人肺腺癌A549细胞系干细胞相关亚群的分离与鉴定

Isolation and identification of side population cells in human lung adenocarcinoma cell line A549

目的分离人肺腺癌A549细胞系中的侧群（SP）细胞亚群并探讨其细胞特性。方法采用免疫组化法检测人肺腺癌A549细胞系中ABCG2蛋白的表达。采用流式细胞术分选A549细胞系中的SP和非SP细胞亚群，并检测两亚群细胞的分化功能。采用逆转录聚合酶链反应（1it—PCR）方法检测两亚群细胞的ABCG2蛋白表达，通过两亚群细胞的生长曲线、细胞分裂指数、细胞周期各时相分布、平板克隆形成实验、体外侵袭和迁移实验、化疗药敏试验、细胞内药物浓度测定以及裸鼠移植瘤实验，比较两亚群细胞的生物学行为，并用RT-PCR和免疫组化方法检测移植瘤组织中ABCG2的表达。结果A549细胞系中ABCG2蛋白的阳性表达率为2．13％。通过流式细胞术能成功分选得到SP细胞和非SP细胞，SP细胞亚群可产生SP及非SP两种细胞，而非SP细胞只能产生非SP细胞。SP细胞表达ABCG2，非SP细胞则不表达。两细胞亚群的增殖、迁移能力相似，吸光度、分裂指数、细胞周期时相分布和细胞体外迁移实验穿过滤膜的细胞数差异均无统计学意义（均P〉0．05），但SP细胞的侵袭能力和成瘤能力强于非SP细胞，细胞体外侵袭实验穿过滤膜的细胞数、体外细胞克隆形成数和移植瘤成瘤率均高于非SP细胞（P〈0．01，P〈0．01，P〈0．05）。SP细胞和非SP细胞对顺铂（DDP）的敏感性和细胞内药物浓度相似，非SP细胞对5-氟尿嘧啶（5-Fu）、依托泊苷（VP-16）、长春瑞滨（NVB）和吉西他滨（GEM）的敏感性及细胞内药物浓度则高于sP细胞（均P〈0．01）。结论人肺腺癌A549细胞系SP细胞亚群富集了肺癌干细胞，通过流式细胞仪分选肺腺癌SP细胞亚群是分离肺腺癌干细胞的有效方法。

Objective To isolate and characterize the side population cells （SP cells） in the lung adenocarcinomas cell line A549. Methods The protein expression of ABCG2 in human lung adenoeareinoma cell line A549 was detected by immunohistochemistry. SP and NSP cells in the cell line A549 were isolated by FACS, and their differentiation was analysed. ABCG2 expression in the two cell subsets was detected by RT-PCR. The cell growth curves, cell division indexes, cell cycles, plate clone formation tests, migration and invasion assays, chemotherapeutic susceptibility tests, tests of the intracellular drug levels, and the tumor cell implantation experiments on nude mice were applied to study the biological properties of the two cell subsets. The expression of ABCG2 in the transplanted tumor in nude mice was detected by immunohistoehemistry and RT-PCR. Results The positive rate of ABCG2 expression in the A549 cells by immunohistoehemistry was 2.13%. SP and NSP cells were isolated by FACS. The SP eells could produce both SP and NSP cells, while NSP cells only produced NSP cells. SP cells expressed ABCG2, but NSP cells did not. The proliferation and migration abilities of the two cell subsets were similar, but the invasion and tumorigenic ability of SP cells was significantly higher than that of NSP cells. The susceptibilities to DDP and its intraeellular levels of the two cell subsets were similar, but the susceptibilities to 5-FU, VP16, NVB and GEM and their intraeellular levels of NSP cells were significantly higher than thoseof the SP cells. Conclusions SP cells in the human lung adenocarcinomas cell line A549 is enriched with tumor stem cells. An effective way to get lung adenocarcinomas stem cells is to isolate SP cells by FACS.