维甲酸对人肝癌细胞凋亡的影响及临床意义

Effects of retinoic acid on human hepatocarcinoma cell lines apoptosis

目的 观察全反式维甲酸 (ATRA)对肝癌细胞凋亡的作用并探讨ATRA的作用机制。方法 在肝癌细胞株SMMC 772 1细胞加入ATRA ,使终浓度为 0 .5 μg/ml。对照组加等剂量的 0 .0 2 %DMSO ,培养 4d。ATRA对SMMC 772 1细胞的影响通过苏木精 伊红染色 ,在光镜下进行细胞形态学观察 ;通过细胞计数绘制生长曲线及计算细胞生长抑制率。流式细胞术分析不同DNA含量的细胞分布 ,计算细胞凋亡百分率。凋亡相关基因Fas、p5 3、Bcl 2蛋白表达的检测亦采用流式细胞仪检测。结果 苏木精 伊红染色可见较多细胞核碎裂 ,胞浆浓缩 ,染色质深染 ,聚集于核膜下 ;部分细胞膜突起呈小泡样 ,小泡脱落形成凋亡小体 ,而对照组无明显的形态学改变。作用 4d后细胞生长抑制率为 47.5 %,与对照组比较 ,差异显著 (P <0 .0 5 )。流式细胞仪分析在G1期前出现亚二倍体凋亡峰 ;凋亡细胞百分比为 16 .0 %,与对照的 6 .9%比较有显著性差异 (P <0 .0 1)。观察发现 ,ATRA对SMMC 772 1细胞Fas、P5 3的表达明显增加 ,并下调Bcl 2的表达。结论 0 .5 μg/ml的ATRA对肝癌细胞的生长有显著抑制效果 ,并可见癌细胞凋亡形态改变。ATRA促进SMMC 72 1肝癌细胞凋亡的机制之一可能是通过调控Fas、P5 3及Bcl 2的表达。

Objective To investgate the growth inhibition and apoptosis induced by all-trans-retinoic acid (ATRA), and its possible molecular mechanism in human hepatocarcinoma cell lines. Methods Human hepatocarcinoma cell lines SMMC-7721 were cultured with ATRA at the concentration of 0.5μg/ml for 4 days. The cells apoptosis and growth inhibition were assessed by cytomorphology and counting the number of cells to calculate the rate of growth-inhibited. Cell cycle, tumor associated genes p53, Fas and Bcl-2 were analyzed with flow cytometry.Results ATRA inhibited the growth of hepatocarcinoma cells SMMC-7721 in vitro. Apoptosis cells of total cells were 16% compared with control 6.9% (P<0.01) and a sub-G 1 phase cell peak was observed. Morphological observation with light microscope revealed apoptosis feature, i.e. chromatin condensation, nucleus fragmentation and cytoplasmic blebbing. There was a significant different rate of growth-inhibited between the two groups.(P<0.005). The expression of p53 and Fas were up-regulated, and Bcl-2 was down-regulated when treated with ATRA in contrast to control.Conclusion ATRA can induce human hepatocarcinoma cell apoptosis. It could be a potential therapeutic agent for hepatocarcinoma. Tumor associated genes such as p53, Fas and Bcl-2 may be involved in molecular mechanism of ATRA effects.