Modulation of cardiomyocyte gene expression by n-3 PUFA supplementation

Despite the recognized protective effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in cardiovascular diseases, and the demonstration of the control of gene expression by polyunsaturated fatty acids (PUFAs), the effects of these n-3 fatty acids on the whole genoma has never been investigated in cardiac cells. Using rat arrays, the effects of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) supplementation on the global gene expression profile were evaluated in cultured neonatal rat cardiomyocytes. Keywords: nutritional intervention, treatment response Primary cardiomyocyte cultures were obtained from the ventricles of newborn Wistar rats and grown in HAM F10 plus 10% fetal calf serum and 10% horse serum medium (controls), or in the same medium supplemented with 60 uM Eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Media were changed every 48 hrs; cells were grown until complete confluence, then they were scraped off in ice cold PBS, and RNA isolation, labeling of complementary RNA (cRNA), hybridization to Agilent 22K-gene arrays (Rat oligo array G4130A) and assessment of expression ratios were performed. About one million cells treated with RNAlater were homogenized and total RNA was extracted by column technology (Rneasy Protect mini kit) and analyzed on both a spectrophotometer and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Only samples with 28S/18S ratio of >2.0 and no evidence of ribosomal peak degradation were included. The cRNA was generated by in vitro transcription with the use of T7 RNA polymerase (Low RNA input fluorescent linear amplification kit) and labeled with Cy3-CTP or Cy5-CTP. Direct comparisons were performed between n-3 PUFAs supplemented cells versus unsupplemented ones (controls); each analysis was replicated swapping the labeling with the two cyanine dyes.

尽管二十碳五烯酸（eicosapentaenoic acid, EPA）与二十二碳六烯酸（docosahexaenoic acid, DHA）在心血管疾病中的保护作用已获公认，且多不饱和脂肪酸（polyunsaturated fatty acids, PUFAs）可调控基因表达的现象已被证实，但目前尚无研究探索这类n-3脂肪酸对心肌细胞全基因组的影响。本研究采用大鼠基因芯片，探究了添加二十碳五烯酸（EPA）与二十二碳六烯酸（DHA）对体外培养的新生大鼠心肌细胞整体基因表达谱的影响。
关键词：营养干预、治疗应答
原代心肌细胞培养物取自新生Wistar大鼠的心室，分别于HAM F10培养基（添加10%胎牛血清与10%马血清，作为对照组），或同培养基中添加60 μM二十碳五烯酸（EPA）或二十二碳六烯酸（DHA）的环境中培养。每48小时更换一次培养基；待细胞完全汇合后，用冰预冷的PBS刮取细胞，随后进行RNA提取、互补RNA（complementary RNA, cRNA）标记、与安捷伦22K基因芯片（大鼠寡核苷酸芯片G4130A）杂交，以及表达比值分析。
使用RNAlater处理的约100万个细胞经匀浆后，采用柱式技术（Rneasy Protect mini kit）提取总RNA，并通过分光光度计与安捷伦2100生物分析仪（Agilent Technologies, 美国加利福尼亚州帕洛阿尔托）进行质量检测。仅纳入28S/18S比值大于2.0且无核糖体峰降解迹象的样本。
采用T7 RNA聚合酶（Low RNA input荧光线性扩增试剂盒）进行体外转录以合成cRNA，并使用Cy3-CTP或Cy5-CTP进行标记。将添加n-3多不饱和脂肪酸的细胞与未添加的对照组细胞进行直接比较；每次分析均通过交换两种菁染料的标记方式进行重复实验。