脂多糖致脑微血管内皮细胞通透性升高的机制研究

发布时间：2018-05-21 15:53

本文选题：紧密连接 + 蛋白激酶C　；参考：《中南大学》2012年博士论文

【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(P0.05),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(P0.05)。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(P0.05),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(P0.05)。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。
[Abstract]:Bacterial meningitis is considered one of the top ten fatal infectious diseases in the world . It is fatal and has a high disability rate . At present , there are still some 30 - 50 % of patients with irreversible nervous system sequelae . Blood - brain barrier ( BBB ) permeability increases vascular - derived brain edema plays a key role in the pathogenesis . The pathogenesis is not clear , and the treatment is difficult . Therefore , it is very important to clarify the change of BBB permeability and control mechanism in bacterial meningitis .

The study shows that brain micro vascular endothelial cells ( BMECs ) and tight junction ( TJ ) are the main bases of BBB structure and function . LPS is the component of the cell wall of Gram - negative bacteria , which is called endotoxin after release of blood , and it has important effect on the function of BBB barrier .

This study is divided into four parts :

In the first chapter , the cell line has the barrier properties of the rat brain microvascular endothelial cells .

Objective : To evaluate the protective barrier and physiological characteristics of cultured mouse brain microvascular endothelial cells ( Bendothelial cells ) in primary cultured rat brain microvascular endothelial cells .

Methods : The rat brain microvascular endothelial cells were inoculated into the cell culture , and the barrier function was measured by the permeability test of transendothelial electrical resistance ( TEER ) and horseradish peroxidase ( HRP ) . Western blot and direct fluorescence staining were used to observe the expression of the related proteins in human brain microvascular endothelial cells , the expression of ZO - 1 and the distribution of F - actin in the cells .

Results : The TEER of Bend . 3 cells increased gradually with culture time , 10d reached 82.33 卤 6.03 惟路 cm ~ 2 , and the difference was statistically significant ( P0.05 ) . Compared with TEER of rat brain microvascular endothelial cells cultured in the same period , there was no significant difference ( P0.05 ) .

Conclusion : The mouse brain microvascular endothelial cell strain Bend . 3 has the barrier property of primary cultured brain microvascular endothelial cells , and its barrier function can reach the ideal state after 10d .

In chapter 2 , the signal molecules of lipopolysaccharide - induced permeability change in brain microvascular endothelial cells were preliminarily selected .

Objective : To demonstrate the increase of permeability of microvascular endothelial cells induced by lipopolysaccharide ( Actin ) recombination , closely linked expression and distribution changes .

Methods : The permeability , F - actin distribution and tight connexin cluaudin - 5 , F - actin distribution and the state of tight connexin cluaudin - 5 were detected by TEER assay , F - actin staining , western blot and immunofluorescence staining . The effects of LPS on the cell barrier function of each group were observed by using calphostin C ( PKC inhibitor ) , C 3 transferase ( inhibitor of tyrosine kinase ) , wortmannin ( inhibitors of tyrosine kinase ) , and the effects of LPS on the barrier function of each group were investigated .

Results : The TEER decreased in time - dependent manner , and the expression of Claudin - 5 protein was down - regulated . The expression of Claudin - 5 protein was changed , and the F - actin of cytoskeletal framework was reduced . PKC and rho inhibitor could improve the decrease of TEER in Bend . 3 cells induced by LPS .
however , that inhibitor of tyrosine kinase can not block the damage of LPS to the function of the Bend . 3 barrier .

Conclusion : The expression and distribution of Actin in brain microvascular endothelial cells induced by lipopolysaccharide ( LPS ) resulted in increased permeability .
PKC and rho are involved in the regulation of this process , while non - PI3 and tyrosine kinases are involved .

In chapter 3 , PKC and RhoA signaling interact to regulate the permeability of microvascular endothelial cells induced by lipopolysaccharide .

Objective : To investigate how PKC isoforms ( 伪 , 尾 and zeta ) participate in the regulation of permeability of microvascular endothelial cells induced by lipopolysaccharide ( LPS ) , and their relationship with RhoA .

Methods : The expression of PKC - 伪 - PKC - 尾 and PKC - 尾 - shRNA was determined by the method of pull - down and immune co - precipitation combined with in vitro enzymatic experiment . The expression of PKC - 伪 PKC - 尾 and PKC - zeta protein was determined by using liposome 2000 . The expression of PKC - 伪 PKC - 尾 and PKC - zeta protein were determined by Western blot . The activation of PKC - 伪 PKC - 尾 and PKC - 伪 - shRNA was investigated by Western blot .
The effects of PKC - 伪 , PKC - 尾 and PKC - zeta on the activation of RhoA were respectively detected by using shRNA , and the relationship between PKC - 伪 , PKC - 尾 and PKC - zeta activation was studied .
In order to further confirm whether PKC - 伪 was the upstream molecular signal of RhoA and inhibited PKC - a activity after LPS - induced BBB function destruction , two groups of Bend . 3 cells stably expressing N19RhoA and vector - 1 plasmid changed TEER values before and after LPS .
In order to clarify the regulatory relationships of PKC - zeta and RhoA in the process of LPS - induced BBB dysfunction , the activity of RhoA in Bend . 3 cells stably expressing PKC - zeta - ShRNA and vector - 2 was inhibited , and the changes of TEER value before and after LPS action were compared .

Results : 1 . All of the subtypes of RhoA and PKC were activated by LPS for 5 min . PKC - 伪 - 魏B , PKC - 尾 , PKC - zeta and N19RhoA were successfully established .

Conclusion : PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a and PKC - zeta , respectively , increased BBB - TJ opening in the presence of PKC - a and PKC - zeta , respectively .

In chapter 4 , RhoA / NF - 魏B / MLCK signal is involved in regulating the permeability of microvascular endothelial cells induced by lipopolysaccharide .

Objective : To investigate whether the signal of RhoA / NF - 魏B / MLCK is involved in the regulation of BBB permeability in LPS - induced BBB .

Methods : The effects of LPS on the activity of RhoA and NF - 魏B in Bend . 3 cells of Bend . 3 cells were determined by using Lipofectamine 2000 . The effects of LPS on the activity of RhoA and NF - 魏B in Bend . 3 cells were determined by using the method of pull down and luciferase reporter gene .

Results : The stable expression of DNMu - I魏B 伪 and N19RhoA plasmid could improve the adhesion of LPS - induced Bend . 3 cells and increase the permeability . LPS acts 5 min for activation of RhoA and activates NF - 魏B in 30min .
LPS could inhibit the activation of RhoA and LPS - induced NF - 魏B activation , but the inhibition of NF - 魏B activation did not affect the activity of RhoA . The level of NF - 魏B activation increased , the level of MLCK transcription increased , the phosphorylation level of 3h MLC was obviously increased , and the above expression was inhibited after blocking NF - 魏B activation .

Conclusion : RhoA / NF - KB / MLCK signal is involved in the regulation of BBB - TJ damage and increase of permeability in LPS - induced BBB - TJ .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R725.1

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