新型肝癌组织的体外三维培养模型

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。

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

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.