白花蛇舌草总黄酮对肝细胞癌干细胞增殖及凋亡的影响  

Effects of total flavone of oldenlandia diffusa on the proliferation and apoptosis of hepatocellular carcinoma stem cell

作　　者：姚博文[1] 李亚昭 廖子君 鲁叶 张祥[4] 马婕群 李倩 张彦兵 YAO Bowen;LI Yazhao;LIAO Zijun;LU Ye;ZHANG Xiang;MA Jiequn;LI Qian;ZHANG Yanbing(Department of Hepatobiliary Surgery,The First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061;Center for Translational Medicine,The First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061;Department of Oncology,Shaanxi Provincial Cancer Hospital,Xi’an 710061;Department of Cardiac Diagnosis,The Affiliated Children Hospital of Xi’an Jiaotong University,Xi’an 710003,China)

机构地区：[1]西安交通大学第一附属医院肝胆外科,陕西西安710061 [2]西安交通大学第一附属医院转化医学中心,陕西西安710061 [3]陕西省肿瘤医院肿瘤内科,陕西西安710061 [4]西安交通大学附属儿童医院心电诊断科,陕西西安710003

出　　处：《西安交通大学学报（医学版）》2023年第3期389-395,共7页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：西安交通大学第一附属医院医院科研发展基金国科金青年储备项目(No.2021QN-04);陕西省重点研发计划基金资助项目(No.2021SF-220)。

摘　　要：目的探讨白花蛇舌草总黄酮(FOD)对Huh7来源的肝细胞癌干细胞增殖及凋亡的影响。方法培养Huh7细胞系,通过流式分选技术筛选出Huh7细胞系中CD133阳性的干细胞(CD133^(+)-Huh7);流式分析术检测分选后CD133阳性细胞比例;Western blotting实验检测分选纯化前后细胞干性指标Nanog、Oct4、Sox2的表达。分别用0、50、100、400μg/mL FOD分别作用CD133^(+)-Huh7肝细胞癌干细胞24、48、72、96 h,应用CCK8(Cell Counting Kit-8)法检测其对细胞增殖的影响;平板克隆实验检测各组细胞增殖能力变化;Annexin V-PE/7-AAD法检测各组细胞凋亡比例,并用Western blotting法检测p53、FAS-FADD、抗凋亡蛋白Bcl-2、促凋亡蛋白Bax及凋亡指标Cleaved-Caspae3等凋亡通路蛋白的表达。结果纯化后的Huh7细胞的干性标志物Nanog、Oct4、Sox2表达更高。100μg/mL FOD刺激CD133^(+)-Huh7干细胞72 h,CCK8实验结果显示其细胞增殖较阴性对照组(0μg/mL处理组)降低(P<0.05),平板克隆实验亦显示其细胞增殖变弱;细胞凋亡比例较阴性对照组显著升高(P<0.05),抗凋亡蛋白Bcl-2表达降低而促凋亡蛋白Bax以及凋亡指标Cleaved-Caspae3的蛋白表达升高,p53、FAS、FADD通路显著上调。结论FOD可显著抑制CD133^(+)-Huh7肝细胞癌干细胞增殖,并显著促进细胞凋亡。Objective To investigate the effects of total flavone of oldenlandia diffusa(FOD)on the proliferation and apoptosis of hepatocellular carcinoma(HCC)stem cells sorted from Huh7.Methods Human HCC cell lines Huh7 was cultured in vitro;CD133 positive(CD133^(+))stem cells in Huh7 cell line were sorted by flow cytometry,and stem cell markers such as Nanog,Oct4 and Sox2 were tested by Western blotting.CD133^(+)-Huh7 was stimulated by different concentrations(0μg/mL,50μg/mL,100μg/mL and 400μg/mL)of FOD for different time(24 h,48 h,72 h and 96 h).CCK8 and plate cell cloning assay were used to detect the effect of FOD on CD133^(+)-Huh7 proliferation while Annexin V-PE/7-AAD was used to detect the effect of FOD on CD133^(+)-Huh7 apoptosis.Western blotting was used to detect the protein expressions of protein 53(P53),factor associated suicide-Fas-associating protein with a novel death domain(Fas-FADD),B-cell lymphoma-2(Bcl-2),Cleaved-Caspase3,and Bcl-2 associated X protein(Bax).Results More than 95%of stem cells were purified for further experiments.Cell proliferation of CD133^(+)-Huh7 was significantly inhibited by FOD,with the significant suppression at the concentration of 100μg/mL for 72 h compared with negative control group(P<0.05).The apoptosis rate was significantly upregulated than that in the negative control group(P<0.05).The protein expression of Bcl2 decreased while Bax and Cleaved-Caspae3 increased via FAS/FADDD and P53 axis.Conclusion FOD can significantly inhibit the proliferation and promote the apoptosis of CD133^(+)-Huh7.

关 键 词：肝细胞癌 干细胞 白花蛇舌草总黄酮(FOD) 增殖 凋亡 

分 类 号：R735.7[医药卫生—肿瘤]