表观遗传学药物对肝内胆管癌的作用

Effects of epigenetic drugs in intrahepatic cholangiocarcinoma cells

目的筛选肝内胆管癌敏感的表观遗传学药物并研究其作用机制。方法采用细胞活力实验筛选对RBE细胞敏感的药物；在多种细胞株上比较其作用；选择最敏感的药物探讨其机制：通过Giemsa染色观察肝内胆管癌细胞经药物处理后的形态学变化，用流式细胞技术检测细胞周期分布，联合流式细胞技术、Westernblot检测细胞的凋亡。结果RBE细胞分别经96种表观遗传学药物处理48h，27μmol／L浓度下22种药物对RBE细胞有抑制作用（细胞活力〈60％），组蛋白去乙酰化酶（HDAC）抑制剂有12种，3trmol／L浓度下仅5种药物对RBE细胞有抑制作用，HDAC抑制剂4种；其中，玉米原斑病菌毒素（HCtoxin）对多种肝内胆管癌细胞株的抑制作用最为突出；与对照组比较，HCtoxin处理CCLP-1细胞48h后，能引起包括小体凋亡等多种细胞形态变化，能将细胞周期阻滞在G。期（82．93％比48．51％），能通过非半胱氨酰天冬氨酸特异性蛋白酶．3（Caspase-3）依赖的凋亡途径诱导细胞凋亡。结论HDAC抑制剂，尤其是HCtoxin，通过多种方式对肝内胆管癌细胞产生显著的抑制作用。

Objective Screening of epigenetic drugs sensitive to intrahepatic cholangiocarcinoma and exploring the mechanism. Methods Screening a 96 - epigenetic library in RBE cells through cell va- lidity assay and comparing the inhibition effect of these sensitive drugs on multiple cell lines ; Giemsa stai- ning was used to observe the cell morphological change and flow cytometry to check the cell cycle distribu- tion ; Morphological observation, flow cytometry technique and Western blotting were combined to check the cell apoptosis. Results Twenty two drugs from the 96 - epigenetic library had certain inhibition on RBE cells, including twelve histone deacetylase （HDAC） targeted species ; HC - toxin＇ s inhibition effect on four intrahepatic cholangiocarcinoma cell lines were the most obvious among the HDAC inhibitors＇ and were superior to that of gemcitabine; cells at G0/G~ phases in HC toxin treated group was much more than that in control group （82.93% vs. 48.51% ） and HC toxin brought about diverse of morphological chan- ges, apoptosis bodies and reduced mitosis included ; flow cytometry indicated intensively apoptosis inducted by HC toxin and it was not associated with Caspase - 3 depended pathways for the Caspase - 3 up - regula- ted in the trial groups tested by Western blotting was not remarkable in statistics. Conclusion HDAC in- hibitors, especially HC toxin, are of great potential in the treatment of intrahepatic cholangiocarcinoma.