新型肝癌组织的体外三维培养模型  

A new 3D culture model of primary hepatocellular carcinoma tissues in vitro

作　　者：尹萌 陈焕鹏 李永超 郭荣平[1] 林小军 刘忠华[4] 余波澜[3] 黄朝峰[2] 赵擎宇[1] YIN Meng;CHEN Huanpeng;LI Yongchao;GUO Rongping;LIN Xiaojun;LIU Zhonghua;YU Bolan;HUANG Zhaofeng;ZHAO Qingyu(Sun Yat-sen University Cancer Center,Guangdong Guangzhou 510060,China;Zhongshan School of Medicine,Sun Yat-sen University,Guangdong Guangzhou 510080,China;the Third Affiliated Hospital of Guangzhou Medical University,Guangdong Guangzhou 510150,China;Animal Experiment Center,South China Agricultural University,Guangdong Guangzhou 510642,China)

机构地区：[1]中山大学肿瘤防治中心,广东广州510060 [2]中山大学中山医学院,广东广州510080 [3]广州医科大学第三附属医院,广东广州510150 [4]华南农业大学动物实验中心,广东广州510642

出　　处：《现代肿瘤医学》2020年第21期3673-3678,共6页Journal of Modern Oncology

基　　金：广东省省级科技计划项目(编号:2015A030302016)。

摘　　要：目的:利用肝癌组织进行体外的三维(three-dimension,3D)组织块培养,通过添加PD98059和霍乱毒素(cholera toxin,CT)优化培养条件,建立一种高活性的肝癌组织体外培养模型。方法:收集中山大学肿瘤防治中心肝胆科的肝癌组织标本,经清洗、冻存、复苏等处理后分为对照组、PD组、CT组、PD+CT组,同时进行体外的二维(two-dimension,2D)和三维培养。各组组织采用DNA断裂的原位末端标记法(TUNEL)检测组织细胞凋亡情况,qRT-PCR检测BCL-2、BID、Caspase 3、Cyclin D1、CDK2、ELK-1、C-FOS、C-MYC、C-JUN等相关基因的mRNA表达水平,免疫荧光染色检测BCL-2和cleaved caspase-3蛋白表达情况,筛选并确定最优的体外培养方法。结果:3D培养组的组织细胞凋亡均少于2D培养组;PD98059通过上调BCL-2蛋白的表达,下调cleaved caspase-3蛋白的表达,激活MEK/ERK下游基因,从而减少体外培养的组织细胞凋亡,增强其抗凋亡能力;霍乱毒素通过上调Cyclin D1、CDK2的基因表达,促进其增殖。结论:我们开发了一种新型肝癌组织的体外三维培养模型,它可以作为进一步的药物试验和人源性异种移植(PDX)模型的工具。Objective:To establish a high-activity three-dimension(3D)model of primary hepatocellular carcinoma tissues in vitro using Matrigel with the culture medium optimized by adding PD98059 and cholera toxin(CT).Methods:Hepatocellular carcinoma tissues were obtained from the hepatobiliary surgery department of the Sun Yat-sen University Cancer Center.After washing,cryopreservation and resuscitation,tissues were divided into control group,PD group,CT group and PD+CT group.Two-dimension(2D)culture and 3D culture in vitro were conducted at the same time.TdT-mediated dUTP Nick-End Labeling(TUNEL)immunofluorescence staining was used for apoptosis detection.Quantitative Real-time Polymerase Chain Reaction(qRT-PCR)was used to observe the gene expressions of human BCL-2,BID,Caspase 3,Cyclin D1,CDK2,ELK-1,C-FOS,C-MYC and C-JUN.The expressions of BCL-2 and cleaved caspase-3 proteins were also detected by immunofluorescence staining.Results:3D culture had less apoptosis than 2D culture.PD98059 reduced the apoptosis of tissue cells and enhanced its anti-apoptosis ability by up-regulating the expression of BCL-2 protein,down-regulating the expression of cleaved caspase-3 protein,as well as activating the MEK/ERK downstream genes.Cholera toxin promoted its proliferation by up-regulating the gene expressions of Cyclin D1 and CDK2.Conclusion:We developed a new 3D culture model of primary hepatocellular carcinoma in vitro,which can serve as a tool for further drug trials and patient-derived xenograft(PDX)models.

关 键 词：肝细胞癌 原代组织 3D培养 PD98059 霍乱毒素 

分 类 号：R735.7[医药卫生—肿瘤]