内皮型一氧化氮合酶基因敲除小鼠心肌细胞对苯福林诱导的过度增殖反应

Phenylephrine-induced excessive hypertrophy responses in cardiomyocytes from endothelial nitric oxide synthase gene knockout mice

目的：通过RNA定量检测和细胞面积测定两种方法，观察内皮型一氧化氮合酶基因敲除与野生型新生小鼠的离体心肌细胞对心肌肥大诱导因子反应性的差异。方法：实验于2007-08在顺德职业技术学院医学系实验研究中心完成。①实验材料：出生1～3d C57BL／6品系内皮型一氧化氮合酶基因敲除小鼠和野生型小鼠，雌雄不拘。②实验方法：对小鼠心肌细胞及成纤维细胞进行培养，72h后分为实验组和对照组，将肾上腺素能受体激动剂苯福林掺入培养心肌细胞，另设空白对照组：在基础培养液中加入与实验组等量的DMEM。③实验评估：采用DNA、RNA定量检测、细胞面积测定方法，在细胞水平观察内皮型一氧化氮合酶基因敲除小鼠和野生型小鼠心肌细胞增殖、肥大反应之间的差异。结果：①培养的心肌细胞在加入苯福林72h后于显微镜下观察两组细胞均呈椭圆形、长菱形。②与对照组相比，实验组心肌细胞与成纤维细胞RNA检测及荧光值均增高（P〈0．05）；空白对照组心肌细胞与成纤维细胞RNA检测荧光值均较实验组和对照组低（P〈0．05）。③与空白对照组相比，实验组和对照组心肌细胞与成纤维细胞面积荧光值均增高（P〈0．05）；其中实验组心肌细胞面积荧光值较对照组高（P〈0．05）。结论：内皮型一氧化氮合酶基因敲除小鼠其离体心肌细胞在心肌肥大诱导因子刺激下产生过度增殖反应。

AIM： The study aimed to observe the difference in hypertrophy responses in cardiomyocytes of cultured neonatal endothelial nitric oxide synthase （eNOS） gene knockout mice and wild mice by RNA quantitation detection and cell area estimation. METHODS： The experiment was performed at the Empirical Study. Center of Medicine Department in Shunde Profession and Technique Institution in August 2007. （1）Male or female C57BL/6 stock neonatal eNOS knockout mice and wild mice aged 1-3 days were selected. （2）Cultivated Mouse cardiomyocytes were divided into experimental and control groups 72 hours later. Adrenergic receptor agonist phenylephrine was cultured with cardiomyocytes. DMEM of the same volume as the experimental group was added in the medium in the blank control group. （3）DNA, RNA quantitation detection and cell area estimation methods was used to observe the responses contrast in cell proliferation and hypertrophy of cardiomyocytes in cultured neonatal eNOS gene knockout mice and wild mice. RESULTS： （1）The oval or rhombus cardiomyocytes were observed 72 hours after phenylephrine incoporation under a microscope （2）RNA fluorescence value increased in cultured neonatal cadiomyocyte and collagenoblast in the experimental group compared to the control group （P 〈 0.05）. The RNA fluorescence intension in cultured neonatal cadiomyocyte and collagenoblast in the blank control group compared to the experimental and control groups （P 〈 0.05）. （3）The fluorescence value in cultured neonatal cadiomyocyte and collagenoblast fluorescence area increased in the experimental and control groups compared to the blank control group （P 〈 0.05）. The cadiomyocyte fluorescence area value was higher in the experimental group compared to the control group （P 〈 0.05）. CONCLUSION： There is an excessive hypertrophy effect in cardiomyocytes from isolated neonatal eNOS gene knockout mice under the stimulation of hypermyotrophy inducing factor.