilitates neutrophil transmigration via myosin light chain kinase-dependent disruption of Occludin, without altering epithelial permeability. Am J Physiol Gastrointest Liver Physiol 302:G343-51.
[40]Legros H, Launay S, Roussel BD, Marcou-Labarre A, Calbo S, Catteau J, Leroux P, Boyer O, Ali C, Marret S, Vivien D, Laudenbach V (2009) Newborn-and adult-derived brain microvascular endothelial cells show age-related differences in phenotype and glutamate-evoked protease release. J Cereb Blood Flow Metab 29:1146-58.
[41]Leitman EM, Tewari A, Horn M, Urbanski M, Damanakis E, Einheber S, Salzer JL, de Lanerolle P, Melendez-Vasquez CV (2011) MLCK regulates Schwann cell cytoskeletal organization, differentiation and myelination. J Cell Sci 124:3784-96.
[42]Liu H, Li M, Wang P, Wang F (2011) Blockade of hypoxia-inducible factor-1 alpha by YC-1 attenuates interferon-gamma and tumor necrosis factor-alpha-induced intestinal epithelial barrier dysfunction. Cytokine 56:581-8.
[43]Lossinsky AS, Shivers RR (2004) Structural pathways for macromolecular and cellular transport across the blood-brain barrier during inflammatory conditions. Review. Histol Histopathol 19:535-64.
[44]Ma TY, Boivin MA, Ye D, Pedram A, Said HM (2005) Mechanism of TNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol 288:G422-30.
[45]Ma TY, Iwamoto GK, Hoa NT, Akotia V, Pedram A, Boivin MA, Said HM (2004) TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation. Am J Physiol Gastrointest Liver Physiol 286:G367-76.
[46]Madara JL (1990) Warner-Lambert/Parke-Davis Award lecture. Pathobiology of the intestinal epithelial barrier. Am J Pathol 137:1273-81.
[47]Mark KS, Davis TP (2002) Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol 282:H1485-94.
[48]Mayhan WG (2001) Regulation of blood-brain barrier permeability. Microcirculation 8:89-104.
[49]Mehta D, Rahman A, Malik AB (2001) Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. J Biol Chem 276:22614-20.
[50]Meng G, Zhao J, Wang HM, Ding RG, Zhang XC, Huang CQ, Ruan JX (2011) Injury of cell tight junctions and changes of actin level in acute lung injury caused by the perfluoroisobutylene exposure and the role of Myosin light chain kinase. J Occup Health 53:250-7.
[51]Meyer TN, Schwesinger C, Ye J, Denker BM, Nigam SK (2001) Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity. J Biol Chem 276:22048-55.
[52]Montaner S, Perona R, Saniger L, Lacal JC (1998) Multiple signalling pathways lead to the activation of the nuclear factor kappaB by the Rho family of GTPases. J Biol Chem 273:12779-85.
[53]Moriez R, Salvador-Cartier C, Theodorou V, Fioramonti J, Eutamene H, Bueno L (2005) Myosin light chain kinase is involved in lipopolysaccharide-induced disruption of colonic epithelial barrier and bacterial translocation in rats. Am J Pathol 167:1071-9.
[54]Mullin JM, Laughlin KV, Ginanni N, Marano CW, Clarke HM, Peralta SA (2000) Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers. Ann N Y Acad Sci 915:231-6.
[55]Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, Innis-Whitehouse W, Eastburn KK, Madara JL (2000) Tight junctions are membrane microdomains. J Cell Sci 113 (Pt 10):1771-81.
[56]Pang H, Bitar KN (2005) Direct association of RhoA with specific domains of PKC-alpha. Am J Physiol Cell Physiol 289:C982-93.
[57]Patil SB, Bitar KN (2006) RhoA-and PKC-alpha-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 290:G83-95.
[58]Peng J, He F, Zhang C, Deng X, Yin F (2011a) Protein kinase C-alpha signals P115RhoGEF phosphorylation and RhoA activation in TNF-alpha-induced mouse brain microvascular endothelial cell barrier dysfunction. J Neuroinflammation 8:28.
[59]Phillips N, Hayward RD, Koronakis V (2004) Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation. Nat Cell Biol 6:618-25.
[60]Polk WW, Ellis ME, Kushleika JV, Simmonds PL, Woods JS (2007) RhoA regulation of NF-kappaB activation is mediated by COX-2-dependent feedback inhibition of IKK in kidney epithelial cells. Am J Physiol Cell Physiol 293:C1160-70.
[61]Rattan R, Giri S, Singh AK, Singh I (2003) Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines:negative regulation of IKKalpha. Free Radic Biol Med 35:1037-50.
[62]Riganti C, Doublier S, Costamagna C, Aldieri E, Pescarmona G, Ghigo D, Bosia A (2008) Activation of nuclear factor-kappa B pathway by simvastatin and RhoA silencing increases doxorubicin cytotoxicity in human colon cancer HT29 cells. Mol Pharmacol 74:476-84.
[63]Rosenberg GA, Yang Y (2007) Vasogenic edema due to tight junction disruption by matrix metalloproteinases in cerebral ischemia. Neurosurg Focus 22:E4.
[64]Rosse C, Linch M, Kermorgant S, Cameron AJ, Boeckeler K, Parker PJ (2010) PKC and the control of localized signal dynamics. Nat Rev Mol Cell Biol 11:103-12.
[65]Rosson D, O'Brien TG, Kampherstein JA, Szallasi Z, Bogi K, Blumberg PM, Mullin JM (1997) Protein kinase C-alpha activity modulates transepithelial permeability and cell junctions in the LLC-PK1 epithelial cell line. J Biol Chem 272:14950-3.
[66]Roux F, Couraud PO (2005) Rat brain endothelial cell lines for the study of blood-brain barrier permeability and transport functions. Cell Mol Neurobiol 25:41-58.
[67]Rubin LL, Staddon JM (1999a) The cell biology of the blood-brain barrier. Annu Rev Neurosci 22:11-28.
[68]Salkeni MA, Lynch JL, Otamis-Price T, Banks WA (2009) Lipopolysaccharide impairs blood-brain barrier P-glycoprotein function in mice through prostaglandin- and nitric oxide-independent pathways. J Neuroimmune Pharmacol 4:276-82.
[69]Schottelius AJ, Baldwin AJ (1999) A role for transcription factor NF-kappa B in intestinal inflammation. Int J Colorectal Dis 14:18-28.
[70]Schwarz BT, Wang F, Shen L, Clayburgh DR, Su L, Wang Y, Fu YX, Turner JR (2007) LIGHT signals directly to intestinal epithelia to cause barrier dysfunction via cytoskeletal and endocytic mechanisms. Gastroenterology 132:2383-94.
[71]Shen Q, Rigor RR, Pivetti CD, Wu MH, Yuan SY (2010) Myosin light chain kinase in microvascular endothelial barrier function. Cardiovasc Res 87:272-80.
[72]Sheth P, Basuroy S, Li C, Naren AP, Rao RK (2003) Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. J Biol Chem 278:49239-45.
[73]Sheth P, Delos SN, Seth A, LaRusso NF, Rao RK (2007) Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 293:G308-18.
[74]Sheth, P., et al.,2003. Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. J Biol Chem.278,49239-45.
[75]Sheth, P., et al.,2007. Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism. Am J Physiol Gastrointest Liver Physiol.293, G308-18.
[76]Shimizu S, Tahara M, Ogata S, Hashimoto K, Morishige K, Tasaka K, Murata Y (2007) Involvement of nuclear factor-kB activation through RhoA/Rho-kinase pathway in LPS-induced IL-8 production in human cervical stromal cells. Mol Hum Reprod 13:181-7.
[77]Siflinger-Birnboim A, Johnson A (2003a) Protein kinase C modulates pulmonary endothelial permeability:a paradigm for acute lung injury. Am J Physiol Lung Cell Mol Physiol 284:L435-51.
[78]Stuart RO, Nigam SK (1995) Regulated assembly of tight junctions by protein kinase C. Proc Natl Acad Sci U S A 92:6072-6.
[79]Sukumaran SK, Prasadarao NV (2002) Regulation of protein kinase C in Escherichia coli K1 invasion of human brain microvascular endothelial cells. J Biol Chem 277:12253-62.
[80]Terry S, Nie M, Matter K, Balda MS (2010) Rho signaling and tight junction functions. Physiology (Bethesda) 25:16-26.
[81]Tiruppathi C, Shimizu J, Miyawaki-Shimizu K, Vogel SM, Bair AM, Minshall RD, Predescu D, Malik AB (2008) Role of NF-kappaB-dependent caveolin-1 expression in the mechanism of increased endothelial permeability induced by lipopolysaccharide. J Biol Chem 283:4210-8.
[82]Turner JR (2006) Molecular basis of epithelial barrier regulation:from basic mechanisms to clinical application. Am J Pathol 169:1901-9.
[83]Verin V, Popowski Y, de Bruyne B, Baumgart D, Sauerwein W, Lins M, Kovacs G, Thomas M, Calman F, Disco C, Serruys PW, Wijns W (2001) Endoluminal beta-radiation therapy for the prevention of coronary restenosis after balloon angioplasty. The Dose-Finding Study Group. N Engl J Med 344:243-9.
[84]Vietor I, Bader T, Paiha K, Huber LA (2001) Perturbation of the tight junction permeability barrier by Occludin loop peptides activates beta-catenin/TCF/ LEF-mediated transcription. Embo Rep 2:306-12.
[85]Vorbrodt AW, Dobrogowska DH (2003) Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers:electron microscopist's view. Brain Res Brain Res Rev 42:221-42.
[86]Wang F, Graham WV, Wang Y, Witkowski ED, Schwarz BT, Turner JR (2005) Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol 166:409-19.
[87]Wang F, Schwarz BT, Graham WV, Wang Y, Su L, Clayburgh DR, Abraham C, Turner JR (2006) IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction. Gastroenterology 131:1153-63.
[88]Webb BL, Hirst SJ, Giembycz MA (2000) Protein kinase C isoenzymes:a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis. Br J Pharmacol 130:1433-52.
[89]Webb RC (2003) Smooth muscle contraction and relaxation. Adv Physiol Educ 27:201-6.
[90]Wells WA, Bonetta L (2005) Endothelial tight junctions form the blood-brain barrier. J Cell Biol 169:378.
[91]Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38:323-37.
[92]Xaio H, Banks WA, Niehoff ML, Morley JE (2001) Effect of LPS on the permeability of the blood-brain barrier to insulin. Brain Res 896:36-42.
[93]Yamamoto M, Ramirez SH, Sato S, Kiyota T, Cerny RL, Kaibuchi K, Persidsky Y, Ikezu T (2008a) Phosphorylation of Claudin-5 and Occludin by rho kinase in brain endothelial cells. Am J Pathol 172:521-33.
[94]Ye D, Ma I, Ma TY (2006) Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol 290:G496-504.
[95]Ye D, Ma TY (2008) Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity. J Cell Mol Med 12:1331-46.
[96]甘娜,尹飞,彭镜,王卫东.溶血磷脂酸促进大鼠体外血脑屏障模型通透性增加机制研究.中华医学杂志,2008,88(6)：12-14.
[97]刘依凌,王凤君,陈传莉,等.Rho激酶在大鼠严重烧伤后肠黏膜通透性改变中的作用及机制研究[J].第三军医大学学报,2008,30(9)：817-819
[98]彭镜,尹飞,甘娜,等.内毒素脂多糖对体外血脑屏障模型通透性的影响及其机制研究[J].中华医学杂志,2006,86(27)：1924-1926.
[99]吴丽文,尹飞,彭镜,王卫东,甘娜。紧密连接蛋白ZO-1、Occludin和Actin参与缺氧缺血诱导的血脑屏障通透性增加[J]。中国当代儿科杂志,2008,10(4)：514-516。
[100]叶增纯.肾小球疾病发病机制的系列研究[D]。中山大学,2010
[101]尹飞,杨于嘉,虞佩兰,等.百日咳菌液致脑水肿时大鼠脑组织细胞因子的变化及机制[J].中风与神经疾病杂志,2001,18(5)：267-269
[1]Abe T, Sugano E, Saigo Y, Tamai M (2003) Interleukin-lbeta and barrier function of retinal pigment epithelial cells (ARPE-19):aberrant expression of junctional complex molecules. Invest Ophthalmol Vis Sci 44:4097-104.
[2]Andreeva AY, Krause E, Muller EC, Blasig IE, Utepbergenov DI (2001a) Protein kinase C regulates the phosphorylation and cellular localization of Occludin. J Biol Chem 276:38480-6.
[3]Balda MS, Gonzalez-Mariscal L, Matter K, Cereijido M, Anderson JM (1993) Assembly of the tight junction:the role of diacylglycerol. J Cell Biol 123:293-302.
[4]Bamforth SD, Kniesel U, Wolburg H, Engelhardt B, Risau W (1999) A dominant mutant of Occludin disrupts tight junction structure and function. J Cell Sci 112 (Pt 12):1879-88.
[5]Banks WA, Erickson MA (2010) The blood-brain barrier and immune function and dysfunction. Neurobiol Dis 37:26-32.
[6]Bazzoni G, Dejana E (2004) Endothelial cell-to-cell junctions:molecular organization and role in vascular homeostasis. Physiol Rev 84:869-901.
[7]Berkes J, Viswanathan VK, Savkovic SD, Hecht G (2003) Intestinal epithelial responses to enteric pathogens:effects on the tight junction barrier, ion transport, and inflammation. Gut 52:439-51.
[8]Boyle EC, Brown NF, Finlay BB (2006) Salmonella enterica serovar Typhimurium effectors SopB, SopE, SopE2 and Sip A disrupt tight junction structure and function. Cell Microbiol 8:1946-57.
[9]Brown RC, Davis TP (2005) Hypoxia/aglycemia alters expression of Occludin and actin in brain endothelial cells. Biochem Biophys Res Commun 327:1114-23.
[10]Bruewer M, Hopkins AM, Hobert ME, Nusrat A, Madara JL (2004) RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin. Am J Physiol Cell Physiol 287:C327-35.
[11]Chen YH, Lu Q, Goodenough DA, Jeansonne B (2002) Nonreceptor tyrosine kinase c-Yes interacts with Occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell 13:1227-37.
[12]Clarke H, Soler AP, Mullin JM (2000) Protein kinase C activation leads to dephosphorylation of Occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets. J Cell Sci 113 (Pt 18):3187-96.
[13]Clarke H, Marano CW, Peralta SA, Mullin JM (2000) Modification of tight junction function by protein kinase C isoforms. Adv Drug Deliv Rev 41:283-301.
[14]Conklin BS, Zhong DS, Zhao W, Lin PH, Chen C (2002a) Shear stress regulates Occludin and VEGF expression in porcine arterial endothelial cells. J Surg Res 102:13-21.
[15]Cordenonsi M, D'Atri F, Hammar E, Parry DA, Kendrick-Jones J, Shore D, Citi S (1999) Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin. J Cell Biol 147:1569-82.
[16]Correale J, Villa A (2009) Cellular elements of the blood-brain barrier. Neurochem Res 34:2067-77.
[17]Cruzalegui FH, Bading H (2000) Calcium-regulated protein kinase cascades and their transcription factor targets. Cell Mol Life Sci 57:402-10.
[18]Date I, Takagi N, Takagi K, Tanonaka K, Funakoshi H, Matsumoto K, Nakamura T, Takeo S (2006) Hepatocyte growth factor attenuates cerebral ischemia-induced increase in permeability of the blood-brain barrier and decreases in expression of tight junctional proteins in cerebral vessels. Neurosci Lett 407:141-5.
[19]DeMaio L, Chang YS, Gardner TW, Tarbell JM, Antonetti DA (2001) Shear stress regulates Occludin content and phosphorylation. Am J Physiol Heart Circ Physiol 281:H105-13.
[20]Duronio V, Scheid MP, Ettinger S (1998) Downstream signalling events regulated by phosphatidylinositol 3-kinase activity. Cell Signal 10:233-9.
[21]Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM (1998) The tight junction protein ZO-1 establishes a link between the transmembrane protein Occludin and the actin cytoskeleton. JBiol Chem 273:29745-53.
[22]Farshori P, Kachar B (1999) Redistribution and phosphorylation of Occludin during opening and resealing of tight junctions in cultured epithelial cells. J Membr Biol 170:147-56.
[23]Feldman GJ, Mullin JM, Ryan MP (2005) Occludin:structure, function and regulation. Adv Drug Deliv Rev 57:883-917.
[24]Fischer S, Wobben M, Marti HH, Renz D, Schaper W (2002) Hypoxia-induced hyperpermeability in brain microvessel endothelial cells involves VEGF-mediated changes in the expression of zonula occludens-1. Microvasc Res 63:70-80.
[25]Fischer S, Wiesnet M, Marti HH, Renz D, Schaper W (2004) Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells. J Cell Physiol 198:359-69.
[26]FitzGerald TJ, Simon E, Meyer J (2004) Prostate carcinoma:opportunities for translational research. J Cell Biochem 91:433-42.
[27]Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1993) Occludin:a novel integral membrane protein localizing at tight junctions. J Cell Biol 123:1777-88.
[28]Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and-2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to Occludin. J Cell Biol 141:1539-50.
[29]Gebbink MF, Kranenburg O, Poland M, van Horck FP, Houssa B, Moolenaar WH (1997) Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein:control of neuronal morphology. J Cell Biol 137:1603-13.
[30]Gonzalez-Mariscal L, Betanzos A, Nava P, Jaramillo BE (2003) Tight junction proteins. Prog Biophys Mol Biol 81:1-44.
[31]Gye MC, Ohsako S (2003) Effects of flutamide in the rat testis on the expression of Occludin, an integral member of the tight junctions. Toxicol Lett 143:217-22.
[32]Hawkins BT, Davis TP (2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57:173-85.
[33]He F, Peng J, Deng XL, Yang LF, Wu LW, Zhang CL, Yin F (2011) RhoA and NF-kappaB are involved in lipopolysaccharide-induced brain microvascular cell line hyperpermeability. Neuroscience 188:35-47.
[34]Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, Tsukita S, Yokoyama M, Staddon JM (2001) Regulation of tight junction permeability and Occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. JBiol Chem 276:10423-31.
[35]Honda M, Nakagawa S, Hayashi K, Kitagawa N, Tsutsumi K, Nagata I, Niwa M (2006) Adrenomedullin improves the blood-brain barrier function through the expression of Claudin-5. Cell Mol Neurobiol 26:109-18.
[36]Howarth AG, Hughes MR, Stevenson BR (1992) Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types. Am J Physiol 262:CA61-9.
[37]Huber JD, Witt KA, Hom S, Egleton RD, Mark KS, Davis TP (2001) Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression. Am JPhysiol Heart Circ Physiol 280:H1241-8.
[38]Ikenouchi J, Furuse M, Furuse K, Sasaki H, Tsukita S, Tsukita S (2005) Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J Cell Biol 171:939-45.
[39]Inoko A, Itoh M, Tamura A, Matsuda M, Furuse M, Tsukita S (2003) Expression and distribution of ZO-3, a tight junction MAGUK protein, in mouse tissues. Genes Cells 8:837-45.
[40]Ishizaki T, Chiba H, Kojima T, Fujibe M, Soma T, Miyajima H, Nagasawa K, Wada I, Sawada N (2003) Cyclic AMP induces phosphorylation of Claudin-5 immunoprecipitates and expression of Claudin-5 gene in blood-brain-barrier endothelial cells via protein kinase A-dependent and -independent pathways. Exp Cell Res 290:275-88.
[41]Kago T, Takagi N, Date I, Takenaga Y, Takagi K, Takeo S (2006) Cerebral ischemia enhances tyrosine phosphorylation of Occludin in brain capillaries. Biochem Biophys Res Commun 339:1197-203.
[42]Kale G, Naren AP, Sheth P, Rao RK (2003) Tyrosine phosphorylation of Occludin attenuates its interactions with ZO-1, ZO-2, and ZO-3. Biochem Biophys Res Commun 302:324-9.
[43]Krause G, Winkler L, Piehl C, Blasig I, Piontek J, Muller SL (2009) Structure and function of extracellular Claudin domains. Ann N Y Acad Sci 1165:34-43.
[44]Krug SM, Amasheh S, Richter JF, Milatz S, Gunzel D, Westphal JK, Huber O, Schulzke JD, Fromm M (2009) Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability. Mol Biol Cell 20:3713-24.
[45]Lechner J, Krall M, Netzer A, Radmayr C, Ryan MP, Pfaller W (1999) Effects of interferon alpha-2b on barrier function and junctional complexes of renal proximal tubular LLC-PK1 cells. Kidney Int 55:2178-91.
[46]Lee DB, Huang E, Ward HJ (2006) Tight junction biology and kidney dysfunction. Am J Physiol Renal Physiol 290:F20-34.
[47]Lee SW, Kim WJ, Choi YK, Song HS, Son MJ, Gelman IH, Kim YJ, Kim KW (2003) SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier. Nat Med 9:900-6.
[48]Li D, Mrsny RJ (2000) Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of Occludin. J Cell Biol 148:791-800.
[49]Liebner S, Fischmann A, Rascher G, Duffner F, Grote EH, Kalbacher H, Wolburg H (2000) Claudin-1 and Claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme. Acta Neuropathol 100:323-31.
[50]Mandel I, Paperna T, Glass-Marmor L, Volkowich A, Badarny S, Schwartz I, Vardi P, Koren I, Miller A (2011) Tight junction proteins expression and modulation in immune cells and multiple sclerosis. J Cell Mol Med.
[51]Mariano C, Sasaki H, Brites D, Brito MA (2011) A look at tricellulin and its role in tight junction formation and maintenance. Eur J Cell Biol 90:787-96.
[52]Mark KS, Davis TP (2002) Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol 282:H1485-94.
[53]McKenzie JA, Riento K, Ridley AJ (2006a) Casein kinase I epsilon associates with and phosphorylates the tight junction protein Occludin. Febs Lett 580:2388-94.
[54]Moolenaar WH (1995) Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem 270:12949-52.
[55]Mooradian AD, Haas MJ, Chehade JM (2003) Age-related changes in rat cerebral Occludin and zonula occludens-1 (ZO-1). Mech Ageing Dev 124:143-6.
[56]Muller SL, Portwich M, Schmidt A, Utepbergenov DI, Huber O, Blasig IE, Krause G (2005) The tight junction protein Occludin and the adherens junction protein alpha-catenin share a common interaction mechanism with ZO-1. J Biol Chem 280:3747-56.
[57]Murakami T, Felinski EA, Antonetti DA (2009) Occludin phosphorylation and ubiquitination regulate tight junction trafficking and vascular endothelial growth factor-induced permeability. J Biol Chem 284:21036-46.
[58]Musch MW, Walsh-Reitz MM, Chang EB (2006) Roles of ZO-1, Occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointesl Liver Physiol 290:G222-31.
[59]Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S (2003) Size-selective loosening of the blood-brain barrier in Claudin-5-deficient mice. J Cell Biol 161:653-60.
[60]Nunbhakdi-Craig V, Machleidt T, Ogris E, Bellotto D, White CR, Sontag E (2002) Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol 158:967-78.
[61]Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, Innis-Whitehouse W, Eastburn KK, Madara JL (2000) Tight junctions are membrane microdomains. J Cell Sci 113(Pt10):1771-81.
[62]Nusrat A, Chen JA, Foley CS, Liang TW, Tom J, Cromwell M, Quan C, Mrsny RJ (2000) The coiled-coil domain of Occludin can act to organize structural and functional elements of the epithelial tight junction. J Biol Chem 275:29816-22.
[63]Peng J, He F, Zhang C, Deng X, Yin F (2011) Protein kinase C-alpha signals P115RhoGEF phosphorylation and RhoA activation in TNF-alpha-induced mouse brain microvascular endothelial cell barrier dysfunction. J Neuroinflammation 8:28.
[64]Persidsky Y, Heilman D, Haorah J, Zelivyanskaya M, Persidsky R, Weber GA, Shimokawa H, Kaibuchi K, Ikezu T (2006a) Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE). Blood 107:4770-80.
[65]Raleigh DR, Boe DM, Yu D, Weber CR, Marchiando AM, Bradford EM, Wang Y, Wu L, Schneeberger EE, Shen L, Turner JR (2011) Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function. J Cell Biol 193:565-82.
[66]Rao R (2009) Occludin phosphorylation in regulation of epithelial tight junctions. Ann N Y Acad Sci 1165:62-8.
[67]Rao RK, Basuroy S, Rao VU, Karnaky JK, Gupta A (2002a) Tyrosine phosp-horylation and dissociation of Occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidative stress. Biochem J 368:471-81.
[68]Ratcliffe MJ, Rubin LL, Staddon JM (1997) Dephosphorylation of the cadherin-associated p100/p120 proteins in response to activation of protein kinase C in epithelial cells. J Biol Chem 272:31894-901.
[69]Ratcliffe MJ, Smales C, Staddon JM (1999) Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli. Biochem J 33S(Pt2):471-8.
[70]Riazuddin S, Ahmed ZM, Fanning AS, Lagziel A, Kitajiri S, Ramzan K, Khan SN, Chattaraj P, Friedman PL, Anderson JM, Belyantseva IA, Forge A, Riazuddin S, Friedman TB (2006) Tricellulin is a tight-junction protein necessary for hearing. Am J Hum Genet 79:1040-51.
[71]Sahai E, Ishizaki T, Narumiya S, Treisman R (1999) Transformation mediated by RhoA requires activity of ROCK kinases. Curr Biol 9:136-45.
[72]Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Inazawa J, Fujimoto K, Tsukita S (1997) Mammalian Occludin in epithelial cells:its expression and subcellular distribution. Eur J Cell Biol 73:222-31.
[73]Sakaguchi T, Gu X, Golden HM, Suh E, Rhoads DB, Reinecker HC (2002) Cloning of the human Claudin-25'-flanking region revealed a TATA-less promoter with conserved binding sites in mouse and human for caudal-related homeodomain proteins and hepatocyte nuclear factor-1 alpha. J Biol Chem 277:21361-70.
[74]Schneeberger EE, Lynch RD (2004) The tight junction:a multifunctional complex. Am J Physiol Cell Physiol 286:C1213-28.
[75]Scudamore CL, Jepson MA, Hirst BH, Miller HR (1998) The rat mucosal mast cell chymase, RMCP-Ⅱ, alters epithelial cell monolayer permeability in association with altered distribution of the tight junction proteins ZO-1 and Occludin. Eur J Cell Biol 75:321-30.
[76]Sheth P, Basuroy S, Li C, Naren AP, Rao RK (2003) Role of phosphatid-ylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. J Biol Chem 278:49239-45.
[77]Simonovic I, Rosenberg J, Koutsouris A, Hecht G (2000) Enteropathogenic Escherichia coli dephosphorylates and dissociates Occludin from intestinal epithelial tight junctions. Cell Microbiol 2:305-15.
[78]Sit ST, Manser E (2011) Rho GTPases and their role in organizing the actin cytoskeleton. J Cell Sci 124:679-83.
[79]Soma T, Chiba H, Kato-Mori Y, Wada T. Yamashita T, Kojima T, Sawada N (2004) Thr(207) of Claudin-5 is involved in size-selective loosening of the endothelial barrier by cyclic AMP. Exp Cell Res 300:202-12.
[80]Steed E, Balda MS, Matter K (2010) Dynamics and functions of tight junctions. Trends Cell Biol 20:142-9.
[81]Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA (1986a) Identification of ZO-1:a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103:755-66.
[82]Subramanian VS, Marchant JS, Ye D, Ma TY, Said HM (2007) Tight junction targeting and intracellular trafficking of Occludin in polarized epithelial cells. Am J Physiol Cell Physiol 293:C1717-26.
[83]Suidan GL, Mcdole JR, Chen Y, Pirko I, Johnson AJ (2008) Induction of blood brain barrier tight junction protein alterations by CD8 T cells. PLoS One 3:e3037.
[84]Takenaga Y, Takagi N, Murotomi K, Tanonaka K, Takeo S (2009) Inhibition of Src activity decreases tyrosine phosphorylation of Occludin in brain capillaries and attenuates increase in permeability of the blood-brain barrier after transient focal cerebral ischemia. J Cereb Blood Flow Metab 29:1099-108.
[85]Tavelin S, Hashimoto K, Malkinson J, Lazorova L, Toth I, Artursson P (2003) A new principle for tight junction modulation based on Occludin peptides. Mol Pharmacol 64:1530-40.
[86]Terry S, Nie M, Matter K, Balda MS (2010) Rho signaling and tight junction functions. Physiology (Bethesda) 25:16-26.
[87]Tiruppathi C, Minshall RD, Paria BC, Vogel SM, Malik AB (2002) Role of Ca2+ signaling in the regulation of endothelial permeability. Vascul Pharmacol 39:173-85.
[88]Wang HR, Zhang Y, Ozdamar B, Ogunjimi AA, Alexandrova E, Thomsen GH, Wrana JL (2003) Regulation of cell polarity and protrusion formation by targeting RhoA for degradation. Science 302:1775-9.
[89]Wang W, Dentler WL, Borchardt RT (2001) VEGF increases BMEC monolayer permeability by affecting Occludin expression and tight junction assembly. Am J Physiol Heart Circ Physiol 280:H434-40.
[90]Wang Y, Zhang J, Yi XJ, Yu FS (2004) Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells. Exp Eye Res 78:125-36.
[91]Ward PD, Klein RR, Troutman MD, Desai S, Thakker DR (2002) Phospholipase C-gamma modulates epithelial tight junction permeability through hyperphosphorylation of tight junction proteins. J Biol Chem 277: 35760-5.
[92]Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38:323-37.
[93]Wolburg H, Wolburg-Buchholz K, Kraus J, Rascher-Eggstein G, Liebner S, Hamm S, Duffner F, Grote EH, Risau W, Engelhardt B (2003) Localization of Claudin-3 in tight junctions of the blood-brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multifo-rme. Acta Neuropathol 105:586-92.
[94]Xu R, Feng X, Xie X, Zhang J, Wu D, Xu L (2012) HIV-1 Tat protein increases the permeability of brain endothelial cells by both inhibiting Occludin expression and cleaving Occludin via matrix metalloproteinase-9. Brain Res 1436:13-9.
[95]Yin F, Watsky MA (2005) LPA and S1P increase corneal epithelial and endothelial cell transcellular resistance. Invest Ophthalmol Vis Sci 46:1927-33.
[96]Youakim A, Ahdieh M (1999) Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin. Am J Physiol 276:G1279-88.
[97]彭镜,尹飞,甘娜,张红媛.内毒素脂多糖对体外血脑屏障模型通透性的影响及其机制研究.中华医学杂志.86(2006)：1924-1926.
[2]Alkon DL, Sun MK, Nelson TJ (2007) PKC signaling deficits:a mechanistic hypothesis for the origins of Alzheimer's disease. Trends Pharmacol Sci 28:51-60.
[3]Anderson JM, Van Itallie CM (1995) Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol 269:G467-75.
[4]Andreeva AY, Krause E, Muller EC, Blasig IE, Utepbergenov DI (2001) Protein kinase C regulates the phosphorylation and cellular localization of Occludin. J Biol Chem 276:38480-6.
[5]Andreeva AY, Piontek J, Blasig IE, Utepbergenov DI (2006) Assembly of tight junction is regulated by the antagonism of conventional and novel protein kinase C isoforms. Int J Biochem Cell Biol 38:222-33.
[6]Asanuma K, Yanagida-Asanuma E, Faul C, Tomino Y, Kim K, Mundel P (2006) Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling. Nat Cell Biol 8:485-91.
[7]Avila-Flores A, Rendon-Huerta E, Moreno J, Islas S, Betanzos A, Robles-Flores M, Gonzalez-Mariscal L (2001) Tight-junction protein zonula occludens 2 is a target of phosphorylation by protein kinase C. Biochem J 360:295-304.
[8]Banan A, Farhadi A, Fields JZ, Mutlu E, Zhang L, Keshavarzian A (2003) Evidence that nuclear factor-kappa B activation is critical in oxidant-induced disruption of the microtubule cytoskeleton and barrier integrity and that its inactivation is essential in epidermal growth factor-mediated protection of the monolayers of intestinal epithelia. J Pharmacol Exp Ther 306:13-28.
[9]Banks WA, Erickson MA (2010) The blood-brain barrier and immune function and dysfunction. Neurobiol Dis 37:26-32.
[10]Birukova AA, Smurova K, Birukov KG, Kaibuchi K, Garcia JG, Verin AD (2004) Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. Microvasc Res 67:64-77.
[11]Bogatcheva NV, Zemskova MA, Kovalenkov Y, Poirier C, Verin AD (2009) Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability. J Cell Physiol 221:750-9.
[12]Brown RC, Morris AP, OT'Neil RG (2007) Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells. Brain Res 1130:17-30.
[13]Bruewer M, Hopkins AM, Hobert ME, Nusrat A, Madara JL (2004) RhoA, Racl, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin. Am J Physiol Cell Physiol 287:C327-35.
[14]Chen YH, Lu Q, Goodenough DA, Jeansonne B (2002) Nonreceptor tyrosine kinase c-Yes interacts with Occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell 13:1227-37.
[15]Clayburgh DR, Barrett TA, Tang Y, Meddings JB, Van Eldik LJ, Watterson DM, Clarke LL, Mrsny RJ, Turner JR (2005) Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J Clin Invest 115:2702-15.
[16]Clayburgh DR, Rosen S, Witkowski ED, Wang F, Blair S, Dudek S, Garcia JG, Alverdy JC, Turner JR (2004) A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability. J Biol Chem 279:55506-13.
[17]Clements RT, Minnear FL, Singer HA, Keller RS, Vincent PA (2005) RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability. Am J Physiol Lung Cell Mol Physiol 288:L294-306.
[18]Cohen G, Rubinstein S, Gur Y, Breitbart H (2004) Crosstalk between protein kinase A and C regulates phospholipase D and F-actin formation during sperm capacitation. Dev Biol 267:230-41.
[19]Colgan OC, Collins NT, Ferguson G, Murphy RP, Birney YA, Cahill PA, Cummins PM (2008) Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function. Brain Res 1193:84-92.
[20]Condliffe AM, Cadwallader KA, Walker TR, Rintoul RC, Cowburn AS, Chilvers ER (2000) Phosphoinositide 3-kinase:a critical signalling event in pulmonary cells. Respir Res 1:24-9.
[21]Cucullo L, Couraud PO, Weksler B, Romero IA, Hossain M, Rapp E, Janigro D (2008a) Immortalized human brain endothelial cells and flow-based vascular modeling:a marriage of convenience for rational neurovascular studies. J Cereb Blood Flow Metab 28:312-28.
[22]Destaing O, Sanjay A, Itzstein C, Home WC, Toomre D, De Camilli P, Baron R (2008) The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts. Mol Biol Cell 19:394-404.
[23]Dohgu S, Banks WA (2008) Lipopolysaccharide-enhanced transcellular transport of HIV-1 across the blood-brain barrier is mediated by the p38 mitogen-activated protein kinase pathway. Exp Neurol 210:740-9.
[24]Drexler HG, Quentmeier H, Dirks WG, MacLeod RA (2002) Bladder carcinoma cell line ECV304 is not a model system for endothelial cells. In Vitro Cell Dev Biol Anim 38:185-6,187.
[25]Fleegal MA, Horn S, Borg LK, Davis TP (2005) Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation. Am J Physiol Heart Circ Physiol 289:H2012-9.
[26]Georgiadis A, Tschernutter M, Bainbridge JW, Balaggan KS, Mowat F, West EL, Munro PM, Thrasher AJ, Matter K, Balda MS, Ali RR (2010) The tight junction associated signalling proteins ZO-1 and ZONAB regulate retinal pigment epithelium homeostasis in mice. PLoS One 5:e 15730.
[27]Ghosh S, May MJ, Kopp EB (1998) NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16:225-60.
[28]Gumbleton M, Audus KL (2001) Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier. J Pharm Sci 90:1681-98.
[29]Harhaj NS, Antonetti DA (2004) Regulation of tight junctions and loss of barrier function in pathophysiology. Int J Biochem Cell Biol 36:1206-37.
[30]Hasegawa H, Fujita H, Katoh H, Aoki J, Nakamura K, Ichikawa A, Negishi M (1999) Opposite regulation of transepithelial electrical resistance and paracellular permeability by Rho in Madin-Darby canine kidney cells. J Biol Chem 274:20982-8.
[31]Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, Tsukita S, Yokoyama M, Staddon JM (2001) Regulation of tight junction permeability and Occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. J Biol Chem 276:10423-31.
[32]Holinstat, M., et al.,2003. Protein kinase Calpha-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement. J Biol Chem. 278,28793-8.
[33]Izumi Y, Hirose T, Tamai Y, Hirai S, Nagashima Y, Fujimoto T, Tabuse Y, Kemphues KJ, Ohno S (1998) An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol 143:95-106.
[34]Jin R, Song Z, Yu S, Piazza A, Nanda A, Penninger JM, Granger DN, Li G (2011) Phosphatidylinositol-3-kinase gamma plays a central role in blood-brain barrier dysfunction in acute experimental stroke. Stroke 42:2033-44.
[35]Jou, T. S., et al.,1998. Structural and functional regulation of tight junctions by Rho A and Racl small GTPases. J Cell Biol.142,101-15.
[36]Kaya, M., et al.,2004. Effects of lipopolysaccharide on the radiation-induced changes in the blood-brain barrier and the astrocytes. Brain Res.1019,105-12.
[37]Kim, K. S.,2003. Pathogenesis of bacterial meningitis:from bacteraemia to neuronal injury. Nat Rev Neurosci.4,376-85.
[38]Konopatskaya O, Poole AW (2010) Protein kinase Calpha:disease regulator and therapeutic target. Trends Pharmacol Sci 31:8-14.
[39]Lapointe TK, Buret AG (2012) Interleukin-18 facilitates neutrophil transmigration via myosin light chain kinase-dependent disruption of Occludin, without altering epithelial permeability. Am J Physiol Gastrointest Liver Physiol 302:G343-51.
[40]Legros H, Launay S, Roussel BD, Marcou-Labarre A, Calbo S, Catteau J, Leroux P, Boyer O, Ali C, Marret S, Vivien D, Laudenbach V (2009) Newborn-and adult-derived brain microvascular endothelial cells show age-related differences in phenotype and glutamate-evoked protease release. J Cereb Blood Flow Metab 29:1146-58.
[41]Leitman EM, Tewari A, Horn M, Urbanski M, Damanakis E, Einheber S, Salzer JL, de Lanerolle P, Melendez-Vasquez CV (2011) MLCK regulates Schwann cell cytoskeletal organization, differentiation and myelination. J Cell Sci 124:3784-96.
[42]Liu H, Li M, Wang P, Wang F (2011) Blockade of hypoxia-inducible factor-1 alpha by YC-1 attenuates interferon-gamma and tumor necrosis factor-alpha-induced intestinal epithelial barrier dysfunction. Cytokine 56:581-8.
[43]Lossinsky AS, Shivers RR (2004) Structural pathways for macromolecular and cellular transport across the blood-brain barrier during inflammatory conditions. Review. Histol Histopathol 19:535-64.
[44]Ma TY, Boivin MA, Ye D, Pedram A, Said HM (2005) Mechanism of TNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol 288:G422-30.
[45]Ma TY, Iwamoto GK, Hoa NT, Akotia V, Pedram A, Boivin MA, Said HM (2004) TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation. Am J Physiol Gastrointest Liver Physiol 286:G367-76.
[46]Madara JL (1990) Warner-Lambert/Parke-Davis Award lecture. Pathobiology of the intestinal epithelial barrier. Am J Pathol 137:1273-81.
[47]Mark KS, Davis TP (2002) Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol 282:H1485-94.
[48]Mayhan WG (2001) Regulation of blood-brain barrier permeability. Microcirculation 8:89-104.
[49]Mehta D, Rahman A, Malik AB (2001) Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. J Biol Chem 276:22614-20.
[50]Meng G, Zhao J, Wang HM, Ding RG, Zhang XC, Huang CQ, Ruan JX (2011) Injury of cell tight junctions and changes of actin level in acute lung injury caused by the perfluoroisobutylene exposure and the role of Myosin light chain kinase. J Occup Health 53:250-7.
[51]Meyer TN, Schwesinger C, Ye J, Denker BM, Nigam SK (2001) Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity. J Biol Chem 276:22048-55.
[52]Montaner S, Perona R, Saniger L, Lacal JC (1998) Multiple signalling pathways lead to the activation of the nuclear factor kappaB by the Rho family of GTPases. J Biol Chem 273:12779-85.
[53]Moriez R, Salvador-Cartier C, Theodorou V, Fioramonti J, Eutamene H, Bueno L (2005) Myosin light chain kinase is involved in lipopolysaccharide-induced disruption of colonic epithelial barrier and bacterial translocation in rats. Am J Pathol 167:1071-9.
[54]Mullin JM, Laughlin KV, Ginanni N, Marano CW, Clarke HM, Peralta SA (2000) Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers. Ann N Y Acad Sci 915:231-6.
[55]Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, Innis-Whitehouse W, Eastburn KK, Madara JL (2000) Tight junctions are membrane microdomains. J Cell Sci 113 (Pt 10):1771-81.
[56]Pang H, Bitar KN (2005) Direct association of RhoA with specific domains of PKC-alpha. Am J Physiol Cell Physiol 289:C982-93.
[57]Patil SB, Bitar KN (2006) RhoA-and PKC-alpha-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 290:G83-95.
[58]Peng J, He F, Zhang C, Deng X, Yin F (2011a) Protein kinase C-alpha signals P115RhoGEF phosphorylation and RhoA activation in TNF-alpha-induced mouse brain microvascular endothelial cell barrier dysfunction. J Neuroinflammation 8:28.
[59]Phillips N, Hayward RD, Koronakis V (2004) Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation. Nat Cell Biol 6:618-25.
[60]Polk WW, Ellis ME, Kushleika JV, Simmonds PL, Woods JS (2007) RhoA regulation of NF-kappaB activation is mediated by COX-2-dependent feedback inhibition of IKK in kidney epithelial cells. Am J Physiol Cell Physiol 293:C1160-70.
[61]Rattan R, Giri S, Singh AK, Singh I (2003) Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines:negative regulation of IKKalpha. Free Radic Biol Med 35:1037-50.
[62]Riganti C, Doublier S, Costamagna C, Aldieri E, Pescarmona G, Ghigo D, Bosia A (2008) Activation of nuclear factor-kappa B pathway by simvastatin and RhoA silencing increases doxorubicin cytotoxicity in human colon cancer HT29 cells. Mol Pharmacol 74:476-84.
[63]Rosenberg GA, Yang Y (2007) Vasogenic edema due to tight junction disruption by matrix metalloproteinases in cerebral ischemia. Neurosurg Focus 22:E4.
[64]Rosse C, Linch M, Kermorgant S, Cameron AJ, Boeckeler K, Parker PJ (2010) PKC and the control of localized signal dynamics. Nat Rev Mol Cell Biol 11:103-12.
[65]Rosson D, O'Brien TG, Kampherstein JA, Szallasi Z, Bogi K, Blumberg PM, Mullin JM (1997) Protein kinase C-alpha activity modulates transepithelial permeability and cell junctions in the LLC-PK1 epithelial cell line. J Biol Chem 272:14950-3.
[66]Roux F, Couraud PO (2005) Rat brain endothelial cell lines for the study of blood-brain barrier permeability and transport functions. Cell Mol Neurobiol 25:41-58.
[67]Rubin LL, Staddon JM (1999a) The cell biology of the blood-brain barrier. Annu Rev Neurosci 22:11-28.
[68]Salkeni MA, Lynch JL, Otamis-Price T, Banks WA (2009) Lipopolysaccharide impairs blood-brain barrier P-glycoprotein function in mice through prostaglandin- and nitric oxide-independent pathways. J Neuroimmune Pharmacol 4:276-82.
[69]Schottelius AJ, Baldwin AJ (1999) A role for transcription factor NF-kappa B in intestinal inflammation. Int J Colorectal Dis 14:18-28.
[70]Schwarz BT, Wang F, Shen L, Clayburgh DR, Su L, Wang Y, Fu YX, Turner JR (2007) LIGHT signals directly to intestinal epithelia to cause barrier dysfunction via cytoskeletal and endocytic mechanisms. Gastroenterology 132:2383-94.
[71]Shen Q, Rigor RR, Pivetti CD, Wu MH, Yuan SY (2010) Myosin light chain kinase in microvascular endothelial barrier function. Cardiovasc Res 87:272-80.
[72]Sheth P, Basuroy S, Li C, Naren AP, Rao RK (2003) Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. J Biol Chem 278:49239-45.
[73]Sheth P, Delos SN, Seth A, LaRusso NF, Rao RK (2007) Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 293:G308-18.
[74]Sheth, P., et al.,2003. Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. J Biol Chem.278,49239-45.
[75]Sheth, P., et al.,2007. Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism. Am J Physiol Gastrointest Liver Physiol.293, G308-18.
[76]Shimizu S, Tahara M, Ogata S, Hashimoto K, Morishige K, Tasaka K, Murata Y (2007) Involvement of nuclear factor-kB activation through RhoA/Rho-kinase pathway in LPS-induced IL-8 production in human cervical stromal cells. Mol Hum Reprod 13:181-7.
[77]Siflinger-Birnboim A, Johnson A (2003a) Protein kinase C modulates pulmonary endothelial permeability:a paradigm for acute lung injury. Am J Physiol Lung Cell Mol Physiol 284:L435-51.
[78]Stuart RO, Nigam SK (1995) Regulated assembly of tight junctions by protein kinase C. Proc Natl Acad Sci U S A 92:6072-6.
[79]Sukumaran SK, Prasadarao NV (2002) Regulation of protein kinase C in Escherichia coli K1 invasion of human brain microvascular endothelial cells. J Biol Chem 277:12253-62.
[80]Terry S, Nie M, Matter K, Balda MS (2010) Rho signaling and tight junction functions. Physiology (Bethesda) 25:16-26.
[81]Tiruppathi C, Shimizu J, Miyawaki-Shimizu K, Vogel SM, Bair AM, Minshall RD, Predescu D, Malik AB (2008) Role of NF-kappaB-dependent caveolin-1 expression in the mechanism of increased endothelial permeability induced by lipopolysaccharide. J Biol Chem 283:4210-8.
[82]Turner JR (2006) Molecular basis of epithelial barrier regulation:from basic mechanisms to clinical application. Am J Pathol 169:1901-9.
[83]Verin V, Popowski Y, de Bruyne B, Baumgart D, Sauerwein W, Lins M, Kovacs G, Thomas M, Calman F, Disco C, Serruys PW, Wijns W (2001) Endoluminal beta-radiation therapy for the prevention of coronary restenosis after balloon angioplasty. The Dose-Finding Study Group. N Engl J Med 344:243-9.
[84]Vietor I, Bader T, Paiha K, Huber LA (2001) Perturbation of the tight junction permeability barrier by Occludin loop peptides activates beta-catenin/TCF/ LEF-mediated transcription. Embo Rep 2:306-12.
[85]Vorbrodt AW, Dobrogowska DH (2003) Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers:electron microscopist's view. Brain Res Brain Res Rev 42:221-42.
[86]Wang F, Graham WV, Wang Y, Witkowski ED, Schwarz BT, Turner JR (2005) Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol 166:409-19.
[87]Wang F, Schwarz BT, Graham WV, Wang Y, Su L, Clayburgh DR, Abraham C, Turner JR (2006) IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction. Gastroenterology 131:1153-63.
[88]Webb BL, Hirst SJ, Giembycz MA (2000) Protein kinase C isoenzymes:a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis. Br J Pharmacol 130:1433-52.
[89]Webb RC (2003) Smooth muscle contraction and relaxation. Adv Physiol Educ 27:201-6.
[90]Wells WA, Bonetta L (2005) Endothelial tight junctions form the blood-brain barrier. J Cell Biol 169:378.
[91]Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38:323-37.
[92]Xaio H, Banks WA, Niehoff ML, Morley JE (2001) Effect of LPS on the permeability of the blood-brain barrier to insulin. Brain Res 896:36-42.
[93]Yamamoto M, Ramirez SH, Sato S, Kiyota T, Cerny RL, Kaibuchi K, Persidsky Y, Ikezu T (2008a) Phosphorylation of Claudin-5 and Occludin by rho kinase in brain endothelial cells. Am J Pathol 172:521-33.
[94]Ye D, Ma I, Ma TY (2006) Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol 290:G496-504.
[95]Ye D, Ma TY (2008) Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity. J Cell Mol Med 12:1331-46.
[96]甘娜,尹飞,彭镜,王卫东.溶血磷脂酸促进大鼠体外血脑屏障模型通透性增加机制研究.中华医学杂志,2008,88(6)：12-14.
[97]刘依凌,王凤君,陈传莉,等.Rho激酶在大鼠严重烧伤后肠黏膜通透性改变中的作用及机制研究[J].第三军医大学学报,2008,30(9)：817-819
[98]彭镜,尹飞,甘娜,等.内毒素脂多糖对体外血脑屏障模型通透性的影响及其机制研究[J].中华医学杂志,2006,86(27)：1924-1926.
[99]吴丽文,尹飞,彭镜,王卫东,甘娜。紧密连接蛋白ZO-1、Occludin和Actin参与缺氧缺血诱导的血脑屏障通透性增加[J]。中国当代儿科杂志,2008,10(4)：514-516。
[100]叶增纯.肾小球疾病发病机制的系列研究[D]。中山大学,2010
[101]尹飞,杨于嘉,虞佩兰,等.百日咳菌液致脑水肿时大鼠脑组织细胞因子的变化及机制[J].中风与神经疾病杂志,2001,18(5)：267-269
[1]Abe T, Sugano E, Saigo Y, Tamai M (2003) Interleukin-lbeta and barrier function of retinal pigment epithelial cells (ARPE-19):aberrant expression of junctional complex molecules. Invest Ophthalmol Vis Sci 44:4097-104.
[2]Andreeva AY, Krause E, Muller EC, Blasig IE, Utepbergenov DI (2001a) Protein kinase C regulates the phosphorylation and cellular localization of Occludin. J Biol Chem 276:38480-6.
[3]Balda MS, Gonzalez-Mariscal L, Matter K, Cereijido M, Anderson JM (1993) Assembly of the tight junction:the role of diacylglycerol. J Cell Biol 123:293-302.
[4]Bamforth SD, Kniesel U, Wolburg H, Engelhardt B, Risau W (1999) A dominant mutant of Occludin disrupts tight junction structure and function. J Cell Sci 112 (Pt 12):1879-88.
[5]Banks WA, Erickson MA (2010) The blood-brain barrier and immune function and dysfunction. Neurobiol Dis 37:26-32.
[6]Bazzoni G, Dejana E (2004) Endothelial cell-to-cell junctions:molecular organization and role in vascular homeostasis. Physiol Rev 84:869-901.
[7]Berkes J, Viswanathan VK, Savkovic SD, Hecht G (2003) Intestinal epithelial responses to enteric pathogens:effects on the tight junction barrier, ion transport, and inflammation. Gut 52:439-51.
[8]Boyle EC, Brown NF, Finlay BB (2006) Salmonella enterica serovar Typhimurium effectors SopB, SopE, SopE2 and Sip A disrupt tight junction structure and function. Cell Microbiol 8:1946-57.
[9]Brown RC, Davis TP (2005) Hypoxia/aglycemia alters expression of Occludin and actin in brain endothelial cells. Biochem Biophys Res Commun 327:1114-23.
[10]Bruewer M, Hopkins AM, Hobert ME, Nusrat A, Madara JL (2004) RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin. Am J Physiol Cell Physiol 287:C327-35.
[11]Chen YH, Lu Q, Goodenough DA, Jeansonne B (2002) Nonreceptor tyrosine kinase c-Yes interacts with Occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell 13:1227-37.
[12]Clarke H, Soler AP, Mullin JM (2000) Protein kinase C activation leads to dephosphorylation of Occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets. J Cell Sci 113 (Pt 18):3187-96.
[13]Clarke H, Marano CW, Peralta SA, Mullin JM (2000) Modification of tight junction function by protein kinase C isoforms. Adv Drug Deliv Rev 41:283-301.
[14]Conklin BS, Zhong DS, Zhao W, Lin PH, Chen C (2002a) Shear stress regulates Occludin and VEGF expression in porcine arterial endothelial cells. J Surg Res 102:13-21.
[15]Cordenonsi M, D'Atri F, Hammar E, Parry DA, Kendrick-Jones J, Shore D, Citi S (1999) Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin. J Cell Biol 147:1569-82.
[16]Correale J, Villa A (2009) Cellular elements of the blood-brain barrier. Neurochem Res 34:2067-77.
[17]Cruzalegui FH, Bading H (2000) Calcium-regulated protein kinase cascades and their transcription factor targets. Cell Mol Life Sci 57:402-10.
[18]Date I, Takagi N, Takagi K, Tanonaka K, Funakoshi H, Matsumoto K, Nakamura T, Takeo S (2006) Hepatocyte growth factor attenuates cerebral ischemia-induced increase in permeability of the blood-brain barrier and decreases in expression of tight junctional proteins in cerebral vessels. Neurosci Lett 407:141-5.
[19]DeMaio L, Chang YS, Gardner TW, Tarbell JM, Antonetti DA (2001) Shear stress regulates Occludin content and phosphorylation. Am J Physiol Heart Circ Physiol 281:H105-13.
[20]Duronio V, Scheid MP, Ettinger S (1998) Downstream signalling events regulated by phosphatidylinositol 3-kinase activity. Cell Signal 10:233-9.
[21]Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM (1998) The tight junction protein ZO-1 establishes a link between the transmembrane protein Occludin and the actin cytoskeleton. JBiol Chem 273:29745-53.
[22]Farshori P, Kachar B (1999) Redistribution and phosphorylation of Occludin during opening and resealing of tight junctions in cultured epithelial cells. J Membr Biol 170:147-56.
[23]Feldman GJ, Mullin JM, Ryan MP (2005) Occludin:structure, function and regulation. Adv Drug Deliv Rev 57:883-917.
[24]Fischer S, Wobben M, Marti HH, Renz D, Schaper W (2002) Hypoxia-induced hyperpermeability in brain microvessel endothelial cells involves VEGF-mediated changes in the expression of zonula occludens-1. Microvasc Res 63:70-80.
[25]Fischer S, Wiesnet M, Marti HH, Renz D, Schaper W (2004) Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells. J Cell Physiol 198:359-69.
[26]FitzGerald TJ, Simon E, Meyer J (2004) Prostate carcinoma:opportunities for translational research. J Cell Biochem 91:433-42.
[27]Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1993) Occludin:a novel integral membrane protein localizing at tight junctions. J Cell Biol 123:1777-88.
[28]Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and-2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to Occludin. J Cell Biol 141:1539-50.
[29]Gebbink MF, Kranenburg O, Poland M, van Horck FP, Houssa B, Moolenaar WH (1997) Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein:control of neuronal morphology. J Cell Biol 137:1603-13.
[30]Gonzalez-Mariscal L, Betanzos A, Nava P, Jaramillo BE (2003) Tight junction proteins. Prog Biophys Mol Biol 81:1-44.
[31]Gye MC, Ohsako S (2003) Effects of flutamide in the rat testis on the expression of Occludin, an integral member of the tight junctions. Toxicol Lett 143:217-22.
[32]Hawkins BT, Davis TP (2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57:173-85.
[33]He F, Peng J, Deng XL, Yang LF, Wu LW, Zhang CL, Yin F (2011) RhoA and NF-kappaB are involved in lipopolysaccharide-induced brain microvascular cell line hyperpermeability. Neuroscience 188:35-47.
[34]Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, Tsukita S, Yokoyama M, Staddon JM (2001) Regulation of tight junction permeability and Occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. JBiol Chem 276:10423-31.
[35]Honda M, Nakagawa S, Hayashi K, Kitagawa N, Tsutsumi K, Nagata I, Niwa M (2006) Adrenomedullin improves the blood-brain barrier function through the expression of Claudin-5. Cell Mol Neurobiol 26:109-18.
[36]Howarth AG, Hughes MR, Stevenson BR (1992) Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types. Am J Physiol 262:CA61-9.
[37]Huber JD, Witt KA, Hom S, Egleton RD, Mark KS, Davis TP (2001) Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression. Am JPhysiol Heart Circ Physiol 280:H1241-8.
[38]Ikenouchi J, Furuse M, Furuse K, Sasaki H, Tsukita S, Tsukita S (2005) Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J Cell Biol 171:939-45.
[39]Inoko A, Itoh M, Tamura A, Matsuda M, Furuse M, Tsukita S (2003) Expression and distribution of ZO-3, a tight junction MAGUK protein, in mouse tissues. Genes Cells 8:837-45.
[40]Ishizaki T, Chiba H, Kojima T, Fujibe M, Soma T, Miyajima H, Nagasawa K, Wada I, Sawada N (2003) Cyclic AMP induces phosphorylation of Claudin-5 immunoprecipitates and expression of Claudin-5 gene in blood-brain-barrier endothelial cells via protein kinase A-dependent and -independent pathways. Exp Cell Res 290:275-88.
[41]Kago T, Takagi N, Date I, Takenaga Y, Takagi K, Takeo S (2006) Cerebral ischemia enhances tyrosine phosphorylation of Occludin in brain capillaries. Biochem Biophys Res Commun 339:1197-203.
[42]Kale G, Naren AP, Sheth P, Rao RK (2003) Tyrosine phosphorylation of Occludin attenuates its interactions with ZO-1, ZO-2, and ZO-3. Biochem Biophys Res Commun 302:324-9.
[43]Krause G, Winkler L, Piehl C, Blasig I, Piontek J, Muller SL (2009) Structure and function of extracellular Claudin domains. Ann N Y Acad Sci 1165:34-43.
[44]Krug SM, Amasheh S, Richter JF, Milatz S, Gunzel D, Westphal JK, Huber O, Schulzke JD, Fromm M (2009) Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability. Mol Biol Cell 20:3713-24.
[45]Lechner J, Krall M, Netzer A, Radmayr C, Ryan MP, Pfaller W (1999) Effects of interferon alpha-2b on barrier function and junctional complexes of renal proximal tubular LLC-PK1 cells. Kidney Int 55:2178-91.
[46]Lee DB, Huang E, Ward HJ (2006) Tight junction biology and kidney dysfunction. Am J Physiol Renal Physiol 290:F20-34.
[47]Lee SW, Kim WJ, Choi YK, Song HS, Son MJ, Gelman IH, Kim YJ, Kim KW (2003) SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier. Nat Med 9:900-6.
[48]Li D, Mrsny RJ (2000) Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of Occludin. J Cell Biol 148:791-800.
[49]Liebner S, Fischmann A, Rascher G, Duffner F, Grote EH, Kalbacher H, Wolburg H (2000) Claudin-1 and Claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme. Acta Neuropathol 100:323-31.
[50]Mandel I, Paperna T, Glass-Marmor L, Volkowich A, Badarny S, Schwartz I, Vardi P, Koren I, Miller A (2011) Tight junction proteins expression and modulation in immune cells and multiple sclerosis. J Cell Mol Med.
[51]Mariano C, Sasaki H, Brites D, Brito MA (2011) A look at tricellulin and its role in tight junction formation and maintenance. Eur J Cell Biol 90:787-96.
[52]Mark KS, Davis TP (2002) Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol 282:H1485-94.
[53]McKenzie JA, Riento K, Ridley AJ (2006a) Casein kinase I epsilon associates with and phosphorylates the tight junction protein Occludin. Febs Lett 580:2388-94.
[54]Moolenaar WH (1995) Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem 270:12949-52.
[55]Mooradian AD, Haas MJ, Chehade JM (2003) Age-related changes in rat cerebral Occludin and zonula occludens-1 (ZO-1). Mech Ageing Dev 124:143-6.
[56]Muller SL, Portwich M, Schmidt A, Utepbergenov DI, Huber O, Blasig IE, Krause G (2005) The tight junction protein Occludin and the adherens junction protein alpha-catenin share a common interaction mechanism with ZO-1. J Biol Chem 280:3747-56.
[57]Murakami T, Felinski EA, Antonetti DA (2009) Occludin phosphorylation and ubiquitination regulate tight junction trafficking and vascular endothelial growth factor-induced permeability. J Biol Chem 284:21036-46.
[58]Musch MW, Walsh-Reitz MM, Chang EB (2006) Roles of ZO-1, Occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointesl Liver Physiol 290:G222-31.
[59]Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S (2003) Size-selective loosening of the blood-brain barrier in Claudin-5-deficient mice. J Cell Biol 161:653-60.
[60]Nunbhakdi-Craig V, Machleidt T, Ogris E, Bellotto D, White CR, Sontag E (2002) Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol 158:967-78.
[61]Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, Innis-Whitehouse W, Eastburn KK, Madara JL (2000) Tight junctions are membrane microdomains. J Cell Sci 113(Pt10):1771-81.
[62]Nusrat A, Chen JA, Foley CS, Liang TW, Tom J, Cromwell M, Quan C, Mrsny RJ (2000) The coiled-coil domain of Occludin can act to organize structural and functional elements of the epithelial tight junction. J Biol Chem 275:29816-22.
[63]Peng J, He F, Zhang C, Deng X, Yin F (2011) Protein kinase C-alpha signals P115RhoGEF phosphorylation and RhoA activation in TNF-alpha-induced mouse brain microvascular endothelial cell barrier dysfunction. J Neuroinflammation 8:28.
[64]Persidsky Y, Heilman D, Haorah J, Zelivyanskaya M, Persidsky R, Weber GA, Shimokawa H, Kaibuchi K, Ikezu T (2006a) Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE). Blood 107:4770-80.
[65]Raleigh DR, Boe DM, Yu D, Weber CR, Marchiando AM, Bradford EM, Wang Y, Wu L, Schneeberger EE, Shen L, Turner JR (2011) Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function. J Cell Biol 193:565-82.
[66]Rao R (2009) Occludin phosphorylation in regulation of epithelial tight junctions. Ann N Y Acad Sci 1165:62-8.
[67]Rao RK, Basuroy S, Rao VU, Karnaky JK, Gupta A (2002a) Tyrosine phosp-horylation and dissociation of Occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidative stress. Biochem J 368:471-81.
[68]Ratcliffe MJ, Rubin LL, Staddon JM (1997) Dephosphorylation of the cadherin-associated p100/p120 proteins in response to activation of protein kinase C in epithelial cells. J Biol Chem 272:31894-901.
[69]Ratcliffe MJ, Smales C, Staddon JM (1999) Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli. Biochem J 33S(Pt2):471-8.
[70]Riazuddin S, Ahmed ZM, Fanning AS, Lagziel A, Kitajiri S, Ramzan K, Khan SN, Chattaraj P, Friedman PL, Anderson JM, Belyantseva IA, Forge A, Riazuddin S, Friedman TB (2006) Tricellulin is a tight-junction protein necessary for hearing. Am J Hum Genet 79:1040-51.
[71]Sahai E, Ishizaki T, Narumiya S, Treisman R (1999) Transformation mediated by RhoA requires activity of ROCK kinases. Curr Biol 9:136-45.
[72]Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Inazawa J, Fujimoto K, Tsukita S (1997) Mammalian Occludin in epithelial cells:its expression and subcellular distribution. Eur J Cell Biol 73:222-31.
[73]Sakaguchi T, Gu X, Golden HM, Suh E, Rhoads DB, Reinecker HC (2002) Cloning of the human Claudin-25'-flanking region revealed a TATA-less promoter with conserved binding sites in mouse and human for caudal-related homeodomain proteins and hepatocyte nuclear factor-1 alpha. J Biol Chem 277:21361-70.
[74]Schneeberger EE, Lynch RD (2004) The tight junction:a multifunctional complex. Am J Physiol Cell Physiol 286:C1213-28.
[75]Scudamore CL, Jepson MA, Hirst BH, Miller HR (1998) The rat mucosal mast cell chymase, RMCP-Ⅱ, alters epithelial cell monolayer permeability in association with altered distribution of the tight junction proteins ZO-1 and Occludin. Eur J Cell Biol 75:321-30.
[76]Sheth P, Basuroy S, Li C, Naren AP, Rao RK (2003) Role of phosphatid-ylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. J Biol Chem 278:49239-45.
[77]Simonovic I, Rosenberg J, Koutsouris A, Hecht G (2000) Enteropathogenic Escherichia coli dephosphorylates and dissociates Occludin from intestinal epithelial tight junctions. Cell Microbiol 2:305-15.
[78]Sit ST, Manser E (2011) Rho GTPases and their role in organizing the actin cytoskeleton. J Cell Sci 124:679-83.
[79]Soma T, Chiba H, Kato-Mori Y, Wada T. Yamashita T, Kojima T, Sawada N (2004) Thr(207) of Claudin-5 is involved in size-selective loosening of the endothelial barrier by cyclic AMP. Exp Cell Res 300:202-12.
[80]Steed E, Balda MS, Matter K (2010) Dynamics and functions of tight junctions. Trends Cell Biol 20:142-9.
[81]Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA (1986a) Identification of ZO-1:a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103:755-66.
[82]Subramanian VS, Marchant JS, Ye D, Ma TY, Said HM (2007) Tight junction targeting and intracellular trafficking of Occludin in polarized epithelial cells. Am J Physiol Cell Physiol 293:C1717-26.
[83]Suidan GL, Mcdole JR, Chen Y, Pirko I, Johnson AJ (2008) Induction of blood brain barrier tight junction protein alterations by CD8 T cells. PLoS One 3:e3037.
[84]Takenaga Y, Takagi N, Murotomi K, Tanonaka K, Takeo S (2009) Inhibition of Src activity decreases tyrosine phosphorylation of Occludin in brain capillaries and attenuates increase in permeability of the blood-brain barrier after transient focal cerebral ischemia. J Cereb Blood Flow Metab 29:1099-108.
[85]Tavelin S, Hashimoto K, Malkinson J, Lazorova L, Toth I, Artursson P (2003) A new principle for tight junction modulation based on Occludin peptides. Mol Pharmacol 64:1530-40.
[86]Terry S, Nie M, Matter K, Balda MS (2010) Rho signaling and tight junction functions. Physiology (Bethesda) 25:16-26.
[87]Tiruppathi C, Minshall RD, Paria BC, Vogel SM, Malik AB (2002) Role of Ca2+ signaling in the regulation of endothelial permeability. Vascul Pharmacol 39:173-85.
[88]Wang HR, Zhang Y, Ozdamar B, Ogunjimi AA, Alexandrova E, Thomsen GH, Wrana JL (2003) Regulation of cell polarity and protrusion formation by targeting RhoA for degradation. Science 302:1775-9.
[89]Wang W, Dentler WL, Borchardt RT (2001) VEGF increases BMEC monolayer permeability by affecting Occludin expression and tight junction assembly. Am J Physiol Heart Circ Physiol 280:H434-40.
[90]Wang Y, Zhang J, Yi XJ, Yu FS (2004) Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells. Exp Eye Res 78:125-36.
[91]Ward PD, Klein RR, Troutman MD, Desai S, Thakker DR (2002) Phospholipase C-gamma modulates epithelial tight junction permeability through hyperphosphorylation of tight junction proteins. J Biol Chem 277: 35760-5.
[92]Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38:323-37.
[93]Wolburg H, Wolburg-Buchholz K, Kraus J, Rascher-Eggstein G, Liebner S, Hamm S, Duffner F, Grote EH, Risau W, Engelhardt B (2003) Localization of Claudin-3 in tight junctions of the blood-brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multifo-rme. Acta Neuropathol 105:586-92.
[94]Xu R, Feng X, Xie X, Zhang J, Wu D, Xu L (2012) HIV-1 Tat protein increases the permeability of brain endothelial cells by both inhibiting Occludin expression and cleaving Occludin via matrix metalloproteinase-9. Brain Res 1436:13-9.
[95]Yin F, Watsky MA (2005) LPA and S1P increase corneal epithelial and endothelial cell transcellular resistance. Invest Ophthalmol Vis Sci 46:1927-33.
[96]Youakim A, Ahdieh M (1999) Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin. Am J Physiol 276:G1279-88.
[97]彭镜,尹飞,甘娜,张红媛.内毒素脂多糖对体外血脑屏障模型通透性的影响及其机制研究.中华医学杂志.86(2006)：1924-1926.