人肺腺癌细胞系(LAC)中SP细胞亚群的分选及其干细胞特性分析

Characterization of side population(SP) of stem-like tumor cells in human lung adenocarcinoma cell line(LAC)

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 explore a feasible method to detect tumor stem-like cells in human lung adenocarcinoma cells （LAC）,and to analyze the properties of the sorted side population （SP）cells.Methods：Fluorescence-activated cell sorting assay（FACS）was performed to sort the SP and non-SP cells of LAC.MTT,colony formation and in vivo tumorigenesis assays were performed to analyze the proliferation capacity of SP and non-SP cells in vitro and in vivo.Flow cytometric assay was performed to detect the cell cycle and differentiation of SP cells.RT-PCR assay was performed to detect the expression of ATP-binding cassette transporter G2（ABCG2） mRNA in SP and non-SP cells.Results：Results from FACS showed that SP cells were detected in approximately 1.88% of LAC.MTT and in vitro colony formation assays showed that the in vitro proliferation capacity of SP cells was significantly stronger than that of non-SP cells.Additionally,the in vivo tumorigenesis capacity of SP cells was significantly stronger than that of non-SP cells.Cell cycle analysis indicated that the cell number of G0/G1 phase in SP cells was higher than that in non-SP cells（P 0.05）.The rates of SP cells in SP and non-SP populations were approximately （1.46±0.08）% and （0.12±0.04）%,respectively.Moreover,the level of ABCG2 mRNA in SP cells was 3.3 times of that in non-SP cells（P 0.05）.Conclusion：The SP cells sorted from LAC possessed characteristics of cancer stem-like cells,and SP cells sorted by FACS could be an effective method used to sort lung cancer stem cells.