摘要目的 检测和分选人卵巢上皮性癌(卵巢癌)细胞系OVCAR-3中的侧群(SP)细胞,鉴定其是否具有肿瘤干细胞的生物学特性.方法 OVCAR-3细胞经DNA染料Hoechst 33342染色后,采用流式细胞仪检测和分选低染(即SP细胞)和高染[即非侧群(NSP)细胞]的两群细胞.通过裸鼠体内的有限稀释成瘤试验对比SP和NSP细胞的成瘤能力[以成瘤比率(成瘤的裸鼠数/接种的裸鼠数)和成瘤时间表示]、自我更新和分化能力.实时PCR技术检测SP和NSP细胞中干性基因(Oct4、Klf4、Nanog基因)和三磷酸腺苷结合盒(ABC)转运蛋白家族重要分子——ABCG2、ABCB1、ABCC2基因的表达.药敏试验检测SP和NSP细胞对4种化疗药物(顺铂、紫杉醇、多柔比星、米托蒽醌)的敏感性.结果 OVCAR-3细胞系中可检测到少量的SP细胞,其比例为(1.13±0.39)％.102个SP细胞即可在裸鼠体内成瘤,成瘤比率为2/5,成瘤时间为52～61 d；而至少104个NSP细胞才能成瘤,成瘤比率为2/5,成瘤时间为64 ～ 98 d.SP细胞的成瘤性至少为NSP细胞的100倍.103个SP细胞接种裸鼠后所形成的瘤组织中SP细胞的比例为(2.09±0.73)％,与分选前的比例相似.SP细胞中干性基因Oct-4和Klf4及ABCG2和ABCC2基因的表达量分别是NSP细胞的(1.95±0.41)、(4.26±0.63)、(3.22 ±0.36)和(1.76±0.26)倍,分别比较,差异均有统计学意义(P＜0.05).在0.25 μg/ml顺铂、0.01 μmol/L紫杉醇、0.25 μmol/L多柔比星和0.05 μg/ml米托蒽醌分别作用下,SP和NSP细胞的相对活性分别为0.757±0.105、0.474±0.035,0.521±0.092、0.384±0.073,0.742±0.051、0.526 ±0.088,0.690±0.096、0.466 ±0.112；相同药物浓度下,SP细胞的相对活性显著高于NSP细胞(P均＜0.05).结论 卵巢癌细胞系OVCAR-3来源的SP细胞与NSP细胞相比,具有显著增强的体内成瘤能力、自我更新和分化能力、高表达干性基因和ABC转运蛋白家族基因以及多药耐药等肿瘤干细胞样细胞的生物学特性.SP表型可考虑作为分选卵巢癌干细胞样细胞的有效方法.

abstractsObjective To identify the presence of side population (SP) cells in human ovarian cancer cell line OVCAR-3 and to investigate whether SP cells have the characteristics of cancer stem cells.Methods SP and non-SP (NSP) cells from OVCAR-3 were isolated by fluorescence-activated cell sorting after being stained by DNA-binding dye Hoechst 33342.Limiting dilution transplantation assay,realtime PCR,and drug sensitivity assay were performed to compare the tumorigenic ability,differentiation ability in vivo,the mRNA expressiou of "stemness" marker (Oct-4,Klf4,and Nanog) and ATP-binding cassette (ABC) transporter (ABCG2,ABCB1,and ABCC2),and response to multiple drugs (cisplatin,paclitaxel,doxorubicin,and mitoxantrone ) between SP and NSP cells.Results A few of SP cells [ ( 1.13 ±0.39) ％ ] which were sensitive to reserpine were identified in OVCAR-3 cells.The injection of as few as 102 SP cells initiated tumors in two of five mice.Tumor latency was 52 -61 days.However,the NSP cells did not generate any tumors in mice until 104 NSP cells were injected (two of five mice).Tumor latency was 64 - 98 days.Tumorigenicity of SP cells was enhanced by at least 100-fold than that of NSP cells.The SP cells regenerated both SP [ ( 2.09 ± 0.73 ) ％ ] and NSP populations in vivo with a fraction size that was comparable to the original population.The mRNA expression of " stemness" genes Oct-4,Klf4 and ABC transporters ABCG2,ABCC2 genes were elevated in SP cells compared to NSP cells,the fold changes were 1.95±0.41 (P＜0.05),4.26 ±0.63 (P＜0.01),3.22±0.36 (P＜0.01),and 1.76±0.26 (P＜0.01 ),respectively.The relative activity of SP and NSP cells were 0.757 ± 0.105 versus 0.474 ± 0.035 (P＜0.01),0.521 ±0.092 versus 0.384 ±0.073 (P＜0.05),0.742 ±0.051 versus 0.526 ±0.088 (P ＜0.01 ),and 0.690 ± 0.096 versus 0.466 ± 0.112 ( P ＜ 0.01 ) when they exposed to 0.25 μg/ml cisplatin, 0.01 μmol/L paclitaxel, 0.25 μmol/L doxorubicin, and 0.05 μg/ml mitoxantrone,respectively.Conclusions SP cells from OVCAR-3 have enhanced self-renewal,differentiation,and tumorinitiating capacity compared to NSP cells.The mRNA expression of stemness genes and ABC transporters are markedly elevated in SP cells,which showed resistance to multiple chemotherapeutic drugs and have characteristics of cancer stem-like cells.Therefore,SP phenotype could be used as a marker to isolate the cancer stem-like cells in ovarian cancer.