高转移潜能肝癌HCCLM3细胞中侧群细胞的分离及其干细胞特性鉴定  

Isolation and stem cell property assessment of side population cells in hepatocellular carcinoma cell lines

作　　者：郭哲[1] 向邦德[1] 姜经航 张俊[2] 钟艳平[3] 苏节[3] 黎乐群[1] 

机构地区：[1]广西医科大学附属肿瘤医院肝胆外科,广西南宁530021 [2]广西医科大学第一附属医院超声科,广西南宁530021 [3]广西医科大学医学实验中心,广西南宁530021

出　　处：《中国肿瘤生物治疗杂志》2015年第2期220-224,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81160262;81260331);国家科技重大专项课题资助项目(No.2012ZX10002010001009)~~

摘　　要：目的:检测高转移潜能肝癌HCCLM3细胞株中侧群(side population,SP)细胞比例,分选SP细胞并探讨其生物学特性。方法:4种肝癌细胞株的转移潜能大小依次为HCCLM3>MHCC97H>MHCC97L>Huh7,分别予以荧光染料Hoechst33342染色,以加入维拉帕米的拮抗组为对照,流式细胞术检测SP细胞比例;从HCCLM3细胞中分选SP细胞和主群(main population,MP)细胞,采用悬浮球培养、Transwell侵袭实验及裸鼠成瘤实验判断HCCLM3 SP细胞是否具备肿瘤干细胞生物学特性;RT-PCR检测SP及MP细胞中干细胞相关基因的表达。结果:HCCLM3细胞中SP细胞比例为(16.9±1.8)%,显著多于MHCC97H、MHCC97L、Huh7中SP细胞比例[(8.4±0.7)%、(4.6±0.5)%、(1.0±0.2)%,均P<0.05]。HCCLM3细胞中,SP细胞悬浮球形成明显高于MP细胞[(25.3±5.1)%vs(11.2±2.6)%,P<0.05],SP细胞的侵袭能力显著高于MP细胞(P<0.05),SP细胞的裸鼠皮下成瘤能力也明显高于MP细胞。HCCLM3细胞的SP细胞中干细胞相关基因ABCG2、CD13、CD44、Nanog、Sox2及Klf4 mRNA平均表达水平分别是MP细胞的5.64、2.26、2.10、3.78、2.37、2.92倍(均P<0.05)。结论:肝癌HCCLM3细胞的SP细胞中富集了肝癌干细胞,这可能与其较高的转移潜能有关。Objective: To isolate and characterize side population( SP) cells in human hepatocellular carcinoma(HCC) cells. Methods: Four HCC cell lines with a metastatic potential in an order of HCCLM3 > MHCC97-H >MHCC97-L > Huh7 were cultivated in routine culture conditions. Single cell suspensions were made for these lines and incubated with different concentrations of Hoechst33342 either alone or with 50 μmol / l verapamil. The proportion of SP cells in these suspensions was analyzed by flow cytometry. SP cells and main population( MP) cells in the single cell suspension of cell line HCCLM3 were sorted. The HCCLM3-derived SP cells and MP cells were then comparatively assessed for tumorsphere formation,migration and invasion in vitro,for tumorigenicity in vivo and for the expression of stem cell-related genes at the mRNA level. Results: The proportion of SP proportion was significantly different among HCCLM3(16. 9 ±1. 8) %,MHCC97-H(8. 4 ± 0. 7) %,MHCC97-L(4. 6 ± 0. 5) % and Huh7(1. 0 ± 0. 2)( P < 0. 05). HCCLM3-derived SP cells but not MP cells displayed many characteristics of cancer stem cells including more sphere formation(25. 3 ± 5. 1)% vs(11. 2 ± 2. 6)%,P < 0. 05),higher invasion ability in vitro and stronger tumorigenicity in nude mice. The mRNA levels of ABCG2,CD13,CD44,Nanog,Sox2 and Klf4 in SP cells were 5. 64,2. 26,2. 10,3. 78,2. 37,2. 92 times of those respectively in MP cells( all P < 0. 05). Conclusion: In the HCCLM3 cell line there is an enriched population of SP cells with liver cancer stem cell-like properties,which may be related to the highly metastatic potential of HCCLM3 cells.

关 键 词：肝癌 HCCLM3细胞 侧群细胞 肿瘤干细胞 转移 

分 类 号：R735.7[医药卫生—肿瘤]