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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。
Aim To investigate the effect of N- (1-alkoxy-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl) -L-alanine- Lg-2) on human hepatocellular carcinoma cell line HepG-2 and its mechanism. Methods The negative control group (HepG-2 cells only), VP-16 treatment group and Lg-2 treatment group were divided into three groups. The effect of Lg-2 on the growth of HepG-2 cells was detected by MTT assay. Flow cytometry Lg-2 on the cell cycle and apoptosis of HepG-2 cells were detected by qRT-PCR. The apoptosis-related genes p53, Bax, Bc1-2, p21, Caspase The expression of Lg-2 in HepG-2 cells was detected by Hoechs33258 staining. The morphological changes of HepG-2 cells were observed. Results MTT assay showed that Lg-2 significantly inhibited the proliferation of HepG-2 cells. After 48 h of treatment, the inhibitory effect was enhanced with the increase of drug concentration (at the same time). The inhibitory rates of HepG-2 in Lg-2 treatment group were Higher than VP-16 treatment group (P <0.05). The inhibition of HepG-2 in Lg-2 treatment group was higher than that in VP-16 treatment group (P <0.05) with the prolongation of drug action time (same concentration). Flow cytometry showed that the proportion of S phase cells in Lg-2 treatment group was significantly higher than that in negative control group and VP-16 treatment group when Lg-2 was applied to HepG-2 cells for 24 and 48 h, while G2 / M phase Cells are significantly reduced. The results of qRT-PCR showed that the expression of Bcl-2 mRNA in Lg-2 treatment group was significantly lower than that in VP-16 treatment group (P <0.05), while the expression of p53, Bax, p21 and Caspase3 mRNA Significantly increased (P <0.05). Hoechst33258 staining showed that the nuclei of Lg-2-treated group were pyknotic and fragmented, showing apoptosis-like changes. Conclusion Lg-2 can inhibit the proliferation of hepatoma cells and block the cells in G2 / M phase, which may be related to the up-regulation of p53, bax, p21, caspase3 expression and down-regulation of bcl-2 gene expression.