| Objective To investigate the effects of fluid shear stress on IL-8 gene in human umbilical vein endothelial cells (HUVECs) and the roles of time course of low shear stress and intensity of fluid shear stress using LightCycler?system;To investigate the gene expression profiles in HUVECs exposed on low shear stress (4.20 dyne/cm2,2 h) and incubated by 17 P -estradiol (10-7 M) + low shear stress (4.20 dyne/cm2,2 h) using the cDNA microarray approach.Methods Endothelial cells were isolated from human umbilical cord veins by collagenase treatment as described by Jaffe and modified. The cells were grown in Ml99 medium and the cells used were mainly prior to passage 3 or 4. Cell culture was maintained in a humidified 5 %/95 % air incubator at 37 . A flow system was established according to the design previously described. The fluid used to shear HUVECs was the cultured medium. Laminar shear stresses were generated by a peristaltic pump. Cells from static controls or from shear stress experiments were washed twice with ice-cold PBS,and total cellular RNA was isolated by TRIzol reagent according to manufacturer's manual. RNA quality was insured by gel visualization and spectrophotometric analysis (OD26o/OD28o). Quantitative RT-PCR assay involves LightCycler?technology. Experimental protocol were performed by instruction manual of LightCycler-RNA Amplification Kit SYBR Green I. Human named genes HGEC-40S were used for differential expression screening. The PCR products of 4 096 genes were spotted on a chemical-material-coated-glass plates in array. The DNAs were then fixed on the glass plate by a serial of treatments. Total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP and total RNA of HUVECs from the paired shear stressed experiment was labeled by Cy5-dCTP. The arrays were then hybridized against the cDNA probe mixture and the fluorescent signals were scanned. The expression ratios reported are the average from the two separate experiments. (1) HUVECs exposed to fluid shear stress (4.20 dyne/cm2) for 1 h,2 h,3 h,4 h,5 h,6 h,7 h,8 h,9 h,10 h,or 12 h,respectively. Normal static cultured HUVECs were selected as a control. Quantitative reversal transcription- polymerase chain reaction (qRT-PCR)using LightCycler?system was employed to assay the expression of IL-8 mRNA. (2) HUVECs exposed to low fluid shear stresses (2.23 dyne/cm2,4.20 dyne/cm2,or 6.08 dyne/cm2) for 1 h,2 h,3 h,4 h,6 h,8 h,10 h,or 12 h,respectively. Normal static cultured HUVECs were selected as a control. Quantitative reversal transcription- polymerase chain reaction (qRT-PCR) using LightCycler?system was employed to assay the expression of IL-8 mRNA. (3) HUVECs exposed to different fluid shear stresses (2.23 dyne/cm2,4.20 dyne/cm2,6.08 dyne/cm2,8.19 dyne/cm2,9.67 dyne/cm2,12.15 dyne/cm2,14.40 dyne/cm2,16.87 dyne/cm2,19.29 dyne/cm2,respectively) for 1 h or 2 h. Normal static cultured HUVECs were selected as a control. Quantitative reversal transcription-polymerase chain reaction (qRT-PCR) using LightCycler?system was employed to assay the expression of IL-8 mRNA. (4) HUVECs exposed to low fluid shear stress (4.20 dyne/cm") for 2 h and incubated by 17 P -estradiol (10-7 M) + low shear stress (4.20 dyne/cm2,2 h). Normal static cultured HUVECs were selected as a control. The complementary DNA microarray approach was used to assay the differential gene expression in endothelial cells.Results (1) EL-8 mRNA did not express in HUVECs untreated with fluid shear stress. The baseline value was 5.261 X 104 copies. IL-8 mRNA expression increased (2.413 X 108 copies) when HUVECs exposed to fluid shear stress for 1 h,and it reached the summit (6.642 X 108 copies) when HUVECs exposed to fluid shear stress for 2 h. Then IL-8 mRNA expression gradually decreased (5.590X 107 copies) at 3 h of stimulation by shear stress and remained at a constant level (3.853 X 107-1.735 X107 copies for 4-6 h and 9.325 X 106-3.812X 106 copies for 7-12 h) throughout the time course of the study. The increase of IL-8 mRNA expression by shear stress was time-depen...
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