全反式维甲酸联合奥曲肽对肝癌细胞-HepG_2增生和凋亡的影响

Effects of all-trans retinoic acid with octreotide on proliferation and apoptosis of hepatoma cell HepG_2

目的:探讨全反式维甲酸联合奥曲肽对肝癌细胞HepG2增生和凋亡的作用.方法:将不同剂量的全反式维甲酸和奥曲肽(1mg/L)直接作用于体外培养的人肝母细胞瘤株HepG2,用MTT比色法分析两种药物联合作用对肝癌细胞生长的影响,流式细胞仪检测细胞凋亡率,细胞周期分布,免疫细胞化学方法检测抑癌基因P21WAF1的表达,台盼兰染色观察药物对细胞毒性作用.结果:小剂量全反式维甲酸(10-6mmol/L)和奥曲肽(1mg/L)连用后,对细胞具有增生抑制作用,与无增生抑制的奥曲肽作用组和对照组相比有显著性差异(P=0.0087P<0.01).细胞免疫化学:全反式维甲酸和奥曲肽药物联合作用48h后,细胞中目的基因P21WAF1/CIP1由胞膜,胞质微弱表达转为核强烈表达.于作用48h后药物合用组对细胞各周期均有影响,奥曲肽作用组可使肝癌细胞HepG2阻滞于S期,且药物合用组于作用72h后,凋亡率为17%,明显高于奥曲肽作用组与对照组.台盼兰染色未发现大片死亡细胞.结论:小剂量全反式维甲酸(10-6mmol/L)联合生长抑素类似物奥曲肽(1mg/L)对肝癌细胞HepG2具有增生抑制和诱导凋亡作用.抑癌基因表达增强说明二者的诱导凋亡和增生抑制作用可能是通过增强抑癌基因P21WAF1/CIP1的表达实现的.从而为在临床上治疗肝癌提供了新的思路.

AIM: To investigate the effects of all-trans retinoic acid combined with octreotide on proliferation and apoptosis of human liver cancer cell HepG2. METHODS: The human liver cancer cell HepG2 cultured in vitro was designed into three groups: combined group, octreotide group and control group. For combined group, HepG2 was treated with different concentrations of all-trans retinoic acid (1×10-6, 5×10-6, 5×105 mmol/L) associated with octreotide (1 mg/L). MTT assays were adopted to determine their effects on cell growth. Cell apoptotic rate and cell cycle were detected by flow cytometry. The expression of tumor supressing gene p21WAF1/CIP1 was detected by immunocytochemical methods. Cytoxicity of the two drugs was observed with typan blue staining. RESULTS: After being treated with the concentration of 1×10-6mmol/L of all-trans retinoic acid and 1mg/L of octreotide, cells' viability was significantly inhibited compared with the octreotide group and controls (P =0.0087 <0.01). No significant difference existed between octreotide group and control group. The expression of the target gene p21WAF1/CIP1 was significantly increased after associated application of all-trans retinoic acid with octreotide. Apoptosis rate of liver cancer cells (17%) in combined group was also significantly higher than those in octreotide and con trol group. No death of great numbers was observed with typan blue staining. CONCLUSION: Proliferation of liver cancer cell HepG2 is inhibited by combination of low concentration of all-trans retinoic acid (1×10-6mmol/L) with octreotide (1 mg/L) while apoptosis is induced. Meanwhile, different periods of cell cycle were also influenced. The effect of the two drugs on proliferating inhibition and apoptotic facilitation may be achieved by increasing the expression of gene p21WAF1/CIP1.