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目的:分选人肺腺癌细胞系(LAC)中SP细胞(side population cells)亚群并分析其具有的干细胞特性。方法:运用流式细胞荧光激活分选技术(FACS)分选LAC中SP细胞亚群和非SP细胞亚群,利用MTT、体外克隆形成和皮下移植瘤试验分析其体内、外增殖能力,流式细胞仪分析细胞周期及细胞分化,RT-PCR方法检测其ATP结合盒转运蛋白(ABCG2)基因的表达情况。结果:流式细胞荧光激活分选结果显示:LAC中含有约1.88%的SP细胞亚群。MTT和体外克隆形成试验表明:SP细胞亚群的体外增殖能力显著强于非SP细胞亚群。同时,裸鼠皮下接种SP细胞亚群,其体内成瘤能力也要显著强于非SP细胞亚群。流式细胞仪分析结果表明:SP细胞亚群的G0/G1期比例显著高于非SP细胞(P<0.05)。细胞分化实验结果表明:两个亚群中SP细胞比率分别为(1.46±0.08)%和(0.12±0.04)%。RT-PCR结果显示:ABCG2基因mRNA在SP细胞亚群中的表达水平是其在非SP细胞亚群中的3.3倍(P<0.05)。结论:利用LAC中SP细胞亚群具有肺癌干细胞的特性,流式细胞荧光激活分选肺腺癌SP细胞亚群可能是分离肺腺癌干细胞的有效方法。
OBJECTIVE: To sort out the subset of side population cells in human lung adenocarcinoma cell line (LAC) and analyze their stem cell properties. Methods: FACS was used to sort out the subpopulation of SP and non-SP in LAC. MTT, in vitro clonogenic assay and subcutaneous xenograft tumor assay were used to analyze the in vitro and in vivo proliferative ability. Flow cytometry Cell cycle and cell differentiation were analyzed by cytometry. The expression of ATP binding cassette transporter (ABCG2) gene was detected by RT-PCR. Results: Fluorescence-activated fluorescence sorting results showed that LAC contained about 1.88% of the SP cell subsets. MTT and in vitro clonogenic assay showed that: SP cell subpopulation in vitro proliferation was significantly stronger than non-SP cell subpopulation. At the same time, nude mice subcutaneously inoculated with SP cell subpopulations, its tumorigenic ability in vivo also significantly stronger than non-SP cell subpopulations. Flow cytometry analysis showed that the proportion of G0 / G1 phase of SP cell subgroup was significantly higher than that of non-SP cell (P <0.05). The results of cell differentiation showed that the ratio of SP cells in the two subpopulations was (1.46 ± 0.08)% and (0.12 ± 0.04)%, respectively. The results of RT-PCR showed that the expression level of ABCG2 mRNA in SP cell subpopulation was 3.3 times higher than that in non-SP cell subgroup (P <0.05). CONCLUSION: Using the characteristics of lung cancer stem cells of SP cell subpopulation in LAC, it may be an effective method to isolate lung adenocarcinoma stem cells by flow cytometry.