Abstract：This paper aims to the research of the impact of fluid shear stress on the adhesion between vascular endothelial cells and leukocyte induced by tumor necrosis factor-α(TNF-α) by microfliudic chip technology. Microfluidic chip was fabricated by soft lithograph; Endothelial microfluidic chip was constructed by optimizing types of the extracellular matrix proteins modified in the microchannel and cell incubation time; human umbilical vein endothelial cells EA.Hy926 lined in the microchannel were exposed to fluid shear stress of 1.68 dynes/cm2 and 8.4 dynes/cm2 respectively. Meanwhile, adhesion between EA.Hy926 cells and leukocyte was induced by TNF-αunder a flow condition. EA. Hy926 cell cultured in the static condition was used as control group. The numbers of fluorescently-labeled leukocyte in microchannel were counted to quantize the adhesion level between EA. Hy926 cells and leukocyte; cell immunofluorescence technique was used to detect the intercellular adhesion molecule (ICAM-1) expression. The constructed endothelial microfluidic chip can afford to the fluid shear stress and respond to exogenous stimulus of TNF-α; compared with the adhesion numbers of leukocyte in control group, adhesion between EA. Hy926 cells exposed to low fluid shear stress and leukocyte was reduced under the stimulus of TNF-α at a concentration of 10 ng/ml(P<0.05); leukocyte adhesion with EA. Hy926 cells exposed to high fluid shear stress was reduced significantly than EA. Hy926 cells in control group and EA.1Hy926 cells exposed to low fluid shear stress(P<0.01); the regulation mechanism of fluid shear stress to the adhesion between EA. Hy926 cells and leukocyte induced by TNF-αwas through the way of ICAM-1. The endothelial microfluidic chip fabricated in this paper could be used to study the functions of endothelial cell in vitro and provide a new technical platform for exploring the pathophysiology of the related cardiovascular system diseases under a flow environment.
. [J]. 中国生物医学工程学报(英文版）, 2015, 23(4): 147-159.
LI Yuan, YANG De-yu, LIAO Juan, GONG Fang, HE Ping, LIU Bei-zhong. Analysis of TNF-α-induced Leukocyte Adhesion to Vascular Endothelial Cells Regulated by Fluid Shear Stress Using Microfluidic Chip-based Technology. Chinese Journal of Biomedical Engineering, 2015, 23(4): 147-159.
[1] Ku DN, Giddens DP, Zarins CK, et al.Pulsatile flow and arherosclerosis in human carotid bifurcation: Positive correlation between plaque location and low and oscillating shear stress[J]. Arteriosclerosis, 1985, 5(3):293-302. [2] Springer TA.Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration[J]. Annal Review of Physiology, 1995, 57:827-872. [3] Luu NT, Rahman M, Stone PC, et al.Responses of endothelial cells from different vessels to inflammatory cytokines and shear stress: evidence for the pliability of endothelial phenotype[J]. Journal of Vascular Research, 2010, 47(5):451-461. [4] Xu H, Gao X, Song J, et al.Peoniflorin prevents the adhesion between inflammatory endothelial cells and leukocytes through inhibiting the activation of MAPKs and NF-κB[J]. Drug Developmerot Research, 2010, 71(5): 275-284. [5] Brown DC, Larson RS.Improvements to parallel plate flow chambers to reduce reagent and cellular requirements[J]. BMC Immunology, 2001, 2:9. [6] Passerini AG, Milsted A, Rittgers SE.Shear stress magnitude and directionality modulate growth factor gene expression in preconditioned vascular endothelial cells[J]. Journal of Vascular Research, 2003, 37(1):182-190. [7] McCormick SM, Eskin SG, McIntire LV, et al. DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(16): 8955-8960. [8] Song JW, Cavnar SP, Walker AC, et al.Microfluidic endothelium for studying the intravascular adhesion of metastatic breast cancer cells[J]. PLoS One, 2009, 4(6): e5756-e5766. [9] Ku CJ, DAmico Oblak T, Spence DM.Interactions between multiple cell types in parallel microfluidic channels:monitoring platelet adhesion to an endothelium in the presence of an anti-adhesion drug[J]. Analytical Chemistry, 2008, 80(19):7543-7548. [10] Rouleau L, Copland IB, Tardif JC, et al.Neutrophil adhesion on endothelial cells in a novel asymmetric stenosis model: effect of wall shear stress gradients[J]. Annals of Biomedical Engineering, 2010, 38(9):2791-2804. [11] Xia Y, Whitesides G M.Soft lithography[J]. Annual Review of Materials Science, 1998, 28(1): 153-184. [12] Prabhakarpandian B, Shen MC, Pant K, et al.Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature[J]. Microvascular Research, 2011,82(3):210-220. [13] Ahn K, Pan S, Beningo K, et al.A permanent human cell line (EA.hy926) preserves the characteristics of endothelin converting enzyme from primary human umbilical vein endothelial cells[J]. Life Sciences,1995, 56(26): 2331-2341. [14] Stepanova OI, L'vova TY, Furaeva KN, et al. Effect of placenta secretory products on migration activity of endothelial EA.Hy926 cells[J]. Bulletin of Experimental Biology and Medicine, 2013, 156(1):156-160. [15] Tanaka Y, Kikukawa Y, Sato K, et al.Culture and leukocyte adhesion assay of human arterial endothelial cells in a glass microchip[J]. Analytical Sciences, 2007, 23(3):261-266. [16] Wang Z, Zhang J, Li B, et al.Resveratrol ameliorates low shear stressinduced oxidative stress by suppressing ERK/eNOSThr495 inendothelial cells[J].Molecular Medicine Reports, 2014, 10(4):1964-1972. [17] Zhang J, Morton H.Friedman. Adaptive response of vascular endothelial cells to an acute increase in shear stress magnitude[J]. American Journal of Physiology-Heart and Circulatory Physiology, 2012, 302(4): H983-H991. [18] Shi Pan.Molecular Mechanisms Responsible for the Atheroprotective Effects of Laminar Shear Stress[J]. Antioxid Redox Signal, 2009, 11(7): 1669-1682. [19] Cicha I, Beronov K, Lopez Ramirez E, et al.Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-α and monocytic cell recruitment in a simplified model of arterial bifurcations[J]. Atherosclerosis,2009, 207(1):93-102. [20] Sawa Y, Sugimoto Y, Ueki T, et al.Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium[J]. Journal of Histochemistry & Cytochemistry, 2007, 55(7):721-733. [21] Sheikh S, Rainger GE, Gale Z, et al.Exposure to fluid shear stress modulates the ability of endothelial cells to recruit neutrophils in response to tumor necrosis factor-alpha: a basis for local variations in vascular sensitivity to inflammation[J]. Blood, 2003, 102(8):2828-2834. [22] Yamawaki H, Pan S, Lee RT.Berk BC Fluid shear stress inhibits vascular inflammation by decreasing thioredoxin-interacting protein in endothelial cells[J]. Journal of the Clinical Investigation, 2005, 115(3):733-738. [23] Song X, Zeng Y, Yu H, et al.The effect of fluid shear stress on ICAM-1 expression of rat brain microvascular endothelial cells[J]. Technology and Health Care, 2001,9(3):287-293. [24] Ando J, Tsuboi H, Korenaga R, et al.Shear stress inhibits adhesion of cultured mouse endothelial cells to lymphocytes by downregulating VCAM-1 expression[J]. American Journal of Physiology, 1994, 267(3 Pt 1):C679-C687. [25] Huang RB, Gonzalez AL, Eniola-Adefeso O.Laminar shear stress elicit distinct endothelial cell E-selectin expression pattern via TNFα and IL-1β activation[J]. Biotechnology and Bioengineering, 2013, 110(3):999-1003.
