鬼臼毒素衍生物Lg-2对人肝癌细胞系HepG-2凋亡的诱导作用  

Induction of Podophyllotoxin Derivatives Lg-2 for Apoptosis in Human Hepatoma Cell Line HepG-2

作　　者：牛海峰[1,2,3] 景鸿恩[3] 惠玲[2] 闫蔚[1,2] 王君[3] 王宏宾[3] 王晓临[3] 周通[3] 张国华[3] 谭大勇[3] 

机构地区：[1]青海大学医学院 [2]兰州军区兰州总医院医学实验中心,兰州730050 [3]青海大学附属医院肝胆胰一科,西宁810001

出　　处：《解放军医药杂志》2013年第8期21-25,共5页Medical & Pharmaceutical Journal of Chinese People’s Liberation Army

基　　金：甘肃省自然科学基金资助项目(1107RJZA106)

摘　　要：目的研究N-(1-烃氧基-2,2,6,6-四甲基-4-哌啶基氧羰基)-L丙氨酸-4'-去甲-4-脱氧鬼臼酯(Lg-2)对人肝癌细胞系HepG-2的诱导凋亡作用及机制。方法分别设阴性对照组(只加HepG-2细胞)、VP-16处理组及Lg-2处理组3组,采用MTT法检测Lg-2对HepG-2细胞生长的影响,流式细胞术检测Lg-2对HepG-2细胞周期和凋亡的影响,荧光定量RT-PCR(qRT-PCR)技术检测Lg-2对HepG-2细胞凋亡相关基因p53、Bax、Bc1-2、p21、Caspase 3表达的影响;设阴性对照组及Lg-2处理组2组,Hoechs33258染色检测Lg-2对HepG-2细胞形态学的影响。结果 MTT法证实Lg-2对HepG-2细胞增殖有明显的抑制作用,药物处理48 h,随药物浓度增加(同一时间时)抑制效应增强,Lg-2处理组各浓度HepG-2抑制率均高于VP-16处理组(P<0.05)。随药物作用时间的延长(同一浓度时)抑制效应增强,Lg-2处理组各作用时间HepG-2抑制率均高于VP-16处理组(P<0.05)。流式细胞术显示,Lg-2作用于HepG-2细胞24和48 h时,Lg-2处理组S期细胞比例与阴性对照组及VP-16处理组比较均明显增高,而G2/M期细胞显著减少。qRT-PCR检测细胞凋亡相关基因,结果表明Lg-2处理组与VP-16处理组相比Bc1-2 mRNA的表达显著降低(P<0.05),而p53、Bax、p21、Caspase3 mRNA的表达显著增高(P<0.05)。Hoechst33258染色显示Lg-2处理组细胞核固缩、碎裂,呈凋亡样改变。结论 Lg-2可抑制肝癌细胞增殖,使细胞阻滞在G2/M期,该过程可能与其上调p53、bax、p21、caspase3表达和下调bc1-2基因的表达有关。Objective To study the effect and mechanism of induced apoptosis of N-（ 1-Oxyl-2,2,6,6-tetram- ethyl-4-piperidinyloxycarbonyl）-L-alanine-4＇-demethyl-4-deoxypodophyllic ester （Lg-2） in human hepatoma cell line HepG-2. Methods A negative control group （added only HepG 2 cell）, VP-16 treatment group and Lg-2 group were established, and the influences of Lg-2 to the HepG 2 cell growth in the three groups were determined by MTT method; the influences of Lg-2 to the HepG 2 cell cycle and apoptosis in the three groups were detected by flow cytometry; the ap- optosis related genes p53, Bax, Bcl-2, p21 and caspase 3 expression were detected by fluorescent quantitative RT-PCR （qRT-PCR） method; a negative control group and Lg-2 treatment group 2 were established, and the Lg-2 influences to HepG 2 cell morphology in the two groups were detected by Hoechs33258 dyeing test. Results MTT method showed that Lg-2 had obvious inhibitory effect to the HepG 2 cell proliferation. After 48 h of drug treatment, inhibitory effects were enhanced with the increase of drug concentration （at the same time） , and inhibition ratios of every concentration in Lg- 2 treatment group were significantly higher than those in VP-16 treatment group （P 〈0.05）. Inhibitory effects were en- hanced with the extension of drug effect time （at the same concentration）, HepG 2 inhibition ratios at every action timein Lg-2 treatment groups were higher than those in VP-16 treatment group （ P 〈 0.05 ）. Flow cytometry showed that Lg-2 affected on the HepG 2 cell for 24 h and 48 h, Sphase cells proportion in Lg-2 treatment groups was significantly in- creased compared with those in negative control group and VP16 group, while the GJM phase cells were significantly re- duced. Apoptosis related gene of QRT-PCR was detected, Bcl-2 mRNA expression in the Lg-2 treatment group was sig- nificantly reduced compared with that in VP-16 group （ P 〈 0.05 ） , while expressions of p53, Bax, p21 and caspase3 mRNA were significantly increa

关 键 词：鬼臼毒素衍生物 N-(1-烃氧基-2 2 6 6-四甲基-4-哌啶基氧羰基)-L丙氨酸-4′-去甲-4-脱氧鬼臼酯 依托泊甙 细胞周期 细胞凋亡 HEPG-2 

分 类 号：R735.7[医药卫生—肿瘤]