摘要
目的探讨Krüpple样因子5(KLF5)对心肌细胞体积、细胞内总蛋白、氧化损伤及Ca^(2+)水平的影响。方法体外分离培养乳鼠心肌细胞,心肌细胞分为以下几组,对照组:不做任何处理,正常培养。模型组:在细胞培养液中添加100 nm的血管紧张素Ⅱ(AngⅡ)。阴性组:心肌细胞中转染siRNA阴性对照。干扰组:心肌细胞中转染KLF5 siRNA。阴性+AngⅡ组:心肌细胞中转染siRNA阴性对照,并在细胞培养液中添加100 nm的AngⅡ。干扰+AngⅡ组:心肌细胞中转染KLF5 siRNA,并在细胞培养液中添加100 nm的AngⅡ。分别检测KLF5的基因和蛋白表达水平。在心肌细胞内转染KLF5 siRNA,同时用AngⅡ处理,测定心肌细胞的体积和心肌细胞内总蛋白的含量,同时检测细胞内肥大基因心房利钠肽(ANP)mRNA的水平,Western blot方法测定细胞内钙调神经磷酸酶(CaN)蛋白水平,激光共聚焦显微镜检测静息状态下Ca^(2+)水平,二硝基苯肼法测定培养液中乳酸脱氢酶(LDH)水平,硫代巴比妥酸法测定丙二醛(MDA)含量,黄嘌呤法测定超氧化物歧化酶(SOD)含量。结果模型组心肌细胞中KLF5基因和蛋白表达水平高于对照组(P<0.05)。干扰组心肌细胞中KLF5水平低于对照组(P<0.05)。阴性组和对照组心肌细胞中KLF5水平没有差异(P>0.05)。模型组心肌细胞的体积增加,细胞总蛋白含量也增加,细胞内肥大基因ANP mRNA的水平也升高,CaN蛋白表达和静息状态下Ca^(2+)水平上调,细胞内生成的MDA增加,SOD活性降低,细胞培养液中LDH水平升高,与对照组相比,差异有统计学意义(P<0.05)。干扰+AngⅡ组心肌细胞体积和蛋白含量下降,细胞内肥大基因ANP mRNA的水平降低,CaN蛋白表达和静息状态下Ca^(2+)水平下降,细胞生成的MDA减少,SOD活性升高,细胞培养液中LDH水平降低,与模型组比较,差异有统计学意义(P<0.05)。阴性+AngⅡ组细胞体积、蛋白含量、细胞内肥大基因ANP mRNA、CaN蛋白、静息状态下Ca^(2+)水平、细胞生成的MDA、SOD活性、细胞培养液中LDH水平与模型组比较没有明显差异(P>0.05)。结论 AngⅡ诱导心肌细胞表达KLF5,KLF5下调可抑制AngⅡ诱导的心肌细胞肥大,其作用机制可能与心肌细胞氧化损伤和Ca^(2+)水平有关。
Objective To investigate the influence of Krüpple-like factor 5(KLF5)on cardiomyocyte volume,the total protein(TP)in cardiomyocytes,oxidative damage and level of calcineurin(CaN).Methods The cardiomyocytes were separated and cultured in vitro,and then divided into control group(without any treatment),model group[100 nm angiotensinⅡ(AngⅡ)added in cultural solution],negative group(transfected with siRNA negative control),interference group(transfected with KLF5 siRNA),negative+AngⅡgroup(transfected with siRNA negative control and 100 nm AngⅡadded in cultural solution),and interference+AngⅡgroup(transfected with KLF5 siRNA and 100 nm Ang II added in cultural solution).The mRNA and protein expressions of KLF5 were detected respectively.The cardiomyocyte volume and TP in cardiomyocytes were determined and meanwhile the level of intracellular hypertrophic gene-atrial natriuretic peptide(ANP)mRNA was detected.The level of CaN was determined by using Western blotting assay,and level of Ca2+at resting state was detected by using laser confocal microscopy.The level of lactic dehydrogenase(LDH)was determined by using dinitrophenylhydrazine(DNPH)method,level of malondialdehyde(MDA)was detected by using thiobarbituric acid(TBA)method,and level of superoxide dismutase(SOD)was determined by using xanthineoxidation(XTO)method.Results The mRNA and protein expressions of KLF5 were higher in model group than those in control group(P<0.05).The level of KLF5 in cardiomyocytes was lower in interference group than that in control group(P<0.05).The level of KLF5 had no difference between negative group and control group(P>0.05).The cardiomyocyte volume,TP in cardiomyocytes and ANP mRNA increased,CaN protein expression and Ca2+level was up-regulated,MDA level increased,SOD activity decreased and LDH level increased in model group compared with control group(P<0.05).The cardiomyocyte volume,TP in cardiomyocytes and ANP mRNA decreased,CaN protein expression and Ca2+level was down-regulated,MDA level decreased,SOD activity increased and LDH level decreased in interference+AngⅡgroup compared with model group(P<0.05).The cardiomyocyte volume,TP in cardiomyocytes,ANP mRNA,CaN protein expression,Ca2+level,MDA level,SOD activity,and LDH level had no significant difference between negative+AngⅡgroup and model group(P>0.05).Conclusion AngⅡcan induce KLF5 expression in cardiomyocytes.When KLF5 is down-regulated,cardiomyocyte hypertrophy induced by AngⅡwill be inhibited,and the mechanism may be related to oxidative damage and Ca2+level in cardiomyocytes.
作者
沈丹凤
王燕
蒋华
Shen Danfeng;Wang Yan;Jiang Hua(Department of Cardiovascular Medicine,Fourth People's Hospital of Sichuan Province,Chengdu 610016,China)
出处
《中国循证心血管医学杂志》
2018年第7期841-845,共5页
Chinese Journal of Evidence-Based Cardiovascular Medicine
