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 increased ( P 〈 0. 05 ). Hoechst33258 dyeing showed Lg-2 treatment group had changes of karyopyknosis, fracture and apoptosis. Conclusion Lg-2 may obviously inhibit cancerous cell multiplication and make cell cycle arrest in G2/M phase, which may be regulated to expressions of p53, bax, p21 and caspase3 up-regulation and down regulation of bcl-2 gene.
出处
《解放军医药杂志》
CAS
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)