肝癌细胞株Bel-7402中侧群细胞的分离和致瘤能力  

Isolation and Tumorigenicity of Side Population Cells Sorted from Hepatocellular Carcinoma Bel-7402 Cell Line

作　　者：陈伟[1] 姜楠[2] 张彤[2] 李华[2] 张琪[2] 陈规划[2] 曾宪成[3,4] 

机构地区：[1]浙江大学医学院附属第二医院普通外科,浙江杭州310009 [2]中山大学附属第三医院肝移植中心,广东广州510630 [3]增城市人民医院 [4]中山大学附属博济医院肝胆外科,广东增城511300

出　　处：《中山大学学报（医学科学版）》2012年第5期561-566,576,共7页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81000959);广东省科技计划项目(2009B030801027);广东省自然科学基金(10451130001004472);增城市科技创新扶持基金(ZC201004)

摘　　要：【目的】分选肝癌Bel-7402细胞中的侧群(SP)细胞并分析其生物学特性。【方法】采用流式细胞荧光激活分选(FACS)技术将Bel-7402细胞分为SP细胞和非侧群(NSP)细胞2亚群。实验分组:SP组与NSP组。细胞生长曲线法和平板克隆法比较SP细胞和NSP细胞的增殖能力和克隆形成能力;RT-PCR法检测SP细胞和NSP细胞的CD133、三磷酸腺苷结合盒转动蛋白G2(ABCG2)表达;流式细胞术分析细胞凋亡率;无血清培养法检测SP细胞及NSP细胞成球率;非肥胖糖尿病/严重联合免疫缺陷(NOD/SCID)小鼠体内成瘤实验比较SP细胞和NSP细胞的致瘤性。【结果】Bel-7402细胞中分选出的SP细胞比例为(2.4±0.0)%。生长曲线表明SP细胞的增殖速度快于NSP细胞(P<0.05)。SP、NSP细胞的克隆形成率分别为(20.5±2.6)%、(5.1±0.9)%,两者差异明显(P<0.05)。SP细胞ABCG2 mRNA和CD133 mRNA平均表达水平分别是NSP细胞的3.03倍(P<0.01)和1.98倍(P<0.01)。SP细胞凋亡率为(0.89±0.23)%,低于NSP细胞(P<0.05)。在无血清培养基中,SP细胞成球率为(14.1±1.9)%,高于NSP细胞(P<0.05)。1×103、104、105数量的SP细胞形成皮下移植瘤(P>0.05,P<0.05,P<0.05),而1×105NSP细胞则还不能成瘤。【结论】肝癌Bel-7402细胞中的SP细胞可能富集了肝癌干细胞。[ Objective ] To study the biological characteristics of side population （SP） cells sorted from hepatocellular Carcinoma Bel-7402 cell line. [Methods] Fluorescence-activated cell sorter （FACS） was used to sort SP cells and non-SP （NSP） cells from Bel- 7402 cell line. Groups were： SP group and NSP group. The colony formation and proliferation ability were compared between SP cells and NSP cells by plate colony and growth curve assay. ATP-binding cassette superfamily G member 2 （ABCG2） and CD133 mRNA levels in SP and NSP cells were examined by RT-PCR. The apoptosis was analyzed by flow cytometry. The sphere rate was counted for SP cells and NSP cells in serum-free medium. The oneogenicity of the SP and NSP cells were analyzed by tumor formation in nonobese diabeti- c/severe combined immune- deficient （NOD/SCID） mice. [Results] （2.4 ± 0.0） % SP cells were sorted from the Bel-7402 cells by FACS. Growth curve assay showed that the proliferation ability of SP cells was higher than NSP cells （P 〈 0.05）. Colony formation efficiency was significantly different （P 〈 0.01 ） between SP （20.5 ± 2.6） % and NSP （5.1 ± 0.9） %. The level of mRNA expression of ABCG2 and CD133 in SP cells were 3.03 （P 〈 0.01 ） and 1.98 （P 〈 0.01 ） times of that in NSP cells, respectively. The apoptotic rate of SP cells were （0.89 ±0.23）% , which were lower than those of NSP cells （P 〈 0.05）. The sphere rate in SP cells was （ 14.1 ± 1.9）%, higher than that in NSP cells in serum-free medium （P 〈 0.05）. With cell numbers of 1× 103, 104, lO5, the SP cells can form subcutaneous transplantation in NOD/SCID mice, but the NSP cells can not form it （P 〉 0.05, P 〈 0.05, and P 〈 0.05）. [ Conclusion ] The SP cells sorted from Bel-7402 cell line may enrich tumor stem ceils.

关 键 词：肝癌 侧群细胞 肿瘤干细胞 BEL-7402细胞 

分 类 号：R730.5[医药卫生—肿瘤]