Fang Shu-Yu, Cui Jie-Jie, Gong Meng-Jia, He Yun, Zhang Jing-Fang, Bi Yang
Laboratory of Stem Cell Biology and Therapy, Children's Hospital of Chongqing Medical University/Ministry of Education Key Laboratory of Child Development and Disorders/International and National Science and Technology Cooperation Base of Child Development and Disorder/Key Laboratory of Pediatrics of Chongqing, Chongqing 400014, China.E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2018 May 20;38(5):527-533. doi: 10.3969/j.issn.1673-4254.2018.05.04.
To investigate the effects of different concentrations of all-trans retinoic acid (ATRA) on the maturation, differentiation and autophagy of Hepa1-6 cells.
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.
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.
ATRA can induce the maturation and differentiation and enhance the level of autophagy in Hepa1-6 cells.
探讨不同浓度的全反式维甲酸（ATRA）对Hepa1-6细胞成熟、分化及自噬的影响。
用0.1、1和10 μmol/L的ATRA处理Hepa1-6细胞，采用实时定量聚合酶链反应（PCR）和蛋白质免疫印迹法检测肝脏特异性标志物表达的变化。采用吲哚菁绿（ICG）和过碘酸希夫（PAS）染色评估Hepa1-6细胞的功能成熟情况，通过透射电子显微镜观察细胞间连接和自噬情况，以确定ATRA处理的最佳浓度。采用蛋白质免疫印迹法检测细胞中自噬相关标志物的表达，利用共聚焦显微镜观察转染ptfLC3质粒的细胞中的自噬流。
与对照细胞相比，经ATRA处理的肝细胞中甲胎蛋白（AFP）表达呈浓度依赖性降低，而白蛋白（ALB）、细胞角蛋白18（CK18）、酪氨酸氨基转移酶（TAT）和载脂蛋白B（ApoB）的表达增加。ICG和PAS染色显示ATRA处理后阳性细胞数量显著增加。ATRA处理后，透射电子显微镜下细胞的紧密连接、细胞骨架和自噬体数量明显增加。ATRA处理导致自噬相关标志物微管相关蛋白1轻链3-II（LC3-II）、Beclin-1、RAB7和P62的表达显著增加，且LC3-II/LC3-I比值升高（P<0.05）。共聚焦显微镜显示ATRA处理后细胞中的绿色和红色斑点明显增加。
ATRA可诱导Hepa1-6细胞成熟和分化，并提高其自噬水平。
