CAJ | 학술논문

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(RT-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.
목적 분리인폐선암A549세포계중적측군(SP)세포아군병탐토기세포특성.방법 채용면역조화법검측인폐선암A549세포계중ABCG2단백적표체.채용류식세포술분선A549세포계중적SP화비SP세포아군,병검측량아군세포적분화공능.채용역전록취합매련반응(RT-PCR)방법검측량아군세포적ABCG2단백표체,통과량아군세포적생장곡선、세포분렬지수、세포주기각시상분포、평판극륭형성실험、체외침습화천이실험、화료약민시험、세포내약물농도측정이급라서이식류실험,비교량아군세포적생물학행위,병용RT-PCR화면역조화방법검측이식류조직중ABCG2적표체.결과 A549세포계중ABCG2단백적양성표체솔위2.13%.통과류식세포술능성공분선득도SP세포화비SP세포,SP세포아군가산생SP급비SP량충세포,이비SP세포지능산생비SP세포.SP세포표체ABCG2,비SP세포칙불표체.량세포아군적증식、천이능력상사,흡광도、분렬지수、세포주기시상분포화세포체외천이실험천과려막적세포수차이균무통계학의의(균P＞0.05),단SP세포적침습능력화성류능력강우비SP세포,세포체외침습실험천과려막적세포수、체외세포극륭형성수화이식류성류솔균고우비SP세포(P＜0.01,P＜0.01,P＜0.05).SP세포화비SP세포대순박(DDP)적민감성화세포내약물농도상사,비SP세포대5-불뇨밀정(5-Fu)、의탁박감(VP-16)、장춘서빈(NVB)화길서타빈(GEM)적민감성급세포내약물농도칙고우SP세포(균P＜0.01).결론 인폐선암A549세포계SP세포아군부집료폐암간세포,통과류식세포의분선폐선암SP세포아군시분리폐선암간세포적유효방법.
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 adenocarcinoma 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 immunohistochemistry and RT-PCR. Results The positive rate of ABCG2 expression in the A549 cells by immunohistochemistry was 2.13%.SP and NSP cells were isolated by FACS.The SP cells 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 intracellular levels of the two cell subsets were similar,but the susceptibilities to 5-FU,VP16,NVB and GEM and their intracellular levels of NSP cells were significantly higher than those of 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.