摘要
目的:观察ATRA对Walker-256肝癌细胞的分化诱导及促进凋亡作用,探讨其作用机制.方法:在肝癌细胞Walker-256细胞株中加入不同浓度的ATRA(100、10、5、1μmol/L),培养2d,荧光倒置显微镜观察细胞形态学变化;MTT比色法检测Walker-256细胞的增殖抑制;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;RT-PCR检测凋亡相关基因Fas、bcl-2mRNA的表达;Western blot检测Caspase3、8蛋白的表达.结果:随着ATRA浓度的递增,Walker-256细胞表现出细胞分化及凋亡的形态学上的改变.ATRA可明显抑制细胞增殖,5和10μmol/LAT R A对细胞增殖抑制最为明显,24、48、72h的增殖抑制率分别为21.86%、57.39%、68.17%和27.23%、80.09%、92.15%,呈浓度及时间依赖性.5和10μmol/L ATRA对细胞的凋亡诱导率与对照组相比,差异有统计学意义(t=9.61,11.38,P<0.05,0.01).ATRA作用Walker-256细胞后上调Fas表达,下调bcl-2的表达,10μmol/L ATRA对Fas及bcl-2的调节与对照组相比,差异有统计学意义(t=12.33,10.78,均P<0.05).与对照组相比,10μmol/LATRA作用48h后对Caspase3及Caspase8酶原剪切活化明显(t=9.76,10.21,均P<0.05).结论:ATRA对Walker-256肝癌细胞具有分化诱导及促进其凋亡的作用.
AIM：To observe differentiation and apoptosis of Walker-256 hepatocarcinoma cells induced by all trans-retinoitc acid（ATRA）,and to explore its possible mechanism. METHODS：Walker-256 hepatocarcinoma cell lines were treated with ATRA at various concen- trations（100,10,5 and 1μmol/L,respectively）. Fortyeight hours later,morphological changes were measured using inverted microscopy;The proliferation effect of Walker-256 cell lines treated with ATRA was determined using MTT as-say;cellular distribution of DNA content and the apoptotic incidence of cells treated with ATRA was measured using flow cytometry（FCM）;the changes of Fas and bcl-2 mRNA expression were determined using RT-PCR,and Caspase 3 and Caspase 8 protein expressions were determined using Western blot. RESULTS：With the increasing concentration of ATRA,Walker-256 cells showed morphological changes in cell differentiation and apoptosis. ATRA inhibited cell proliferation of Walker-256, with the maximum inhibitory effects at con- centrations of 5 and 10μmol/L.Proliferation rates at 24,48 and 72 h at concentrations of 5 and 10μmol/L were 21.86%,57.39%,68.17%, and 27.23%,80.09%,92.15%,respectively,show- ing a concentration-and time-dependent rela- tionship.There was significance in apoptosis incidence among 5,10μmol/L groups and the control group（t=9.61,t=11.38,all P〈0.05）;For Walker-256 cells treated with ATRA,the Fas ex- pression mRNA was significantly up-regulated and the bcl-2 mRNA was significantly down-reg- ulated at the concentration of 10μmol/L ATRA, compared with the control group（t=12.33,t= 10.78,P〈0.05）.After treatment with 10μmol/L ATRA for 48 h,the Caspase 3 and Caspase 8 zy- mogen were significantly activated,compared with the control group（t=9.76,t=10.21,P〈 0.05）. CONCLUSION：ATRA induces Walker-256 he- patocarcinoma cell differentiation and apoptosis.
出处
《世界华人消化杂志》
CAS
北大核心
2008年第26期2929-2934,共6页
World Chinese Journal of Digestology
基金
湖北省自然科学基金资助项目
No.2007ABA284~~
