摘要
目的研究亚致死温度射频消融(RFA)对肝癌干细胞(LCSC)产生及其相关转录因子表达的影响。方法利用小鼠Hep1-6肝癌细胞株和肝细胞癌(HCC)患者临床样品,检测LCSC相关标志物和转录因子的表达。结果不同温度分别刺激Hep1-6细胞后发现,45℃是不能诱导细胞死亡的亚致死性温度。流式细胞仪(FCM)检测显示45℃处理可引起CD13^+、CD44^+、CD90^+、CD133^+Hep1-6细胞水平明显上调,提示45℃温度导致Hep1-6细胞中以上各型LCSC产生增加;实时定量聚合酶链反应(RT-q PCR)检测显示45℃温度导致CD13、CD90、CD133 m RNA水平明显上调。CD13 m RNA水平在5例HCC患者复发肝癌组织中均明显上调,CD133 m RNA在4例复发肝癌中上调,CD90 m RNA仅在1例复发肝癌中上调;FCM检测显示CD13^+LCSC水平在4例复发肝癌中明显上调,CD133^+LCSC水平仅在1例复发肝癌中上调,提示CD13^+LCSC水平上调与45℃温度关系更密切。RT-q PCR检测显示4例CD13^+LCSC上调的复发肝癌患者13个LCSC相关转录因子中Sox2、Stat1明显上调,FCM检测显示45℃处理Hep1-6细胞后Sox2、Stat1 m RNA明显上调。用Sox2、Stat1 si RNA分别沉默了Sox2、Stat1基因,表明Sox2、Stat1均参与了45℃温度诱导的CD13^+LCSC产生。结论 RFA治疗中45℃亚致死性温度所致CD13^+LCSC水平增高与Sox2、Stat1表达有关。该结果对肝癌复发研究有一定借鉴意义。
Objective To study the effect of radiofrequency ablation （RFA） with sublethal temperature on the production of liver cancer stem cells （LCSCs） and on the expression of LCSCs-related transcriptional factors. Methods Mouse hepl-6 hepatoma cell line and clinical samples of patients with hepatocellular carcinoma （HCC） were used to test the expressions of LCSCs-related markers and transcriptional factors. Results Different temperatures were used to stimulate Hepl-6 cells, and it was proved that the temperature of 45℃ was a sublethal temperature that could not induce cell death. Flow cytometry testing showed that treatment with 45℃ could obviously increase CD13^＋, CD44^＋, CD90 and CD133^＋ Hepl-6 cells, suggesting that treatment with 45℃ could increase the production of above mentioned types of LCSCs in hep1-6 cells. Real-time quantitative polymerase chain reaction （RT-qPCR） assay indicated that the temperature of 45% could cause significant increase in CD13, CD90 and CD133 mRNA. In all 5 HCC patients, CD13 mRNA in the recurrent HCC lesions was remarkably increased, CD133 mRNA was increased in 4 patients with recurrent HCC, and CD90 mRNA was increased in only one patient with recurrent HCC. Flow cytometry testing revealed that CD13^＋ LCSCs were strikingly increased in 4 recurrent HCC patients, while CD133^＋ LCSC was increased in only one patient, suggesting that more close correlation existed between the increase of CD13^＋ LCSCs and the temperature of 45℃. RT-qPCR assay showed that in 4 recurrent HCC patients with increased CD13^＋ LCSC, the Sox2 and Statl among 13 LCSCs-related transcriptional factors were obviously increased. Flow cytometry testing showed that 45℃ treatment also increased the expression of Sox2 and Statl mRNA in Hep1-6 cells. Finally, Sox2 and Stat1 could be knockdown by siRNAs, indicating that both Sox2 and Statl transcriptional factors were involved in 45℃-induced production of CD13^＋ LCSCs in Hep1-6 ceils. Conclusion In RFA therapy, the use of sublethal temperature of 45℃ can increase CD13^＋ LCSCs, which is related to the promotion of Sox2 and Statl expression. The results of this study can be used for reference in the research of liver cancer recurrence.
作者
郝美君
刘凯
郭向华
欧阳雅博
乔录新
石英
陈德喜
郑加生
HAO Meijun LIU Kai GUO Xianghua OUYANG Yabo QIAO Luxin SHI Ying CHEN Dexi ZHENG Jiasheng(Affiliated Beijing You ＇an Hospital of Capital Medical University, Beijing 100069, China)
出处
《介入放射学杂志》
CSCD
北大核心
2017年第7期636-640,共5页
Journal of Interventional Radiology
基金
国家自然科学基金(81402556
81272266)
NSFC-NIH国际合作项目(81361120401)
首都发展基金(首发2014-1-1151)
北京市肝病研究所流式平台基金(BJIH-01602)
关键词
肝细胞癌
射频消融
亚致死温度
肝癌干细胞
hepatocellular carcinoma
radiofrequency ablation
sublethal temperature
liver cancer stem cell
