三维与二维培养肝癌HepG2细胞的药物敏感性及生物学特性比较

目的：观察肝癌HepG2细胞株在不同培养模式下的生物学性质、耐药性。方法：采用琼脂糖预铺底法建立体外三维细胞模型,通过倒置显微镜、扫描电镜等方法证实三维细胞模型的成功建立;采用透射电镜、流式细胞术等技术方法,比较二维培养及三维培养细胞中细胞间连接、细胞周期、细胞凋亡的差异;采用MTT法获得不同培养模式下药物5-FU、DDP作用的细胞抑制率。结果：三维培养HepG2细胞第7天可形成直径160μm左右的多细胞球,悬浮生长;二维培养的HepG2细胞贴壁生长。三维培养与二维培养相比,存在细胞周期阻滞：G1期细胞增多[（59.23±1.28）%vs（39.45±1.21）%],G2期细胞减少[（12.47±0.14）%vs（36.15±1.09）%],早期自发凋亡细胞比例减少[（1.62±0.50）%vs（6.29±1.61）%],具有统计学差异（P〈0.05）;S期细胞比例变化不大[（28.30±0.93）%vs（24.40±0.96）%,P〉0.05],晚期自发凋亡坏死细胞比例变化不大[（0.65±0.17）%vs（0.66±0.29）%,P〉0.05]。二维培养细胞间连接少见,三维培养细胞连接结构丰富,可见桥粒连接、中间连接及紧密连接结构;三维培养下5-FU、DDP药物的细胞抑制率明显降低（与二维培养相比）。结论：经倒置显微镜和扫描电镜观察证实肝癌HepG2细胞体外三维培养模型成功建立,在三维培养条件下HepG2细胞对5-FU、DDP表现出多细胞耐药;与二维培养相比,三维培养的细胞间的相互作用有明显增强,且多细胞球生物学特性主要表现为细胞周期阻滞和早期凋亡减少,推测其可能与多细胞耐药有关。

HepG2 cells cultured in three-dimensional model or monolayer

Objective：We chose the cell line HepG2 of liver cancer to establish a three dimensional culture model,and compare the biological character,drug resistance of the cells in different culture mode.Methods：To establish the 3D cell culture system in vitro with liquid overlay technique,and the cells were identified by inverted microscope and scanning electron microscope.Compare the cell junction,cell cycle,cell apoptosisand between 2D and 3D cultured cells by means of flow cytometry,transmission electro-microscope .Compare the cell inhibition ratio of 5-FU,DDP between 2D and 3D cultured cells.Results：Multicellular spheroids were floating in the medium,the diameter of spheroids was approximately 160μm 7 days later.When HepG2 cells were cultured as two dimension,cells were adherent to the bottom of flask.Cells in G1 phase increased[（59.23±1.28）% vs （39.45±1.21）%],in G2 decreased [（12.47±0.14）% vs （36.15±1.09）%],earlier period apoptosis rate decresed[（1.62±0.50）% vs （6.29±1.61）%] were found in 3D cells,these differences between 2D and 3D cells were statistically significant（P〈0.05）.The modify of S phase and advanced stage apoptosis rate were not statistically significant[（28.30±0.93）% vs （24.40±0.96）%],[（0.65±0.17）% vs （0.66±0.29）%] （P〉0.05）.The structures of cell junction were different in both culture modes.Compared with 2D culture,the inhibition ratio to 5-FU,DDP of 3D cells was much lower.Conclusion：3D cell culture system in vitro was successfully established.Compared with 2D culture,there was remarkable decline in susceptibility to drugs including 5-FU,DDP in cells of 3D culture.There is cell junction in 3D culture mode,which shows a significant enhancement on intercellular interaction; and the biological characters are different between 3D and 2D,as has been demonstrated in cell phase block and decreased earlier period apoptosis; we presume there are relations among the biological characters,the cell junctions and MCR.