寡聚核苷酸微阵列技术应用于脉动流剪应力作用下人体动脉平滑肌细胞基因差异表达的研究  

Application of Oligo-microarray in an In Vitro Study of the Effects of Pulsatile Fluid Shear Stress on Gene Expression of Human Smooth Muscle Cells

作　　者：田佳[1] 樊瑜波[1] 陈君楷[1] 

机构地区：[1]四川大学生物医学工程实验室

出　　处：《生物医学工程学杂志》2004年第2期208-211,共4页Journal of Biomedical Engineering

基　　金：国家自然科学基金重点资助项目 (10 13 2 0 2 0 );教育部跨世纪优秀人才计划

摘　　要：采用本实验室自行设计和研制的 Flow Cham ber血液流动剪切装置 ,从细胞生物力学的角度 ,采用寡聚核苷酸微阵列技术 ,探讨人体脐动脉平滑肌细胞 (VSMC)在脉动流剪应力作用下的基因差异性表达 ,以及相关基因表达的调控 ;比较脉动流和定常流两种剪应力作用下 VSMC基因表达的差异。通过提取细胞总 RNA ,逆转录合成单链 c DNA,双链 c DNA,体外转录合成生物素标记的 c DNA与基因进行芯片杂交 ,经抗体的检测标记荧光染料Cy3,用基因芯片扫描仪进行基因芯片的图像扫描。结果发现 ,与对照组比较 ,经脉动流剪应力作用后 ,血管平滑肌细胞有 1330个基因出现差异表达 ;经定常流剪应力作用后 ,与对照组比较 ,有 2 6 76个基因出现差异表达 ;比较定常流和脉动流两组的血管平滑肌细胞基因 ,有 2 2 97个基因存在表达差异。实验表明 ,近生理状态的脉动流与定常流两种剪应力 ,对体外培养的人体血管平滑肌细胞的基因表达 ,在数量、范围和调控水平上有着很大的差异。提示在生理或病理情况下 ,血液流体力学的变化 ,可诱导血管平滑肌细胞基因在 m RNA表达水平出现不同的应力响应。We assessed the effects of pulsatile flow shear stress on the gene expression profiles of human umbilical artery smooth muscle cells (HUASMCs) in vitro using the Express Chip TM DNA microarray method and investigated the difference between pulsatile and steady shear stress on differentially expressed genes of HUASMCs. In a modified pulsatile flow chamber system, HUASMCs were exposed to pulsatile and steady fluid shear stress (5.52 dyne/cm 2) for 6 h respectively, and normal static cultured HUASMCs were selected as a control. The total cellular RNA was extracted by TRIzol Reagent (Life Technologies, Inc) according to the manufacturer's manual. Conversion of mRNA to single strand cDNA and double strand cDNA template was synthesized by Reverse Transcription from the total RNA. cRNA probe was transcribed with biotin labeling. After hybridization of probe with microarray, the binding of streptavidin to biotin was performed and amplified with the first antibody and further amplified with Cy3 conjugated second antibody. Then detection of Cy3 dye was carried out with ScanArray 5000. The results showed that a total of 1,330 genes revealed differential expression in HUASMCs exposed on pulsatile shear stress (5.52 dyne/cm 2, 6 h); however, 2,676 genes revealed differential expression in HUASMCs exposed on steady shear stress.Comparsion of HUASMCs exposed to pulsatile with the HUASMCs exposed to steady shear stress showed there were 2,297 genes revealing differential expression. The transcriptional profile of fluidally induced genes in HUASMCs suggested a different response to pulsatile and steady shear stress.

关 键 词：寡聚核苷酸 微阵列技术 人体动脉 动脉平滑肌细胞 基因差异 基因表达 脉动流剪应力 基因芯片 

分 类 号：R318.01[医药卫生—生物医学工程]