【作者】 何芳；

【导师】 尹飞；

【作者基本信息】 中南大学 ， 儿科学， 2012， 博士

【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。更多还原

【Abstract】 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 cellsObjective 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 hyperpermeabilityObjective 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 permeabilityObjective 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 hyperpermeabilityObjective 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.更多还原