机械牵张对人肺血管内皮细胞通透性的影响及其分子机制

doi:10.3877/cma.j.issn.2096-1537.2019.02.013

中华重症医学电子杂志 ›› 2019, Vol. 05 ›› Issue (02) : 151 -158. doi: 10.3877/cma.j.issn.2096-1537.2019.02.013

所属专题：文献；

基础研究

机械牵张对人肺血管内皮细胞通透性的影响及其分子机制

王亚¹, 兰媛¹, 傅威¹, 刘晓青¹, 黄勇波¹, 毛璞², 黎毅敏¹^,^†(

)

^1. 510120　广州医科大学第一附属医院，广州呼吸健康研究院，呼吸疾病国家重点实验室
^2. 510120　广州医学院第一附属医院医院感染管理科

收稿日期:2019-02-19 出版日期:2019-05-28
通信作者: 黎毅敏
基金资助:
国家自然科学基金(81770079); 广东省自然科学基金(2017A030313781和2017A030313712); 广东省科技计划(2016A020215170)

Effect of mechanical stretch on permeability of human pulmonary vascular endothelial cells and underlying molecular mechanism

Ya Wang¹, Yuan Lan¹, Wei Fu¹, Xiaoqing Liu¹, Yongbo Huang¹, Pu Mao², Yimin Li¹^,^†()

^1. The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, State Key Laboratory of Respiratory Diseases, Guangzhou 510120, China
^2. Department of Infection Management, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China

Received:2019-02-19 Published:2019-05-28
Corresponding author: Yimin Li
About author:
Corresponding author: Li Yimin, Email: dryiminli@vip.163.com

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引用本文：

王亚, 兰媛, 傅威, 刘晓青, 黄勇波, 毛璞, 黎毅敏. 机械牵张对人肺血管内皮细胞通透性的影响及其分子机制[J]. 中华重症医学电子杂志, 2019, 05(02): 151-158.

Ya Wang, Yuan Lan, Wei Fu, Xiaoqing Liu, Yongbo Huang, Pu Mao, Yimin Li. Effect of mechanical stretch on permeability of human pulmonary vascular endothelial cells and underlying molecular mechanism[J]. Chinese Journal of Critical Care & Intensive Care Medicine(Electronic Edition), 2019, 05(02): 151-158.

目的

观察机械牵张对人肺动脉内皮细胞（HPAEC）和人肺微血管内皮细胞（HPMVEC）通透性的影响及对通透性关键蛋白VE-cadherin、Claudin-5和Caveolin-1表达的调控。

方法

采用机械牵张装置Flexcell FX-5000T对HPAEC（ATCC）和HPMVEC（ATCC）施加0.5 Hz频率10%或20%牵张应力。应用qRT-PCR及Western Blot方法检测机械牵张前后内皮通透性关键蛋白VE-cadherin、Claudin-5、Caveolin-1 mRNA和蛋白含量表达变化。采用细胞动态分析仪以及Transwell小室/异硫氰酸荧光素（FITC）-白蛋白法检测机械牵张后的HPAEC细胞和HPMVEC细胞通透性改变。

结果

20%机械牵张后，与对照组比较，HPAEC细胞通透性关键蛋白VE-cadherin mRNA和蛋白表达、Claudin-5 mRNA和蛋白表达和Caveolin-1 mRNA表达均下降（P<0.05），而Caveolin-1蛋白水平较对照组无明显变化；HPMVEC细胞通透性关键蛋白VE-cadherin mRNA和蛋白表达，Caveolin-1 mRNA和蛋白表达均下降（P<0.05），而Claudin-5 mRNA和蛋白水平较对照组无明显变化。

结论

机械牵张会导致肺血管内皮通透性增高，是通过下调通透性关键蛋白的表达，并且两种细胞的通透性影响机制有差异，HPAEC牵涉紧密连接，HPMVEC牵涉跨内皮细胞途径。

关键词: 机械牵张, 人肺动脉内皮细胞, 细胞通透性, 内皮通透性关键蛋白VE-cadherin, Claudin-5, Caveolin-1

Objective

To observe the effect of mechanical stretch on the permeability of human pulmonary artery endothelial cells (HPAECs) and human pulmonary microvascular endothelial cells (HPMVECs) and to examine whether the expression of the key permeability-related proteins VE-cadherin, Claudin-5, and Caveolin-1 is involved in this process.

Methods

The mechanical stretch device Flexcell FX-5000T was used to apply 10% or 20% tensile stress to HPAECs and HPMVECs at a frequency of 0.5 Hz. qRT-PCR and Western blot were used to detect the mRNA and protein expression of VE-cadherin, Claudin-5, and Caveolin-1 in cells before and after mechanical stretch. Mechanical stretched HPAEC cells and HPMVEC cells were monitored by a cell dynamic analyzer and Transwell chamber/FITC-albumin assay to detect their permeability.

Results

In HPAECs after mechanical stretch, the expression of VE-cadherin mRNA and protein, Claudin-5 mRNA and protein, and Caveolin-1 mRNA decreased significantly compared with control cells (P<0.05), while the level of Caveolin-1 protein did not change significantly compared with the control group. In HPMVECs after mechanical stretch, the expression of VE-cadherin mRNA and protein and Caveolin-1 mRNA and protein decreased significantly compared with control cells (P<0.05), while the levels of Claudin-5 mRNA and protein did not change significantly compared with the control group.

Conclusion

Mechanical stretch leads to increased permeability of pulmonary vascular endothelial cells, which is caused by down-regulation of the expression of permeability key proteins, but the permeability mechanisms of the two cell types are different: HPAECs involve tight junctions and HPMVECs involve the transendothelial pathway.

Key words: Mechanical stretch, Human pulmonary artery endothelial cells, Human pulmonary microvascular endothelial cells, VE-cadherin, Claudin-5, Caveolin-1

表1 PCR引物设计情况

基因名称	引物序列	产物大小（bp）
GAPDH	F：5′-TCCTCCACCTTTGACGCT-3′	177
?	R：5′-TCTTCCTCTTGTGCTCTTGC-3′	?
VE-cadherin	F：5′-CCTCACGGATAATCACGATAACA-3′	99
?	R：5′-GAGATGACCACGGGTAGGAAGT-3′	?
Claudin-5	F：5′-AGCGGGTGTCAGACTGAGGA-3′	162
?	R：5′-CCCTGCCGATGGAGTAAAGA-3′	?
Caveolin-1	F：5′-CGACCCTAAACACCTCAACG-3′	154
?	R：5′-GGCAGACAGCAAGCGGTAA-3′	?

表1 PCR引物设计情况

基因名称	引物序列	产物大小（bp）
GAPDH	F：5′-TCCTCCACCTTTGACGCT-3′	177
?	R：5′-TCTTCCTCTTGTGCTCTTGC-3′	?
VE-cadherin	F：5′-CCTCACGGATAATCACGATAACA-3′	99
?	R：5′-GAGATGACCACGGGTAGGAAGT-3′	?
Claudin-5	F：5′-AGCGGGTGTCAGACTGAGGA-3′	162
?	R：5′-CCCTGCCGATGGAGTAAAGA-3′	?
Caveolin-1	F：5′-CGACCCTAAACACCTCAACG-3′	154
?	R：5′-GGCAGACAGCAAGCGGTAA-3′	?

图1 机械牵张HPAEC和HPMVEC预处理组及对照组给予50 nmol/L Thrombin刺激后电阻值变化。图a为HPAEC 20%机械牵张24 h与对照组比较；图b为HPMVEC 20%机械牵张24 h与对照组比较

图2 机械牵张HPAEC和HPMVEC预处理组及对照组对于FITC-albumin的通透性变化。图a为HPAEC 20%机械牵张24 h与对照组比较；图b为HPMVEC 20%机械牵张24 h与对照组比较

图3 机械牵张对HPAEC通透性关键蛋白VE-cadherin、Claudin-5和Caveolin-1 mRNA表达的影响

表2 机械牵张对HPAEC细胞VE-cadherin、Claudin-5和Caveolin-1 mRNA表达的影响（±s）

关键蛋白	10%机械牵张				20%机械牵张
关键蛋白	1 h	2 h	4 h	6 h	1 h	2 h	4 h	6 h
VE-cadherin	0.77±0.58	0.96±0.42	0.63±0.24	0.66±0.37	0.42±0.15	0.61±0.15	0.47±0.20	0.75±0.37
Claudin-5	1.15±0.34	1.33±0.09	1.33±0.15	1.06±0.11	0.80±0.01	1.22±0.24	1.28±0.09	1.10±0.18
Caveolin-1	0.44±0.12	0.77±0.30	0.77±0.25	0.67±0.53	0.50±0.06	0.61±0.15	0.69±0.12	0.98±0.35

表2 机械牵张对HPAEC细胞VE-cadherin、Claudin-5和Caveolin-1 mRNA表达的影响（±s）

关键蛋白	10%机械牵张				20%机械牵张
关键蛋白	1 h	2 h	4 h	6 h	1 h	2 h	4 h	6 h
VE-cadherin	0.77±0.58	0.96±0.42	0.63±0.24	0.66±0.37	0.42±0.15	0.61±0.15	0.47±0.20	0.75±0.37
Claudin-5	1.15±0.34	1.33±0.09	1.33±0.15	1.06±0.11	0.80±0.01	1.22±0.24	1.28±0.09	1.10±0.18
Caveolin-1	0.44±0.12	0.77±0.30	0.77±0.25	0.67±0.53	0.50±0.06	0.61±0.15	0.69±0.12	0.98±0.35

图4 机械牵张对HPMVEC通透性关键蛋白VE-cadherin、Claudin-5和Caveolin-1 mRNA表达的影响

表3 机械牵张对HPMVEC细胞VE-cadherin、Claudin-5和Caveolin-1 mRNA表达的影响（±s）

关键蛋白	10%机械牵张				20%机械牵张
关键蛋白	1 h	2 h	4 h	6 h	1 h	2 h	4 h	6 h
VE-cadherin	1.10±0.09	0.76±0.12	0.98±0.20	0.88±0.02	1.15±0.13	0.55±0.03	0.54±0.02	0.94±0.18
Claudin-5	0.88±0.17	1.43±0.21	1.17±0.18	1.21±0.15	1.25±0.21	1.28±0.15	1.14±0.20	1.25±0.13
Caveolin-1	0.72±0.06	0.82±0.10	0.87±0.21	0.81±0.10	0.68±0.05	0.74±0.05	0.74±0.02	1.07±0.04

表3 机械牵张对HPMVEC细胞VE-cadherin、Claudin-5和Caveolin-1 mRNA表达的影响（±s）

关键蛋白	10%机械牵张				20%机械牵张
关键蛋白	1 h	2 h	4 h	6 h	1 h	2 h	4 h	6 h
VE-cadherin	1.10±0.09	0.76±0.12	0.98±0.20	0.88±0.02	1.15±0.13	0.55±0.03	0.54±0.02	0.94±0.18
Claudin-5	0.88±0.17	1.43±0.21	1.17±0.18	1.21±0.15	1.25±0.21	1.28±0.15	1.14±0.20	1.25±0.13
Caveolin-1	0.72±0.06	0.82±0.10	0.87±0.21	0.81±0.10	0.68±0.05	0.74±0.05	0.74±0.02	1.07±0.04

图5 机械牵张对HPAEC通透性关键蛋白VE-cadherin、Claudin-5和Caveolin-1表达的影响

表4 机械牵张对HPAEC细胞VE-cadherin、Claudin-5和Caveolin-1蛋白表达的影响（±s）

关键蛋白	10%机械牵张				20%机械牵张
关键蛋白	3 h	6 h	12 h	24 h	3 h	6 h	12 h	24 h
VE-cadherin	0.84±0.27	0.86±0.05	1.00±0.16	0.93±0.24	0.72±0.25	0.58±0.12	0.49±0.16	0.51±0.09
Claudin-5	0.61±0.17	0.82±0.47	0.88±0.17	0.90±0.19	0.84±0.13	0.71±0.11	0.40±0.24	0.60±0.15
Caveolin-1	0.70±0.17	0.67±0.28	0.82±0.21	0.91±0.15	0.82±0.32	0.91±0.23	0.75±0.12	0.80±0.09

表4 机械牵张对HPAEC细胞VE-cadherin、Claudin-5和Caveolin-1蛋白表达的影响（±s）

关键蛋白	10%机械牵张				20%机械牵张
关键蛋白	3 h	6 h	12 h	24 h	3 h	6 h	12 h	24 h
VE-cadherin	0.84±0.27	0.86±0.05	1.00±0.16	0.93±0.24	0.72±0.25	0.58±0.12	0.49±0.16	0.51±0.09
Claudin-5	0.61±0.17	0.82±0.47	0.88±0.17	0.90±0.19	0.84±0.13	0.71±0.11	0.40±0.24	0.60±0.15
Caveolin-1	0.70±0.17	0.67±0.28	0.82±0.21	0.91±0.15	0.82±0.32	0.91±0.23	0.75±0.12	0.80±0.09

图6 机械牵张对HPMVEC通透性关键蛋白VE-cadherin、Claudin-5和Caveolin-1蛋白表达的影响

表5 机械牵张对HPMVEC细胞VE-cadherin、Claudin-5和Caveolin-1蛋白表达的影响（±s）

关键蛋白	10%机械牵张				20%机械牵张
关键蛋白	3 h	6 h	12 h	24 h	3 h	6 h	12 h	24 h
VE-cadherin	0.80±0.25	0.68±0.27	0.68±0.28	0.74±0.17	0.83±0.19	0.91±0.21	0.89±0.38	0.39±0.23
Claudin-5	0.89±0.16	0.74±0.09	0.66±0.22	1.12±0.54	0.97±0.26	0.88±0.23	0.88±0.28	0.68±0.48
Caveolin-1	0.91±0.12	0.93±0.23	0.82±0.10	0.83±0.14	0.86±0.21	0.88±0.29	0.85±0.27	0.54±0.12

表5 机械牵张对HPMVEC细胞VE-cadherin、Claudin-5和Caveolin-1蛋白表达的影响（±s）

关键蛋白	10%机械牵张				20%机械牵张
关键蛋白	3 h	6 h	12 h	24 h	3 h	6 h	12 h	24 h
VE-cadherin	0.80±0.25	0.68±0.27	0.68±0.28	0.74±0.17	0.83±0.19	0.91±0.21	0.89±0.38	0.39±0.23
Claudin-5	0.89±0.16	0.74±0.09	0.66±0.22	1.12±0.54	0.97±0.26	0.88±0.23	0.88±0.28	0.68±0.48
Caveolin-1	0.91±0.12	0.93±0.23	0.82±0.10	0.83±0.14	0.86±0.21	0.88±0.29	0.85±0.27	0.54±0.12

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Effect of mechanical stretch on permeability of human pulmonary vascular endothelial cells and underlying molecular mechanism

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