全反式维甲酸诱导肝癌细胞Hepa1-6成熟和分化过程中的自噬

摘要

Objective To investigate the effects of different concentrations of all-trans retinoic acid (ATRA) on the maturation, differentiation and autophagy of Hepa1-6 cells.Method Hepa1-6 cells were treated with 0.1,1,and 10 μmol/L ATRA,and the changes in the expressions of hepatic specific markers were detected using real-time PCR and Western blotting.Indocyanine green(ICG)and periodic acid-schiff(PAS)staining was used to assess the functional maturation of Hepa1-6 cells,and the cell-cell junction and autophagy were observed under transmission electron microscopy to determine the optimal concentration of ATRA for treatment. The expressions of autophagy-related markers in the cells were detected using Western blotting, and confocal microscopy was used to observe the autophagic flow in the cells transfected with ptfLC3 plasmid.Results Compared with the control cells,the hepatocytes treated with ATRA showed a concentration-dependent decrease in AFP expression and increase in the expressions of ALB, CK18, TAT and ApoB. ICG and PAS staining revealed significantly increased number of positive cells after ATRA treatment. Following ATRA treatment, the cells exhibited obviously increased tight junctions, cytoskeleton and number of autophagosomes under transmission electron microscopy. ATRA treatment resulted in significantly increased the expressions of autophagy-related markers LC3-II, Beclin-1, RAB7 and P62 and also an increased ratio of LC3-Ⅱ/LC3-Ⅰ(P<0.05).Confocal microscopy revealed obviously increased green and red spots in the cells after ATRA treatment. Conclusion ATRA can induce the maturation and differentiation and enhance the level of autophagy in Hepa1-6 cells.%目的 探讨不同浓度全反式维甲酸(ATRA)对小鼠肝癌细胞Hepa1-6成熟分化及细胞自噬水平的影响.方法 以小鼠肝癌细胞Hepa1-6为研究对象,分别给予0.1、1、10 μmol/L浓度的ATRA处理,荧光定量PCR和Western blot检测肝细胞相关标志基因的表达,吲哚菁绿(ICG)及过碘酸-希夫(PAS)染色检测小鼠肝癌细胞Hepa1-6的成熟功能,透射电镜观测细胞连接及自噬小体,选择最适的ATRA作用浓度;Western blot检测自噬相关标志蛋白的表达,ptfLC3质粒转染细胞后共聚焦观察自噬流变化,检测自噬流是否通畅.结果 相较于空白对照组,ATRA可呈浓度依赖性抑制肝前体标志蛋白AFP的表达,并促进肝细胞成熟标志ALB、CK18、TAT和ApoB的表达,ICG及PAS染色提示阳性细胞数明显增多;透射电镜结果显示ATRA诱导组的紧密连接和细胞骨架明显增多,可见高尔基复合体以及较多的自噬小体和自噬溶酶体.均以10 μmol/L组最为显著,作为最适终浓度.自噬相关标志蛋白LC3-II、Beclin-1、RAB7、P62的表达不同程度增高,LC3-Ⅱ/LC3-Ⅰ的比值明显增加(P<0.05),激光共聚焦可见10 μmol/L ATRA处理组细胞胞浆内绿色亮点的自噬小体及红色亮点的自噬溶酶体均较对照组明显增多.结论 ATRA可诱导小鼠肝癌细胞Hepa1-6的成熟分化,并促进细胞自噬水平增高.