黄连素抑制人喉癌Hep-2细胞增殖及机制研究

李蕾,丁小青,周建业,周卫东,汤夏冰*

(南京医科大学附属无锡人民医院，江苏 无锡 214023)

摘要：目的探讨黄连素对人喉癌Hep-2细胞增殖的抑制作用及其可能机制。方法体外培养Hep-2细胞，使用不同浓度黄连素诱导不同时间，应用MTT法检测细胞增殖抑制率；流式细胞术测定细胞周期及凋亡；实时荧光定量PCR和蛋白质免疫印迹(Western blot)法分别检测细胞周期蛋白D(CyclinD)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)基因和蛋白表达水平。结果与0 μmol/L黄连素对照组比较，2.5、5.0、10.0、20.0 μmol/L黄连素作用Hep-2细胞24、48 h，可明显抑制细胞增殖，且呈浓度时间依赖性(P<0.05)；2.5、5.0、10.0 μmol/L黄连素作用 Hep-2细胞48 h，细胞早期凋亡率明显升高，呈浓度依赖性(P<0.05)；G0/G1期细胞增多(P<0.05)，诱导细胞阻滞于G0/G1期；10μmol/L黄连素作用Hep-2细胞48 h，Bax基因和蛋白表达水平明显升高(P<0.05)，CyclinD、Bcl-2基因和蛋白表达水平明显下降(P<0.05)。结论黄连素能抑制喉癌细胞增殖，引起G0/G1期阻滞，并诱导其凋亡，其作用机制可能与Bax基因表达上调及CyclinD、Bcl-2基因表达下调有关。

关键词：黄连素；喉癌；细胞增殖

DOI:10.13463/j.cnki.cczyy.2015.06.004

中图分类号：R767文献标志码：A

文章编号：2095-6258(2015)06-1109-03

基金项目:南京医科大学科技发展基金(2013NJMU161, 2013NJMU167)。

作者简介:李蕾(1986-)，女，硕士研究生，主要从事喉癌的基础与临床研究。

收稿日期:(2015-04-20)

*通信作者:汤夏冰，电话-15861598912,电子信箱-txbny@sina.com

Berberine on proliferation of human laryngeal Hep-2 carcinoma cells

LI Lei, DING Xiaoqing, ZHOU Jianye, ZHOU Weidong, TANG Xiabing*

(Wuxi People’s Hospital Affiliated to Nanjing Medical University,Wuxi 214023,Jiangsu Province,China)

Abstract:ObjectiveTo investigate the effect of berberine on proliferation of human laryngeal Hep-2 carcinoma cells and its possible mechanism. MethodsThe Hep-2 cells were treated with different concentrations of berberine for different hours, the proliferation inhibitory rates were detected by MTT assay; the cell cycle and apoptosis were analyzed by flow cytometry; the levels of CyclinD, Bcl-2 and Bax were measured by real-time PCR and Western blot. ResultsThe proliferation inhibition rates of Hep-2 cells treated with 2.5、5.0、10.0、20.0 μmol/L berberine for 24 and 48 hours were increased compared with 0μmol/L group in concentration-and time-dependent manners (P<0.05). The early apoptotic rate was increased in concentration-dependent manner (P<0.05), and the proportion of cells in G0/G1 phase was increased after treated with 2.5, 5.0, 10.0 μmol/L berberine for 48 hours (P<0.05). The levels of Bax mRNA and protein expression significantly increased (P<0.05), while the levels of CyclinD and Bcl-2 expression significantly decreased (P<0.05). ConclusionBerberine can inhibit proliferation, and induct cell cycle G0/G1 arrest and apoptosis of Hep-2 cells probably via upregulation of Bax and downregulation of CyclinD and Bcl-2 mRNA and protein expression.

Keywords:berberine; laryngeal carcinoma; cell proliferation

黄连素又称小檗碱，属于异喹啉类生物碱，是一种传统的中医消炎及抗菌药物。研究[1-12]表明，黄连素在肝癌、肺癌、乳腺癌、鼻咽癌等多种肿瘤中有抗癌作用。但在喉癌发生、发展中的作用及调控机制尚未见报道。本研究以人喉癌Hep-2细胞作为研究对象，体外研究黄连素对Hep-2细胞增殖的影响及其可能机制，为黄连素的临床应用提供理论依据。

1材料与方法

1.1材料人喉癌细胞株Hep-2购自中国典型培养物保藏中心；MEM培养液购自Hyclone公司；黄连素、MTT购自Sigma公司；细胞周期、Annexin V-FITC、细胞凋亡检测试剂盒、蛋白质免疫印迹(Western blot)相关试剂购自碧云天公司；Trizol、逆转……