| Objective To investigate the effects of pulsatile flow shear stress on the gene expression profiles of human umbilical artery smooth muscle cells (HUASMCs) in vitro by using the ExpressChipæ© NA microarray method and the role of HUASMCs in the reconstruction to suit shear stress; to investigate the difference between pulsatile and steady flow shear stress on differentially expressed genes of HUASMCs ; and to study the biological characteristics of HUASMCs cultured in vitro.Methods Smooth muscle cells were isolated from human umbilical cord arteries, as described by Campbell, and modified. The cells were grown in MCDB131 medium and the cells used were mainly prior to passage 2 or 3. Cell culture was maintained in a humidified 5% / 95% air incubator at 37"C. A modified flow chamber system, which was driven by a straight motor and a peristaltic pump respectively, can accurately adjust the drive force, frequency and pressure, and the temperature of the system to generate pulsatile and steady fluid shear stress. The fluid used to shear HUASMCs was the cultured medium. HUASMCs from pulsatile and steady shear stress experiment or from static control were washed a couple of times with B solution, and the total cellular RNA was extracted by TRIzol Reagent (Life Technologies, Inc) according to the manufacturer's manual. RNA quality was insured by gel visualization and spectrophotometric analysis (OD26o/OD28o). Conversion of mRNA to single stand cDNA and double stand cDNA template was synthesized by Reverse Transcription from the total RNA. cRNA probe was transcribed with biotin labeling. After hybridization of probe with microarray, binding of streptavidin to biotin was performed to be amplificated with the first antibody, and further amplificated with Cy3-conjugated second antibody. Then1 This work is supported by th National Nature Science Foudation (Grant No.10132020).detection of Cy3 dye was carried with ScanArray 5000. (a) HUASMCs were exposed to pulsatile fluid shear stress (5.52 dyne/cm2) for 6h and normal static cultured HUASMCs were selected as a control. ExpressChipæ© NA microarray system was employed to assay the differential gene expression in vascular smooth muscle cells induced by pulsatile fluid, (b) HUASMCs were exposed to steady fluid shear stress (4.60 dyne/cm2) for 6h and normal static cultured HUASMCs were selected as a control. The ExpressChipæ© NA microarray system was employed to assay the differential gene expression in vascular smooth muscle cells induced by steady fluid. Results (1) Highly purified primary HUASMCs were gained and can be propagated about 10 population doublings(PD). After PD 4, the cells lost the proliferation ability gradually. The effect of MCDB131 medium with 10~20%(V/V)newborn calf serum (fetal bovine serum) on the proliferation of primary culture and subculture of HUASMCs was greater than that of other cultured mediums. Umbilical artery smooth muscle cells have been used as an economical alternative for the study of morphological and structural changes in the development of atherosclerosis1121. Identification of smooth muscle cells was shown specific a -actin expression by immunohistochemistry from an explanted culture. (2) After bioinformatics analysis, we identified a total of 1,330 genes (approximately 10.39%) which revealed differential expression, 669 genes were up-regulated and 661 were down-regulated in HUASMCs exposed on pulsatile shear stress (5.52 dyne/cm2, 6 h); however, a total of genes (approximately 20.91%) revealed differential expression, 1,660 genes were up-regulated and 1,610 were down-regulated in HUASMCs exposed on steady shear stress (5.52 dyne/cm2, 6 h). Conclusion (1) Highly purified primary HUASMCs were gained and the MCDB131 medium with 10~20%(V/V) newborn calf serum (fetal bovine serum) on the proliferation of primary cultures of HUASMCs was greatest in the cultured mediums. (2) There are 1,330 genes of 12,800 revealed differential expressions (^2-fold or 22-fold) in HUASMCs exposed on pulsatile shear stress (5.52 dyne/cm2, 6 h) and fr...
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