灵芝酸A对非小细胞肺癌PC9细胞糖酵解及其关键限速酶的影响  

Effect of ganoderic acid A on glycolysis and its key rate-limiting enzymes of non-small cell lung cancer PC9 cells

作　　者：任爱华[1] 董彦伯 苗润芝 吕俞娇 刘岩峰[1] REN Aihua;DONG Yanbo;MIAO Runzhi;LYU Yujiao;LIU Yanfeng(Department of Anatomy,School of Basic Medicial Sciences,Beihua University,Jilin 132013,China;Department of Surgery,School of Clinical Medical Sciences,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学基础医学院解剖教研室,吉林吉林132013 [2]北华大学临床医学院外科教研室,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2024年第3期682-688,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省教育厅科学技术研究项目(JJKH20220069KJ,JJKH20180355KJ);北华大学省级大学生创新创业训练计划项目(202310201135)。

摘　　要：目的:探讨不同剂量灵芝酸A(GAA)对非小细胞肺癌(NSCLC) PC9细胞的生物活性、糖酵解及其限速酶表达的影响,并阐明其作用机制。方法:体外培养NSCLC PC9细胞,将PC9细胞分为空白对照组、低剂量(25μmol·L^(-1)) GAA组、中剂量(50μmol·L^(-1)) GAA组和高剂量(100μmol·L^(-1)) GAA组。采用噻唑蓝(MTT)法检测各组PC9细胞存活率,Transwell小室实验检测各组PC9细胞迁移细胞数,葡萄糖检测试剂盒检测各组PC9细胞葡萄糖摄取量,三磷酸腺苷(ATP)检测试剂盒检测各组PC9细胞中ATP水平,乳酸检测试剂盒检测各组PC9细胞中乳酸水平,实时荧光定量PCR(RT-qPCR)法检测各组PC9细胞中己糖激酶2(HK2)和M2型丙酮酸激酶(PKM2) mRNA表达水平,Westernblotting法检测各组PC9细胞中HK2和PKM2蛋白表达水平。结果:MTT法,培养24、48和72 h时,与空白对照组比较,中和高剂量GAA组PC9细胞存活率明显降低(P<0.05);培养72 h时,与低剂量GAA组比较,高剂量GAA组细胞存活率明显降低(P<0.05)。Transwell小室实验,与空白对照组比较,中和高剂量GAA组细胞迁移数明显减少(P<0.05);与低剂量GAA组比较,高剂量GAA组细胞迁移数明显减少(P<0.05)。与空白对照组比较,中和高剂量GAA组PC9细胞中葡萄糖摄取量和乳酸水平均明显降低(P<0.05),高剂量GAA组PC9细胞中ATP水平明显降低(P<0.05)。RT-qPCR法,与空白对照组比较,中和高剂量GAA组PC9细胞中HK2和PKM2 mRNA表达水平均明显降低(P<0.05)。Western blotting法,与空白对照组比较,中和高剂量GAA组PC9细胞中HK2和PKM2蛋白表达水平明显降低(P<0.05)。结论:中和高剂量GAA可抑制PC9细胞增殖及迁移的生物活性,降低糖酵解作用,其作用机制可能与抑制关键限速酶HK2和PKM2的表达有关。Objective:To discuss the effects of different doses of ganoderic acid A(GAA) on the biological activities,glycolysis,and the expression of rate-limiting enzymes of non-small cell lung cancer(NSCLC) PC9 cells,and to clarify the mechanism.Methods:The NSCLC PC9 cells were cultured in vitro and divided into blank control group,low dose(25 μmol·L~(-1)) of GAA group,medium dose(50 μmol·L~(-1)) of GAA group,and high dose(100 μmol·L~(-1)) of GAA group.The methylthiazolydiphenyltetrazolium(MTT) assay was used to detect the survival rates of the PC9 cells in various groups;Transwell chamber assay was used to detect the number of migration cells of the PC9 cells in various groups;the glucose uptakes of the PC9 cells in various groups were detected by glucose assay kit;the levels of ATP in the PC9cells in various groups were detected by ATP assay kit;the levels of lactic acid in the PC9 cells in various groups were detected by lactate assay kit;the expression levels of hexokinase 2(HK2) and pyruvate kinase M2(PKM2) mRNA in the PC9 cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR) method;the expression levels of HK2 and PKM2 proteins in the PC9 cells in various groups were detected by Western blotting method.Results:The MTT assay results showed that,at 24,48,and 72 h of culture,compared with blank control group,the survival rates of the cells in medium and high doses of GAA groups were significantly decreased(P<0.05).At 72 h of culture,compared with low dose of GAA group,the survival rate of the cells in high dose of GAA group was significantly decreased(P<0.05).The Transwell chamber assay results showed that compared with blank control group,the numbers of migration cells in medium and high doses of GAA groups were significantly decreased(P<0.05);compared with low doses of GAA group,the number of migration cells in high dose of GAA group was significantly decreased(P<0.05).Compared with blank control group,the glucose uptakes and levels of lactic acid in the cells in medium and h

关 键 词：灵芝酸A 癌 非小细胞肺 己糖激酶2 M2型丙酮酸激酶 糖酵解 

分 类 号：R734.2[医药卫生—肿瘤]