Effects of glucose transporter expression on VSMC. Rattus norvegicus

Hypothesis: Overexpression of the GLUT1 facilitative glucose transporter, in A7r5 vascular smooth muscle cells, is sufficient and/or necessary to induce alterations in gene expression which influence apoptosis, growth, and proliferation. Overall design: Scientific Approach: A7r5 rat embryonic vascular smooth muscle cells (VSMCs) (ATCC, CRL-1444) were infected for 72 hours with adenoviral vectors containing human GLUT1 or empty vector controls at a multiplicity of infection of 10 (MOI of 1 = 100 particles/cell) and grown in both high glucose (25mM) and low glucose (5.5mM) media. The human GLUT1 cDNA adenoviral vector and empty vector controls were a gift from Dr Arno Kumagai (University of Michigan). Total RNA was isolated using TRIzol (Invitrogen Life Technologies, Carlsbad, CA), followed by extraction with phenol/chloroform until the interface was clear. Total RNA was further purified using Qiagen RNeasy Mini Kit (Qiagen 74106). Immunoblot analysis was conducted to confirm GLUT1 overexpression at the time of Total RNA isolation (72 hours post-infection) for each condition. Number of chips: Total RNA was isolated from each of 2 conditions (control cells in low glucose (5.5mM), and GLUT1 overexpressing cells in low glucose), on three separate days and pooled to obtain one sample for each condition. In total we submitted two samples.

研究假说：在A7r5血管平滑肌细胞中，易化型葡萄糖转运蛋白1（GLUT1 facilitative glucose transporter）的过表达，足以且/或必要诱导影响细胞凋亡、生长与增殖的基因表达改变。
实验设计：
科学研究方案：将A7r5大鼠胚胎血管平滑肌细胞（vascular smooth muscle cells，简称VSMCs，ATCC，CRL-1444）以感染复数（multiplicity of infection，简称MOI）为10（MOI=1即每细胞100个病毒颗粒）的腺病毒载体（携带人源GLUT1或空载体对照）感染72小时，随后分别置于高糖（25mM）与低糖（5.5mM）培养基中培养。本研究所用的人GLUT1 cDNA腺病毒载体及空载体对照由密歇根大学Arno Kumagai博士惠赠。
采用TRIzol试剂（Invitrogen生命科技公司，加利福尼亚州卡尔斯巴德）分离总RNA，随后经酚-氯仿抽提直至界面澄清；再使用Qiagen RNeasy Mini试剂盒（Qiagen 74106）对总RNA进行进一步纯化。
在各实验组总RNA分离的时刻（即感染后72小时），通过免疫印迹分析验证GLUT1的过表达水平。
芯片实验样本设计：分别在三个独立实验日内，从两种实验条件（低糖培养的对照细胞、低糖培养的GLUT1过表达细胞）中分离总RNA并混合，以制备得到每种条件的一份样本；最终共提交两份样本。