顺铂诱导多倍体肿瘤巨细胞模型的构建

Construction of a cisplatin-induced polyploid giant cancer cell model

目的:探讨顺铂在体外诱导肿瘤细胞构建多倍体肿瘤巨细胞(PGCC)模型的方法,并对构建的细胞模型特性进行考察。方法:分别采用1.5、3、6、12、24μg/mL的顺铂处理A549细胞24 h,以及3μg/mL的顺铂处理3、4、5 d,流式细胞术检测其DNA含量变化,选择DNA含量最高的组合作为顺铂诱导A549、HepG2、SK-OV-3细胞形成PGCC的浓度和时间;Giemsa染色观察3种已处理细胞的表面积和DNA核面积变化;逆转录定量PCR检测3种已处理细胞的干性基因Nanog、Sox-2、OCT-4、c-Myc的mRNA表达情况,流式细胞术检测3种已处理细胞的干性表面标志物CD44、CD133的表达情况;β-半乳糖苷酶染色检测3种已处理细胞的衰老情况;MTT法检测0.75、1.5、3、6、12、24、48、96μg/mL顺铂作用于3种已处理细胞72 h的细胞活力。结果:3μg/mL顺铂处理3 d可有效诱导3种细胞系形成多倍体(>4N)细胞。诱导后的细胞表现出以下特征:细胞和核面积显著增大;干性标志物CD44、CD133表达不同程度上调,部分干性基因表达增加;部分A549细胞呈现衰老表型;与对照组相比,源于A549和SK-OV-3细胞的PGCC对顺铂的耐受性增强(P<0.01)。结论:成功建立了顺铂诱导的源于A549细胞的PGCC模型,该模型诱导的A549 PGCC具有增大的细胞表面积和核面积,表达干性基因Nanog、Sox-2、OCT-4,对顺铂的耐受度增加,为进一步研究肿瘤耐药机制提供了有效的实验平台。

OBJECTIVE:To investigate the characteristics and mechanisms of cisplatin-induced polyploid giant cancer cells(PGCCs)in vitro.METHODS:Cultured cells were treated with cisplatin at concentrations of 1.5,3,6,12,and 24μg/mL for 3,4,and 5 days.DNA content was analyzed by flow cytometry to determine the optimal combination of concentration and duration for PGCC induction in A549,HepG2,and SK-OV-3 cells.Morphological changes and DNA nuclear area were examined through morphological observation and Giemsa staining.Expression of stemness genes(Nanog,Sox-2,OCT-4,c-Myc)was evaluated by RT-qPCR,while stemness surface markers(CD44,CD133)were analyzed by flow cytometry.Cell senescence was assessed byβ-galactosidase staining.After 72-hour treatment with cisplatin at concentrations ranging from 0.75 to 96μg/mL,cell viability was measured by the MTT assay.RESULTS:Treatment with 3μg/mL cisplatin for 3 d effectively induced polyploidy(>4N)in all three cell lines.The induced cells exhibited significantly increased cell and nuclear areas;varying degrees of upregulation in stemness markers CD44 and CD133,along with partial increase in stemness gene expression;partial senescence phenotype in A549 cells;and enhanced cisplatin tolerance in A549 and SK-OV-3 PGCCs compared to controls.CONCLUSION:The cisplatin-induced PGCC model in A549 cells demonstrated enlarged cell and nuclear areas,expressed stemness genes(Nanog,Sox-2,OCT-4),and showed increased cisplatin tolerance.The model may be useful for investigating,drug resistance mechanisms.