网格蛋白和微囊在LPS致血管通透性增高中的作用及机制

发布时间：2018-05-11 13:56

本文选题：血管通透性增高 + LPS　；参考：《第三军医大学》2014年博士论文

【摘要】：血管通透性增高是严重创伤、脓毒症患者的重要病理改变，表现为血管内皮屏障受损，对液体、血浆蛋白、大分子物质的通透性增高，导致组织水肿，促进内环境紊乱等合并症发生，研究阐明血管通透性增高的发生机制有助于维持内环境稳定、改善疗效和提高存活率。脂多糖(lipopolysaccharide, LPS)是脓毒症的重要启动因子，目前认为LPS引起血管通透性增高主要有两条途径：“细胞间”途径和“跨细胞”途径。其中，“细胞间”途径开放主要由内皮细胞张力丝形成并收缩引起，并认为是调节血管通透性增高的主要途径。然而近来研究发现，细胞张力丝聚合发生在LPS作用后的早期，4h后细胞骨架解聚重构，张力丝消失，提示LPS诱导血管通透性增高还存在其他调节机制。根据基础研究，黏附连接是血管内皮细胞间(除血脑屏障外)的主要连接方式，其主要结构蛋白血管内皮钙粘蛋白(vascular endothelial cadherin, VE-cad)被胞吞可以引起黏附连接强度降低、细胞间隙开放和血管通透性增高。以往研究表明，VE-cad在细胞质膜的表达取决于被胞吞的多少，而VE-cad的胞吞主要受网格蛋白介导，但近来有研究发现，上皮钙粘蛋白(epithelial cadherin, E-cad)的胞吞可以通过非网格蛋白途径——细胞质膜微囊(caveolae，简称微囊)途径完成，例如在人表皮样癌细胞和胰腺导管癌上皮细胞，微囊可以胞吞E-cad，引起上皮细胞间黏附连接破坏、细胞分离和肿瘤转移。那么，网格蛋白介导和微囊介导的VE-cad胞吞是否参与了LPS诱导的血管通透性增高，机制如何？据此，我们以CRL-2922内皮细胞株为研究对象，研究内容为以下三部分：①观察网格蛋白介导和微囊介导的VE-cad胞吞在LPS诱导血管通透性增高中的作用；②探讨网格蛋白介导和微囊介导的VE-cad胞吞导致LPS作用后不同程度的血管通透性增高的机制；③探讨LPS作用后网格蛋白介导和微囊介导的VE-cad胞吞途径转换的机制。主要实验方法：第一部分网格蛋白介导和微囊介导的VE-cad胞吞在LPS诱导血管通透性增高中的作用 (一)网格蛋白介导的VE-cad胞吞在LPS诱导血管通透性增高中的作用 1.采用人血管内皮细胞株CRL-2922，检测LPS (10μg/mL)作用不同时间(1h、2h、4h和6h)后VE-cad质膜蛋白表达、单层细胞通透性，观察变化规律。 2.采用人血管内皮细胞株CRL-2922，观察LPS作用不同时间(1h、2h、4h和6h)后网格蛋白与VE-cad的免疫共沉淀和共定位。 3.采用网格蛋白胞吞抑制剂CPZ (100μmol/L)和网格蛋白重链siRNA (50nmol/L)，观察其对LPS作用(1h和4h)后网格蛋白与VE-cad的免疫共沉淀、VE-cad质膜蛋白表达，以及单层细胞通透性的影响。 (二)微囊介导的VE-cad胞吞在LPS诱导血管通透性增高中的作用 1.采用人血管内皮细胞株CRL-2922，观察LPS作用不同时间(1h、2h、4h和6h)后Cav1与VE-cad的免疫共沉淀和共定位。 2.采用微囊抑制剂filipin (5μg/mL)和Cav1siRNA (50nmol/L)，观察其对LPS作用(1h和4h)后Cav1与VE-cad的免疫共沉淀、VE-cad质膜蛋白表达，以及单层细胞通透性的影响。 3.采用人血管内皮细胞株CRL-2922，观察LPS作用不同时间(1h、2h、4h和6h)后微囊主要结构蛋白Cav1的蛋白表达和磷酸化(Tyr14)，以及Src的蛋白表达的变化，并观察Src抑制剂SU6656(2μmol/L)和TLR4抑制剂CLI-095(5μg/mL)对LPS作用(1h和4h)后的Cav1磷酸化(Tyr14)、Cav1与VE-cad的免疫共沉淀、P-Cav1与VE-cad的免疫共沉淀、VE-cad质膜蛋白表达，以及单层细胞通透性的影响。第二部分网格蛋白介导和微囊介导的VE-cad胞吞导致LPS作用后不同程度的血管通透性增高的机制 1.采用人血管内皮细胞株CRL-2922，观察LPS作用不同时间(1h、2h、4h和6h)后VE-cad与Rab11(循环内颗粒标志物)的免疫共沉淀、VE-cad与LAMP2(次级内颗粒/溶酶体标志物)的免疫共沉淀的变化。 2.采用网格蛋白胞吞抑制剂CPZ和微囊抑制剂filipin，观察其对LPS作用(1h和4h)后VE-cad与Rab11的免疫共沉淀、VE-cad与LAMP2的免疫共沉淀的影响。第三部分LPS作用后网格蛋白介导和微囊介导的VE-cad胞吞途径转换的机制 1.采用人血管内皮细胞株CRL-2922，观察LPS作用不同时间(1h、2h、4h和6h)后细胞骨架的动态变化。 2.采用细胞骨架解聚剂Cyt D (2μmol/L)和细胞骨架稳定剂Jasp (1μmol/L)，观察其对LPS作用(1h和4h)后网格蛋白与VE-cad的免疫共沉淀、Cav1与VE-cad的免疫共沉淀、VE-cad与Rab11的免疫共沉淀、VE-cad与LAMP2的免疫共沉淀、VE-cad质膜蛋白表达，以及单层细胞通透性的影响。主要结果：一、网格蛋白介导和微囊介导的VE-cad胞吞在LPS诱导血管通透性增高中的作用 (一)网格蛋白介导的VE-cad胞吞在LPS诱导血管通透性增高中的作用 1.正常对照组中VE-cad在质膜蛋白表达高，LPS (10μg/mL)作用后VE-cad质膜蛋白表达逐渐降低(P0.05)，VE-cad的总蛋白表达也逐渐降低(P0.05)。LPS作用后单层细胞通透性呈时间依赖性的增高(P0.05)。 2. LPS作用后网格蛋白的表达逐渐降低(P0.05)；网格蛋白与VE-cad的免疫共沉淀在LPS作用1h后增高(P0.05)，然后逐渐降低；免疫组合激光共聚焦显微镜观察到网格蛋白与VE-cad的共定位在LPS作用1h后增高，在LPS作用4h后降低。 3.网格蛋白胞吞抑制剂CPZ (100μmol/L)和网格蛋白重链siRNA (50nmol/L)可以显著降低LPS作用1h后网格蛋白与VE-cad的免疫共沉淀(P0.05)，增高LPS作用1h后VE-cad质膜蛋白表达(P0.05)，改善LPS作用1h后的单层细胞通透性(P0.05)；但对LPS作用4h后网格蛋白与VE-cad的免疫共沉淀、VE-cad质膜蛋白表达，以及单层细胞通透性没有显著影响。 (二)微囊介导的VE-cad胞吞在LPS诱导血管通透性增高中的作用 1. LPS作用后，Cav1与VE-cad的免疫共沉淀在正常对照组中几乎未见，随着LPS作用时间延长而逐渐增高(P0.05)，免疫组化激光共聚焦显微镜观察其共定位也发现在LPS作用4h后有明显的共定位。 2.微囊抑制剂非律平(5μg/mL)和Cav1siRNA (50nmol/L)可以显著降低LPS作用4h后Cav1与VE-cad的免疫共沉淀，并增高VE-cad质膜蛋白表达，以及改善单层细胞通透性(P0.05)。 3. LPS作用后，微囊主要的蛋白成分Cav1的蛋白表达无显著变化，但其Tyr14位点磷酸化水平却逐渐增高(P0.05)，Src蛋白表达呈时间依赖性的增高(P0.05)，TLR4抑制剂CLI-095(5μg/mL)可显著降低LPS作用4h后增高的Src蛋白表达。Src抑制剂SU6656(2μmol/L)和TLR4抑制剂CLI-095(5μg/mL)可显著降低LPS作用4h后的Cav1磷酸化(Tyr14)，减少Cav1与VE-cad的免疫共沉淀以及P-Cav1与VE-cad的免疫共沉淀(P0.05)，增加VE-cad质膜蛋白表达(P0.05)，以及单层细胞通透性显著降低(P0.05)。二、网格蛋白介导和微囊介导的VE-cad胞吞导致LPS作用后不同程度的血管通透性增高的机制 1. LPS作用后，VE-cad与Rab11的免疫共沉淀在1h增高(P0.05)，随后逐渐降低；VE-cad与LAMP2的免疫共沉淀在正常时几乎未见，但随LPS作用时间延长，呈时间依赖性的增高(P0.05)。 2. LPS作用1h后增高的VE-cad与Rab11免疫共沉淀可以被网格蛋白胞吞抑制剂CPZ显著抑制(P0.05)，，但不受微囊抑制剂非律平的影响；LPS作用4h后增高的VE-cad与LAMP2的免疫共沉淀可以被微囊抑制剂显著抑制(P0.05)，但不受网格蛋白胞吞抑制剂的影响。三、LPS作用后网格蛋白介导和微囊介导的VE-cad胞吞途径转换的机制 1.正常对照组中，肌动蛋白呈均匀散在分布，细胞骨架无明显的聚合；LPS作用1h后，肌动蛋白聚集呈点片状，发生明显聚合，可见细胞中细长的张力丝形成；4h后肌动蛋白重新显示出散在分布的趋势，细胞骨架解聚，张力丝几近消失。 2.细胞骨架解聚剂Cyt D可显著抑制LPS作用1h后增高的网格蛋白与VE-cad以及VE-cad与Rab11免疫共沉淀(P0.05)，显著增高Cav1与VE-cad以及VE-cad与LAMP2的免疫共沉淀(P0.05)，显著降低VE-cad质膜蛋白表达(P0.05)，并加重LPS作用1h后的单层细胞通透性增高(P0.05)。在LPS作用1h后给予细胞骨架稳定剂Jasp处理，可以显著降低LPS作用4h后的Cav1与VE-cad以及VE-cad与LAMP2的免疫共沉淀(P0.05)，增高LPS作用4h后的VE-cad质膜蛋白表达(P0.05)，并改善LPS作用4h后的单层细胞通透性(P0.05)。结论： 1.网格蛋白介导和微囊介导的VE-cad胞吞均参与了LPS诱导的血管通透性增高，网格蛋白介导的VE-cad胞吞主要发生在LPS作用的早期(1～2h)，而微囊介导的VE-cad胞吞主要发生在LPS作用后期(4h)，并由LPS-TLR4-Src信号途径激活。 2. VE-cad经网格蛋白介导的胞吞后位于循环内颗粒中，导致LPS作用早期(1～2h)VE-cad质膜蛋白表达丢失和单层细胞通透性有限的增高，而VE-cad经微囊介导的胞吞后位于次级内颗粒/溶酶体中，导致后期(4h)严重的VE-cad质膜蛋白表达丢失和单层细胞通透性增高。 3. LPS作用后细胞骨架先发生聚合然后解聚，这种动态变化调节VE-cad胞吞由网格蛋白介导向微囊介导转换。
[Abstract]:The increase of vascular permeability is a serious trauma. The important pathological changes of patients with sepsis are the damage of the vascular endothelial barrier, the increase in the permeability of the liquid, plasma protein and macromolecules, the edema of the tissue and the disturbance of the internal environment. The study of the mechanism of the increase of the permeability of the blood tube helps to maintain the stability of the internal environment. Lipopolysaccharide (LPS) is an important promoter of sepsis. It is believed that there are two main ways of increasing vascular permeability by LPS: "intercellular" pathway and "cross cell" pathway. Among them, the opening of "intercellular" pathway is mainly formed by the formation and contraction of endothelial cell tension filament. It is considered to be the main way to regulate the increase of vascular permeability. However, recent studies have found that the polymerization of cell tension filament occurs at the early stage after the action of LPS. After 4h, the cytoskeleton disintegration and the disappearance of the tension filament, suggesting that there are other regulatory mechanisms for the increase of vascular permeability induced by LPS.
According to basic research, adhesion connection is the main connection between vascular endothelial cells (except the blood brain barrier). The main structural protein vascular endothelial cadherin (VE-cad) is swallowed, which can cause the adhesion strength to decrease, the intercellular space opening and the vascular permeability increase. The expression of cell plasmalemma depends on the number of endocytosis, and VE-cad's endocytosis is mainly mediated by grulin, but recent studies have found that the endocytosis of epithelial cadherin (E-cad) can be accomplished through the non grid protein pathway, the cell membrane microcapsule (caveolae, for short), for example, in human epidermoid cancer cells And the epithelial cells of pancreatic ductal carcinoma, microcapsules can endocytic E-cad, cause adhesion failure, cell separation and tumor metastasis in epithelial cells. Then, is grid protein mediated and microencapsulated VE-cad endocytosis involved in LPS induced increased vascular permeability, and how is the mechanism?
According to this, we take the CRL-2922 endothelial cell line as the research object. The following three parts are studied: (1) to observe the role of gridin mediated and microcapsule mediated VE-cad endocytosis in the increase of vascular permeability induced by LPS; secondly, to explore the different degree of vascular permeability induced by LPS in the action of grid protein mediated and microcapsule mediated VE-cad endocytosis. The mechanism of increase is discussed. The mechanism of LPS mediated cytosolic mediated and microencapsulated VE-cad endocytosis pathway is discussed.
The main experimental methods:
Part I the role of clathrin mediated and microencapsulated VE-cad endocytosis in LPS induced vascular permeability enhancement
(1) the role of clathrin mediated VE-cad endocytosis in increasing vascular permeability induced by LPS
1. the human vascular endothelial cell strain CRL-2922 was used to detect the expression of the plasma membrane protein (1H, 2h, 4H and 6h), and the permeability of the monolayer cells after different time (1H, 2h, 4H and 6h), and to observe the changes of the changes.
2. human endothelial cell line CRL-2922 was used to observe the co immunoprecipitation and co localization of clathrin and VE-cad after LPS treatment at different time (1H, 2h, 4H and 6h).
3. CPZ (100 mol/L) and gridin heavy chain siRNA (50nmol/L) were used to observe the effects of the immunoprecipitation, the expression of VE-cad plasma membrane protein and the permeability of the monolayer cells after LPS action (1H and 4h).
(two) the role of microencapsulation mediated VE-cad endocytosis in LPS induced vascular permeability enhancement
1. human endothelial cell line CRL-2922 was used to observe the immunoprecipitation and co localization of Cav1 and VE-cad after LPS treatment at different time (1H, 2h, 4H and 6h).
2. the effects of microcapsule inhibitor filipin (5 g/mL) and Cav1siRNA (50nmol/L) on the immunoprecipitation of Cav1 and VE-cad after LPS action (1H and 4h), the expression of VE-cad plasma membrane protein, and the permeability of monolayer cells were observed.
3. the human vascular endothelial cell line CRL-2922 was used to observe the protein expression and phosphorylation (Tyr14) of the major structural protein Cav1 of the microcapsules (Tyr14) and the changes in the expression of Src in different time (1H, 2h, 4H and 6h), and to observe the phosphorylation of the Src inhibitor SU6656 (2 micron) and the inhibitory agent (5 micron). The co immunoprecipitation of Cav1 and VE-cad, the immunoprecipitation of P-Cav1 and VE-cad, the expression of plasma membrane protein of VE-cad, and the permeability of monolayer cells.
The second part is the mechanism of vascular permeability enhanced by clathrin mediated and microencapsulated VE-cad endocytosis after LPS.
1. the human vascular endothelial cell strain CRL-2922 was used to observe the immunoprecipitation of VE-cad and Rab11 (the granule markers in circulation), and the changes of co precipitation between VE-cad and LAMP2 (the secondary granules / lysosome markers) after the action of LPS at different time (1H, 2h, 4H and 6h).
2. the effects of the immunoprecipitation of VE-cad and Rab11 after the action of LPS (1H and 4h) and the immunoprecipitation of VE-cad and LAMP2 were observed by using the trellis endocytosis inhibitor CPZ and the microcapsule inhibitor filipin.
The third part is the mechanism of LPS mediated mesocytosis and microencapsulated VE-cad endocytosis pathway.
1. human endothelial cell line CRL-2922 was used to observe the dynamic changes of cytoskeleton after LPS treatment at different time (1H, 2h, 4H and 6h).
2. the cytoskeleton depolymerization agent Cyt D (2 mu mol/L) and cytoskeleton stabilizer Jasp (1 mu mol/L) were used to observe the immunoprecipitation of the gridin and VE-cad after the action of LPS (1H and 4h), the immunoprecipitation of Cav1 and VE-cad, the immunoprecipitation of VE-cad and immunization, the expression of plasma membrane protein, and the monolayer cells. The influence of permeability.
Main results:
First, the role of clathrin mediated and microencapsulated VE-cad endocytosis in LPS induced vascular permeability enhancement.
(1) the role of clathrin mediated VE-cad endocytosis in increasing vascular permeability induced by LPS
1. in the normal control group, the expression of VE-cad in the plasma membrane protein was high, and the expression of VE-cad plasma membrane protein decreased gradually after the action of LPS (10 g/mL). The total protein expression of VE-cad decreased gradually (P0.05) after the action of.LPS, and the permeability of the monolayer was time dependent (P0.05).
After the action of 2. LPS, the expression of gridin was gradually reduced (P0.05); the immunoprecipitation of gridin and VE-cad increased (P0.05) after 1h (P0.05), and then decreased gradually. The co localization of gridin and VE-cad increased after LPS action 1H and decreased after LPS action 4H.
3. grid protein endocytosis inhibitor CPZ (100 mu mol/L) and gridin heavy chain siRNA (50nmol/L) can significantly reduce the immunoprecipitation (P0.05) of the gridin and VE-cad after LPS action 1H, and increase the expression of VE-cad plasma membrane protein after LPS action 1H (P0.05). Immunoprecipitation with VE-cad showed no significant effect on VE-cad plasma membrane protein expression and permeability of monolayer cells.
(two) the role of microencapsulation mediated VE-cad endocytosis in LPS induced vascular permeability enhancement
After the action of 1. LPS, the immunoprecipitation of Cav1 and VE-cad was hardly seen in the normal control group, and gradually increased with the prolongation of the time of LPS action (P0.05). The co localization of the immuno confocal laser scanning microscope also found that there was a clear co location after the action of LPS on 4H.
2. microcapsule inhibitor (5 g/mL) and Cav1siRNA (50nmol/L) can significantly reduce the immunoprecipitation of Cav1 and VE-cad after 4h, and increase the expression of VE-cad plasma membrane protein, and improve the permeability of monolayer cells (P0.05).
After the action of 3. LPS, the protein expression of the main protein component of the microcapsule, Cav1, had no significant changes, but the phosphorylation level of the Tyr14 site increased gradually (P0.05), the expression of Src protein showed a time dependent increase (P0.05), and the TLR4 inhibitor CLI-095 (5 micron) could significantly reduce the LPS as the Src protein expression inhibitor (2 mu) and the increase of Src protein expression (2 mu). The R4 inhibitor CLI-095 (5 mu g/mL) can significantly reduce the Cav1 phosphorylation (Tyr14) after LPS action 4h, reduce the immunoprecipitation of Cav1 and VE-cad, and P-Cav1 and VE-cad immunoprecipitation (P0.05), increase the expression of plasma membrane protein, and decrease the permeability of monolayer cells significantly.
Two, the mechanism of increased permeability of vascular endothelial cells induced by LPS mediated by clathrin and microencapsulation mediated VE-cad endocytosis.
After the action of 1. LPS, the immunoprecipitation of VE-cad and Rab11 increased in 1H (P0.05), and then gradually decreased, and the immunoprecipitation of VE-cad and LAMP2 was hardly seen at normal time, but with the prolongation of LPS action time, it showed a time dependent increase (P0.05).
2. LPS and Rab11 immunoprecipitation could be significantly inhibited by the VE-cad and Rab11 immunoprecipitation (P0.05), but not affected by the microcapsule inhibitor (P0.05), but the immune coprecipitation of VE-cad and LAMP2 after LPS action 4H could be significantly suppressed by microcapsule inhibitors (P0.05), but not by the trellis endocytosis inhibitors. Influence.
Three, the mechanism of LPS mediated mesocytosis and microencapsulated VE-cad endocytosis pathway.
1. in the normal control group, the actin was distributed evenly and the cytoskeleton was not obviously polymerized; after the LPS action 1H, the actin aggregation was a bit flaky, obviously polymerized, and the elongated ttene formed in the cells; after 4h, actin reshowed the distribution of the trend, the cytoskeleton depolymerized and the tension filament nearly disappeared.
2. cytoskeleton depolymerization agent Cyt D significantly inhibited the increase of LPS after 1h and VE-cad, VE-cad and Rab11 immunoprecipitation (P0.05), significantly increased Cav1 and VE-cad as well as VE-cad and LAMP2 immunoprecipitation, significantly reduced the expression of plasma membrane protein, and increased the permeability of monolayer cells after the action. P0.05). The cytoskeleton stabilizer Jasp treatment after the action of 1H can significantly reduce the immune coprecipitation (P0.05) of Cav1 and VE-cad as well as VE-cad and LAMP2 after LPS action 4h, and increase the expression of plasma membrane protein after LPS, and improve the permeability of monolayer cells after the action of LPS.
Conclusion:
1. grid protein mediated and microcapsule mediated VE-cad endocytosis participated in the increase of vascular permeability induced by LPS. The VE-cad endocytosis mediated by gridin occurred mainly in the early stage of LPS (1 to 2H), while microcapsule mediated VE-cad endocytosis occurred mainly in the late stage of LPS (4h) and activated by the LPS-TLR4-Src signal pathway.
2. VE-cad, which is located in the inner circulatory particles after the mesocytosis of the grid protein, leads to the loss of the expression of VE-cad plasma membrane protein and the limited permeability of the monolayer cells in the early stage of the LPS action, and VE-cad is located in the secondary granules / lysosomes after the endocytosis of the microcapsule, leading to the loss of the late (4h) expression of the VE-cad plasma membrane protein and the single VE-cad protein expression. The permeability of layer cells increased.
After 3. LPS, the cytoskeleton first polymerized and then depolymerized. This dynamic change regulates VE-cad cytosis mediated by clathrin mediated microencapsulation.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R459.7

【共引文献】