qPCR array检测高糖对胆固醇合成基因表达的影响

目的:高血糖易引起胆固醇在体内积聚,增加糖尿病合并动脉粥样硬化性心血管疾病的患病风险。本文通过建立稳定的实时定量PCR芯片(Real-time quantitative polymerasechain reaction array,qPCR array)检测方案,研究高糖对小鼠肝癌细胞Hepa1-6胆固醇合成基因表达的影响,探讨胆固醇合成基因在糖尿病大血管并发症发展中的作用机制。方法:以不同浓度葡萄糖(5、15、30mmo/L)和不同时间(0、6、12、18、24 h),刺激肝癌细胞Hepa1-6,利用qPCR array检测其胆固醇合成基因的表达差异。结果:与5mmol/L相比,高糖组(15、30 mmo/L)处理细胞18 h后,胆固醇合成基因CYP51、EBP、NSDHL、SQLE、FDFT1和PMVK的表达上调(P0.05),呈现剂量依赖性。与0 h相比,15 mmol/L高糖处理细胞12 h,CYP51、EBP和SQLE mRNA表达量上调(P0.01)。至24 h,CYP51、EBP降至0 h水平,而SQLE的表达量继续增加;NSDHL在12 h表达无差异,至18 h表达量发生上调(P0.05)。结论:该qPCR array检测方案能特异性检测胆固醇合成基因的表达量。高糖能够促进胆固醇合成基因的表达,使细胞内胆固醇积聚,这可能是糖尿病患者容易发生动脉粥样硬化的原因。这提示我们将胆固醇合成基因作为药物靶点可能延缓糖尿病动脉粥样硬化进展。

Effect of High Glucose on the Expression of Cholesterol Biosynthesis Geneby qPCR Array

Objective:An elevation in blood glucose concentration leads to increased risk of developing diabetes-associatedatherosclerotic cardiovascular disease due to an excessive accumulation of cholesterol. To investigate the effect of high glucose on theexpression of cholesterol biosynthesis gene in mouse hepatoma cells (Hepa1-6), a stable and accurate real-time quantitative polymerasechain reaction array method was established in order to inquire the mechanism of cholesterol biosynthesis gene on diabeticatherosclerosis.Methods:Hepa1-6 cells were cultured with different concentrations (5, 15 and 30 mmol/L) of glucose and different time(0, 6, 12, 18 and 24 h). qPCR array method was used to examine the mRNA expression of cholesterol biosynthesis gene.Results:Highglucose (15, 30 mmo/L) can up-regulate the mRNA levels of CYP51, EBP, NSDHL, SQLE, FDFT1 and PMVK in aconcentration-dependent manner compared to control cells cultured with 5 mmol/L glucose (P<0.05). As compared to control (0 h), themRNA levels of CYP51, EBP and SQLE increased by 15 mmol/L glucose in 12 h. However, CYP51, EBP in the cells treated for 24 hdecreased to approximately control group, but SQLE continued to increase (P<0.01). High glucose did not affect the mRNA levels ofNSDHL in 12 h, but NSDHL expression was increased in 18 h (P<0.05).Conclusion:qPCR array can specifically detect the cholesterolbiosynthetic gene expression. Hyperglycemia may promote the accumulation of the cholesterol for the improvement of gene expression inHepa1-6. The reason may be that more people on diabetes have atherosclerosis than normal. Cholesterol biosynthesis gene which wasregarded as the targets of drugs can delay the process of diabetic atherosclerosis.