3D培养的非小细胞肺癌多倍体巨细胞模型构建及其生物学特性的研究

Construction of a polyploid giant cell model of non-small cell lung cancer in 3D culture and its biological properties

目的探索三维(3D)人非小细胞肺癌多倍体巨细胞与二维(2D)模型的基因表达差异。方法人非小细胞肺癌细胞系NCI-H1299购自中国科学院上海细胞库。培养非小细胞肺癌NCl-H1299细胞球体,加入多西紫杉醇(Doc)2.5μmol/L、5.0μmol/L、10.0μmol/L诱导,24 h后取出药物继续在新鲜培养基中培养72 h,收集细胞,另设未加药组作为对照组。Hochest染色观察细胞核的变化,活-死细胞染色试剂盒观察细胞活性,流式细胞术检测细胞凋亡,流式细胞术检测细胞倍性,RT-PCR检测上皮间充质转化相关基因以及干性相关基因的表达差异。结果3D培养的NCl-H1299细胞经Doc处理后细胞核增大,Calcein AM/PI染色显示Doc处理组细胞的死亡情况与加药浓度成正相关。5μmol/L组细胞多倍体细胞比例(35.2±5.88)明显高于对照组(17.26±2.26),3D培养的Doc处理组的早期凋亡[(9.06±1.24)%]低于对照组[(16.2±1.82)%],晚期凋亡[(19.73±1.98)%]低于对照组[(26.13±1.36)%],2D培养的Doc处理组早期凋亡[(23.96±1.82)%]高于对照组[(0.94±0.41)%],晚期凋亡[(23.91±0.96)%]高于对照组[(2.66±0.84)%],差异均有统计学意义(P<0.05)。3D多倍体肿瘤巨细胞模型上皮间质转化相关基因相较2D模型表达降低,3D培养细胞干性相关基因经Doc处理后与在2D培养中趋势相反。结论本研究成功构建了非小细胞肺癌多倍体巨细胞3D培养模型,并证明其在上皮间充质转化与干性相关基因表达与2D培养模型上有一定的差异,该模型可更好地模拟体内肿瘤的发展变化。

Objective To investigate gene expression differences between three-dimensional(3D)human non-small cell lung cancer polyploid giant cells and two-dimensional(2D)models.Methods The human non-small cell lung cancer cell line NCI-H1299 was purchased from Shanghai Cell Bank,Chinese Academy of Sciences.Non-small cell lung cancer NCl-H1299 cell spherores were cultured,adding docetaxel(Doc)2.5μmol/L,5.0μmol/L,10.0μmol/L.After 24 h,the drug was removed and cultured in fresh medium for 72 h.The cells were collected,and another unmedicated group was used as the control group.Changes in nuclei were observed by Hochest staining,cell activity by live-dead cell staining kit,apoptosis by flow cytometry,cell ploidy by flow cytometry,and differential expression of genes related to epithelial mesenchymal transition by RT-PCR.Results 3D cultured NCl-H1299 cells treated with Doc had an enlarged nucleus,and Calcein AM/PI staining showed that cell death in the Doc-treated group was positively correlated with drug addition concentration.The proportion of polyploid cells in the 5μmol/L group(35.2±5.88)was significantly higher than that in the control group[(17.26±2.26)],early apoptosis[(9.06±1.24)%]was lower than that in the control group[(16.2±1.82)%],late apoptosis[(19.73±1.98)%]was lower than that of the control group[(26.13±1.36)%],early apoptosis in the 2D cultured Doc-treated group[(23.96±1.82)%]was higher than that in the control group[(0.94±0.41)%],late apoptosis[(23.91±0.96)%]was higher than that in the control group[(2.66±0.84)%],the differences were statistically significant(P<0.05).The expression of genes related to epithelial stromal transformation in 3D polyploid tumor giant cells was decreased compared with 2D model,and the stemness related genes in 3D cultured cells showed the opposite trend to that in 2D culture.Conclusions This study successfully constructed a 3D culture model of polyploid giant cell in non-small cell lung cancer and demonstrated that there were some differences in the gene expression of epithelial mesenchymal transition and stemness and the 2D culture model,which can better mimic the changes in tumor development in vivo.