褪黑素对IL-lβ诱导的人脐静脉内皮细胞通透性的影响

发布时间：2018-05-24 20:07

本文选题：褪黑素 + 白介素-1β　；参考：《北京协和医学院》2011年博士论文

【摘要】：目的：第二部分：为了探讨褪黑素(MEL)对血管单层内皮细胞屏障的保护作用,以培养在Transwell系统内的单层人脐静脉内皮细胞(HUVECs)为模型,观察MEL对IL-1β诱导的单层内皮细胞通透性的影响。第二部分：观察MEL对IL-1β诱导的HUVECs间黏附连接和细胞骨架的影响。第三部分：探讨MEL对单层内皮细胞屏障保护作用可能的受体途径和信号通路。方法：第一部分：原代分离培养HUVECs,并使用包被小鼠抗人CD31单克隆抗体的免疫磁珠对原代分离的HUVECs进一步纯化。以小鼠抗人vWF单克隆抗体及FITC标记的山羊抗小鼠荧光二抗鉴定纯化后的HUVECs。将纯化后的HUVECs接种在Transwell系统,通过检测不同干预条件下透过单层内皮的荧光标记的葡聚糖来反映内皮通透性的改变。第二部分：用免疫荧光显微镜和激光共聚焦显微镜观察不同干预条件下内皮细胞黏附连接蛋白VE钙粘素(VE-cadherin)和细胞骨架蛋白的变化情况。使用Western blot检测VE-cadherin的表达变化情况。第三部分：检测MEL和IL-1p对影响内皮细胞间连接和细胞骨架的重要的信号通路Rho GTPase家族中Rac蛋白的影响；应用MEL受体阻滞剂Luz、Rac的抑制剂NSC-23766,探讨MEL发挥HUVECs屏障保护作用的受体途径和信号通路。结果：第二部分：(1)成功分离培养原代HUVECs,免疫磁珠筛选后能继续贴壁生长并传代,倒置显微镜下HUVECs呈鹅卵石状,vWF免疫荧光染色证实细胞确为HUVECs。细胞呈梭形、圆形或多角形。(2)与空白对照组相比,5、10和20ng/mL的IL-1β分别使HUVECs通透性由1.5±0.1升高为4.8±0.3、5.2±0.4和9.8±0.8(P0.01)；10和100μmol/L的MEL分别使10ng/mL的IL-1β诱导的通透性从5.2±0.4降至2.8±0.3和2.7±0.3(P0.01)。此结果与“时间-过程”通透性实验结果一致。第三部分：(1)荧光显微镜观察：正常融合后的内皮细胞表达VE-cadherin呈现出连续不间断的状态；IL-1β(10ng/ml)破坏了细胞间的黏附连接,使VE-cadherin在细胞膜上的表达下调,并出现断裂。MEL(10μmol/L)预处理后,VE-cadherin的表达状态得到了改善。(2)激光共聚焦显微镜下观察：IL-1β(10ng/ml)明显的破坏了细胞间的黏附连接,使正常状态下分布在细胞边缘的肌动蛋白转变为跨细胞体的应力纤维,原本融合的细胞间出现明显的缝隙。MEL(10μmol/L)预处理后,没有出现明显的黏附连破坏现象,细胞中心应力纤维的表达减少,边缘部位的肌动蛋白增加,细胞间缝隙变小。(3)Western Blot结果表明：与对照组相比,IL-1p使HUVECs的VE-cadherin表达量下调。MEL可抑制IL-18诱导的VE-cadherin下调,但仍低于对照组。第三部分：(1)Luz阻断了MEL对内皮屏障的保护作用；阻断了MEL对内皮黏附连接和细胞骨架的影响,导致细胞连接破坏和应力纤维增加,细胞间出现缝隙。(2)MEL预处理抑制了IL-1β诱导的Rac的失活,而Luz可以阻断MEL的这种作用。(3)NSC-23766抑制了MEL对内皮屏障的保护作用。结论：(1)IL-1β诱导HUVECs黏附连接蛋白VE-cadherin表达降低,应力纤维增加,细胞间出现缝隙,通透性升高。(2)MEL通过激活Rac信号途径改善了内皮细胞黏附连接和细胞骨架的状态,抑制了IL-1β诱导的内皮通透性的升高。
[Abstract]:Purpose :

The second part : To investigate the protective effect of melatonin ( MEL ) on vascular endothelial cell barrier , the effect of MEL on the permeability of single - layer endothelial cells induced by IL - 1尾 was observed in vitro .

The second part : To observe the effect of MEL on IL - 1尾 - induced intercellular adhesion and cellular skeleton .

The third part : To investigate the possible receptor pathway and signal pathway of MEL on the protective effect of single - layer endothelial cells .

Method :

In the first part , the primary isolated culture was isolated and further purified by immunomagnetic beads coated with mouse anti - human CD31 monoclonal antibody . The mouse anti - human monoclonal antibody and FITC - labeled goat anti - mouse fluorescent antibody were used for the identification and purification . After purification , the purified culture was inoculated in the Transwell system , and the changes of endothelial permeability were reflected by detecting the fluorescent labeled glucan of the single - layer endothelium under different intervention conditions .

In the second part , we observed the changes of E - cadherin and E - cadherin in endothelial cells under different intervention conditions by immunofluorescence microscopy and laser confocal microscope . The expression of VE - cadherin was detected by Western blot .

The third part : The effects of MEL and IL - 1p on the expression of Rac protein in the family of signaling pathways , such as endothelial cell and cell skeleton , were examined .
The receptor pathway and signal pathway of the protective effects of MEL were investigated by using the inhibitor NSC - 23766 of the MEL receptor blocker , Luz , Rac .

Results :

The second part : ( 1 ) The cells were isolated and cultured in primary culture . After the screening of immune magnetic beads , the adherent growth was continued and the cells were passaged . The cells were fusiform , round or polygonal , and IL - 1尾 in 5 , 10 and 20 ng / mL increased from 1.5 卤 0.1 to 4.8 卤 0.3 , 5.2 卤 0.4 and 9.8 卤 0.8 respectively ( P0.01 ) .
In MEL of 10 and 100 渭mol / L , the permeability of IL - 1尾 induced by 10 ng / mL decreased from 5.2 卤 0.4 to 2.8 卤 0.3 and 2.7 卤 0.3 respectively ( P0.01 ) .

The third part : ( 1 ) Fluorescence microscopy observation : The expression of VE - cadherin in endothelial cells after normal fusion showed a continuous uninterrupted state ;
After pretreatment with MEL ( 10 渭mol / L ) , the expression of E - cadherin was improved .

The third part : ( 1 ) Luz blocked the protective effect of MEL on endothelial barrier ;
( 2 ) MEL pretreatment inhibited the inactivation of IL - 1尾 - induced Rac and Luz blocked MEL ' s effect . ( 3 ) NSC - 23766 inhibited the protective effect of MEL on endothelial barrier .

Conclusion : ( 1 ) IL - 1尾 - induced expression of E - cadherin decreased , stress fiber increased , gap between cells increased , and permeability increased . ( 2 ) MEL improved the adhesion of endothelial cells and the state of cellular skeleton by activating Rac signaling pathway , and inhibited the increase of endothelial permeability induced by IL - 1尾 .
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

【共引文献】