肝癌细胞株Bel-7402中侧群细胞的分离和致瘤能力

【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 × 105 NSP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?

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

【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 oncogenicity 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, 105, 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 cells.