Assessment of the role of TLR-4 in shear-stress-induced IL-8 gene transcription activation in vascular endothelial cells by gene mutation and gene transfection technology.
- Author:
Feng LIANG
1
;
Ning HUANG
;
Boyao WANG
;
Huaiqing CHEN
Author Information
1. Research Unit of Infection and Immunity, West China Medical Center, Sichuan University, Chengdu 610041.
- Publication Type:Journal Article
- MeSH:
Cells, Cultured;
Endothelium, Vascular;
cytology;
physiology;
Gene Expression Regulation;
Humans;
Interleukin-8;
genetics;
Membrane Glycoproteins;
genetics;
physiology;
Mutation;
Receptors, Cell Surface;
genetics;
physiology;
Stress, Mechanical;
Toll-Like Receptor 4;
Toll-Like Receptors;
Transcription, Genetic;
physiology;
Transfection;
Umbilical Veins;
cytology
- From:
Journal of Biomedical Engineering
2002;19(4):667-672
- CountryChina
- Language:Chinese
-
Abstract:
This study is to evaluate the role of TLR-4 in lower laminar shear stress-induced interleukin-8 gene transcription activation in vascular endothelial cells by using gene mutation and transfection techniques. RT-PCR, Northern hybridization and immunocytochemical fluorescent staining showed that TLR-4 was expressed in human umbilical vein endothelial cells(HUVEC). When stimulated with 4.2 dyne/cm2 shear stress for 1 hour, an increase of TLR-4 mRNA expression was observed in HUVECs detected by RT-PCR and Northern hybridization. The intracellular domain deletion mutant TLR-4 cDNA (lacking the 155 COOH terminal amino acids of the wild type (TLR-4) and -102 -61 bp DNA sequence in 5'-flanking region of IL-8 gene (IL-8USCS) were isolated from endothelial cells by RT-PCR and PCR. These two DNA fragments were cloned into pcDNA3 and pEGFP1 respectively to construct TLR-4 dominant negative mutant pcDNA3-mTLR4 and IL-8 reporter gene pEGFP1-IL8USCS. ECV304 cells were transfected with the pEGFP1-IL8USCS or co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 by Dosper liposome transfectional reagent and selected by G418, then stimulated with 4.2 dyne/cm2 shear stress for 3 hours. Flow cytometric analysis showed that when exposed to 4.2 dyne/cm2 shear stress for 3 hours, there was a marked increase in the green fluorescent protein expression in pEGFP1-IL8USCS-transfected ECV304 cells. In contrast, there was almost no change in the green fluorescent protein expression in the cells co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 after the stimulation, suggesting the TLR-4 mutant depressed TLR-4 signaling. These experiments suggest that the inflammatory TLR-4/NF-kappa B signaling pathway would probably be involved in flow shear stress-induced IL-8 gene expression in human vascular endothelial cells.
