摘要
目的探讨表观遗传学药物对2D和3D胆管癌系统性药效毒理学研究。方法采用将单层培养的TFK-1细胞接种于2％polyHEMA100μ1包被的u型底-96孔板中制备TFK-13D肿瘤细胞球。采用wsT法和FDA／PI荧光染色法检测表观遗传学药物作用后2D和3D胆管癌肿瘤模型中肿瘤细胞的存活率。采用甲基化特异性PCR检测药物作用前后APC、E—Cadherin和p16INK4A基因甲基化程度的改变。结果3D培养方法能形成外观圆形内部紧密的肿瘤小球，球体的平均直径为350-400μm。TFK-1胆管癌细胞3D模型比2D模型对药物治疗抵抗更明显。与2D肿瘤模型相比，高剂量的药物才能逆转3D模型中肿瘤细胞的高甲基化状态，但是2D模型表现的剂量相关的去甲基化特点在3D模型中并未出现。结论实验成功建立胆管癌上皮细胞3D模型。立体的3D肿瘤模型比单细胞层的2D模型生物学特点更为复杂，该特点能影响表观遗传学药物的药效。实验证实，3D人胆管上皮细胞模型可作为表观遗传学药物治疗胆管癌研究中的一种有效可靠的模型。
Objective To investigate the effects of epigenetic drugs on 2D- and 3D-cultured cholangiocarcinoma cells in vitro. Methods In this study, we have built compact and round TFK-1 spheroid in poly-HEMA coated 96-well plate and determined the effects of epigenetical drugs on 2D and 3D cultured cholangiocarcinoma cells ： TFK-1. Viability of 2D and 3D cells model was examined by WST assay and FDA/PI staining. Using methylation-specific PCR analysis, we demonstrated the changes of methylation status of promoters regarding three tumor suppressor genes APC, E-Cadherin, and p16 INK4A. Results The average diameters of compact and round TFK-1 spheroids were in the range of 350- 400 μm. The TFK-1 spheroid cells were more resistant to the epigenetic drugs and demonstrated a 11. 2155-fold higher IC50 values for hydralazine and valproic acid than the same cells grown as monolayer. Higher doses of epigenetic drugs were needed to reverse the hypermethylation status in 3D cultured cells than 2D eells; however, the parallel dosage - dependent characteristic did not show in the 3D spheroid group. Conclusions Taken together, we established a 3D culture model of human cholangiocarcinoma epithelial spheroid. The 3D spheroid cells are more complex than the 2D monolayer cells and their unique characteristics are able to affect the consequences of epigenetie therapy. The 3D spheroid is a promising model for the research of epigenetic therapy.
出处
《中华肝胆外科杂志》
CAS
CSCD
北大核心
2012年第2期139-144,共6页
Chinese Journal of Hepatobiliary Surgery
基金
中、德合作项目“Nanocancer,0312026A”和“Nano-Biocomp,0312025A”资助
