全反式维甲酸诱导HepG2细胞分化和降低软琼脂克隆形成能力及其机制的初步探讨
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摘要
目的研究全反式维甲酸(ATRA)体外对人肝癌细胞株(HepG2)分化、凋亡及软琼脂克隆形成能力的影响;并初步探讨ATRA调控骨桥蛋白(OPN)表达、诱导HepG2细胞分化的分子机制。方法ATRA处理HepG2细胞,光镜下观察细胞形态;酶动力学法检测γ-谷氨酰转肽酶(γ-GT)比活性、甲胎蛋白(AFP)分泌量的变化;MTT法分析ATRA对HepG2增殖的影响;软琼脂培养法观察HepG2的克隆形成能力;Hoechst染色法检测ATRA作用前后的细胞凋亡情况;Western blot检测HepG2细胞中ERK2的磷酸化水平及OPN、NF-κB蛋白表达量。结果ATRA显著抑制HepG2细胞在体外的增殖并诱导细胞分化和凋亡,使代表肝细胞恶变的γ-GT比活性、AFP分泌量明显下降(P<0.05);软琼脂克隆形成实验结果表明未经ATRA处理的HepG2可在软琼脂上长成克隆,但经ATRA刺激后,其形成克隆变小且数目明显减少;western blot结果显示,未经ATRA处理的HepG2细胞ERK2的磷酸化水平及OPN的表达量均很高,经ATRA处理5d后,ERK2的磷酸化及OPN的表达明显减少,并具显著的时间剂量依赖性,10μmol/L ATRA作用70d后ERK2的磷酸化及OPN的表达几乎为零,但ERK2、NF-κB蛋白表达无变化。结论ATRA是HepG2有效的分化诱导剂,可诱导细胞分化、凋亡并可降低HepG2的克隆形成能力;ATRA诱导HepG2细胞分化,降低软琼脂克隆形成能力,抑制细胞增殖及恶性转化的分子机制可能与ATRA抑制ERK/MAPK信号转导通路中ERK的磷酸化并进一步抑制OPN的表达相关,为ATRA诱导分化治疗肝癌提供了实验依据。
Objective To study the differentiation、apoptosis、the ability of colony formation and the expression of osteopontin(OPN) of human hepatoma HepG2 cells in vitro and its mechanism induced by all-trans-retinoic acid(ATRA). Methods HepG2 was treated with 1 or 10μmol/L ATRA.The morphology of HepG2 was observed by light microscope and the diversity ofγ-glutamyl transpeptidase(γ-GT) and alpha-fetoprotein(AFP) were detected by kinetics analysis.The cell proliferation of HepG2 was analyzed by MTT assay and the ability of colony formation was assay by soft agar.The cell apoptosis was measured with Hoechst dyeing.The phosphorylation of ERK2 and the expression of OPN and NF-κB were studied by western blot. Results The proliferation of HepG2 significantly inhibited,the differentiation and apoptosis was induced by ATRA,moreover the specific activities ofγ-GT and the secretary amount of AFP significantly decreased.The HepG2 cells could grow to clone on soft agar,but the size and quantity distinguished diminish after stimulated by ATRA.The western blot indicated the phosphorylation of ERK2 and the expression of OPN down-regulation significantly in HepG2 cells treated with ATRA for 5d.When the HepG2 cells was treated with 10μmol/L ATRA for 70d,the phosphorylation of ERK2 and the expression of OPN was not found,while no change of ERK2 and NF-κB. Conclusion ATRA was effective to induce differentiation of HepG2 cells . ATRA could induce differentiation,inhibit proliferation and broke down the ability of colony formation by inhibiting the phosphorylation of ERK2 and the expression of OPN.
Objective To study the differentiation、apoptosis、the ability of colony formation and the expression of osteopontin(OPN) of human hepatoma HepG2 cells in vitro and its mechanism induced by all-trans-retinoic acid(ATRA). Methods HepG2 was treated with 1 or 10μmol/L ATRA.The morphology of HepG2 was observed by light microscope and the diversity ofγ-glutamyl transpeptidase(γ-GT) and alpha-fetoprotein(AFP) were detected by kinetics analysis.The cell proliferation of HepG2 was analyzed by MTT assay and the ability of colony formation was assay by soft agar.The cell apoptosis was measured with Hoechst dyeing.The phosphorylation of ERK2 and the expression of OPN and NF-κB were studied by western blot. Results The proliferation of HepG2 significantly inhibited,the differentiation and apoptosis was induced by ATRA,moreover the specific activities ofγ-GT and the secretary amount of AFP significantly decreased.The HepG2 cells could grow to clone on soft agar,but the size and quantity distinguished diminish after stimulated by ATRA.The western blot indicated the phosphorylation of ERK2 and the expression of OPN down-regulation significantly in HepG2 cells treated with ATRA for 5d.When the HepG2 cells was treated with 10μmol/L ATRA for 70d,the phosphorylation of ERK2 and the expression of OPN was not found,while no change of ERK2 and NF-κB. Conclusion ATRA was effective to induce differentiation of HepG2 cells . ATRA could induce differentiation,inhibit proliferation and broke down the ability of colony formation by inhibiting the phosphorylation of ERK2 and the expression of OPN.
引文
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