Capsazepine诱导人肝癌HepG2细胞凋亡机制研究  

Mechanism of Capsazepine inducing apoptosis of human hepatocellular carcinoma cell line HepG2

作　　者：韩璐[1,2,3,4] 狄翠霞[1,2,3] 毛爱红[1,2,3,4] 颜家玮 张红[1,2,3] 

机构地区：[1]中国科学院近代物理研究所,甘肃兰州730000 [2]中国科学院重离子束辐射生物医学重点实验室,甘肃兰州730000 [3]甘肃省重离子束辐射医学应用基础重点实验室,甘肃兰州730000 [4]中国科学院大学生命科学学院,北京100049

出　　处：《中华肿瘤防治杂志》2015年第10期764-769,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金联合基金重点项目(U1432248);国家自然科学基金青年科学基金(11205219);"西部之光"人才培养计划(Y260230XB0)

摘　　要：目的研究瞬时感受器电位离子通道(transient receptor potential vani1loid 1,TRPVl)阻滞剂Capsazepine对人肝癌HepG2细胞凋亡的机制。方法采用MTT法检测浓度分别为0.01、0.1、1、25、100μmol/L Capsazepine作用于HepG2细胞24h后对细胞生长的影响;Hoechst 33258染色法观察0.1和10μmol/L Capsazepine作用于HepG2细胞24h后细胞的形态学改变;流式细胞术观察0.1和10μmol/L Capsazepine作用于HepG2细胞24h后细胞的凋亡率和周期的变化;实时荧光定量PCR技术(real time-PCR)、蛋白质印迹法和激光共聚焦法检测0.1和10μmol/L Capsazepine作用于HepG2细胞24h后,细胞中离子通道受体蛋白TRPV1,以及凋亡相关因子p53,Bcl-2和Bax在mRNA和蛋白水平的表达情况。结果随着药物浓度的增大,Capsazepine对体外培养的HepG2细胞有抑制增殖作用,且具有剂量相关性;0.01、0.1、1、25、100μmol/L的Capsazepine作用于HepG2细胞24h后,HepG2细胞存活率分别为84.77%、73.83%、70.58%、61.42%和48.48%,F=176.523,P<0.001。流式细胞术检测显示,Capsazepine可致HepG2细胞阻滞于G0/G1期,并诱导其凋亡;0.1与10μmol/L的Capsazepine作用于HepG2细胞24h,与对照组相比,G0/G1期细胞比例分别为(65.48±0.04)%和(72.14±0.30)%,F=187.791,P<0.001;对照组与Capsazepine处理组细胞凋亡率分别为(3.25±1.08)%和(21.50±4.85)%,t=0.000 26,P=0.026。Hoechst荧光染色法可见,随着药物浓度的增大,细胞的生长变慢,胞质变疏松,细胞核皱缩,出现典型的细胞凋亡形态。蛋白质印迹法检测可见,对照组与0.1和10μmol/L的Capsazepine作用于HepG2细胞24h,离子通道受体蛋白TRPV1的表达量分别为0.62±0.03、0.58±0.04和0.38±0.04,随着Capsazepine浓度的增加,TRPV1的表达量显著下降,差异有统计学意义,F=38.926,P<0.001。在对照组与实验组中,p53蛋白表达量分别为0.25±0.007、0.26±0.005和0.31±0.01,Bax蛋白表达量分别为1.49±0.16、2.87±0.06和3.12±0.15,Bcl-2蛋白表达量分别为0.70±0.08、0.35±OBJECTIVE To investigate the mechanism of apoptosis induced by Capsazepine in human hepatoma cell line HepG2. METHODS Different concentrations of Capsazepine （0.01,0. 1,1,25 and 100 μol/L） were used to treat HepG2 cell for 24 h, and cell proliferation was measured by MTT assay. After treatment in different concentrations of Capsazepine （0.1 and 10 μmol/L） for 24 h,the morphological changes of apoptosis were observed by Hoeehst 33258 stai- ning. Cell cycle distribution and apoptotic rate were evaluated by flow cytometry （FCM） after treatment in different con- centrations of Capsazepine （0.1 and 10 μmol/L） for 24 h. Quantitative real-time RT-PCR,Western blotting analysis and immunofuorescence were used to detect the mRNA and protein expressions of TRPV1, and apoptosis related factors Bcl-2, Bax and p53 with 0.1 and 10 μmol/L concentrations of Capsazepine. RESULTS Capsazepine could inhibit the prolifera- tion of HepG2 cells in a dose dependent manner. Cell viability of HepG2 cells with different concentration of Capsazepine treatment （0.01,0.1,1,25 and 100 μmol/L） after 24 hours were 84.77% ,73.83% ,70. 58% ,61.42% and 48.48% （F= 176. 523,P〈0. 001）. Remarkable morphological changes of apoptosis including cytoplasmic and nuclear condensation and partition of cytoplasm were observed by Hoechst 33 258 staining. According to FCM analysis, HepG2 cells were arrested in G0/G1 phase and theapoptotic rate were increased from （3. 25±1.08）% to（21. 50±4. 85）% （t=0. 000 26,P= 0. 026）. G0/G1 phase cell proportion were （65.48±0.04）% and（72.14±0.30）%（F= 187. 791 ,P〈0. 001）. Capsazepine could inhibit TRPV1 expression in protein level （F= 38. 926, P〈0. 001）. Both protein expressions of Bax and p53 were up-regulated （F values were 50. 968 and 129. 002,P〈0. 001） while Bel-2 expression was down-regulated （F=42. 626, P〈0. 001）. CONCLUSION Capsazepine can inhibit the proliferation and induce cell cycle arrest and apoptosis of human hepatoma cell line HepG2. Fur

关 键 词：瞬时感受器电位离子通道 CAPSAZEPINE HEPG2细胞 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]