Objective :To explore side population ( SP ) phenotypes in gastric cancer cell line and high invasive sub - cell lines and to observe the relationship between SP phenotype and invasion coupled with spontaneous lung metastasis. Methods : High invasive cells were selected from gastric cancer cell line SNU5 using Transwell chamber in vitro. Cancer cells suspension was stained with Hoechst33342 and propidiumiodide ( PI ) with absence or presence of verapamil. Then SP was analyzed by fluorescence activated cell sorter. The ability of sphere formation, migration and invasion was tested by serum - free culture method, wound healing assay and Transwell assay. We also inoculated with SP phenotype and non - SP cell phenotype of spontaneous to detect the future lung metastasis. Gene chip was applied to analyze the different gene expression profile of SP and non - SP phenotype cells. Results : After three rounds of screening we estahlished a highly invasive subcellular SNU5 - P3. SP cells accounted for 1. 6％ of the SNU5 - P3 cells. SNU5 - P3 - SP cells were cultured in serum - free medium and formed 104 ± 19 sphere cells formation, which was more than sphere cells formed by SNU5 - P3 or SNU5 - P3 - non - SP. The invasive assay showed that the invasive ability of SNU5 - P3 - SP had 2. 5 times of than SNU5 - P3 or SNU5 - P3 - non - SP cells. The wound healing assay showed increased migration capacity of SNU5 - P3 - SP compared with SNU5 - P3 or SNU5 - P3 - non - SP cells. The ability of metastasis of three cells showed that SNU5 - P3 - SP had obvious lung metastasis. The visible lung metastasis of SNU5 - P3 - SP was 100％ , SNU5 was 50％ and SNU5 - P3 - non - SP was only 16. 7％ . Microarray analysis showed that we ohtained 733 different genes, of which 450 genes were up - regulated and 36％ of them were invasion or metastasis related genes. Conclusion : SP phenotype cells had stem cell characteristics and play roles in the invasion of gastric cancer and lung metastases.%目的:观察人胃癌SNU5肿瘤细胞系及其高侵袭细胞亚系中是否含有SP细胞,并验证该表型细胞与侵袭转移的关系.方法:采用Transwell小室,体外筛选出高侵袭细胞;经Hoechst33342染色,应用流式检测并分选出SP表型以及非SP表型细胞;应用无血清培养方法检测SNU5亲本细胞、SP表型以及非SP表型细胞在无血清培养中的成球能力.将分选出的SP表型以及非SP表型细胞进行侵袭实验、划痕迁移实验.裸鼠皮下接种,检测SP表型以及非SP表型细胞的自发性肺转移能力.基因芯片的方法分析SP表型以及非SP表型细胞的基因表达谱差异.结果:经过三轮筛选建立了稳定的高侵袭亚细胞群SNU5-P3.SP分选检测发现,SNU5-P3的SP细胞比例为1.6%;无血清培养发现,SNU5-P3-SP细胞成球数为104±19,显著强于SNU5以及SNU5-P3-non-SP细胞.侵袭实验检测发现,SNU5-P3-SP细胞的侵袭能力比SNU5、SNU5-P3-non-SP细胞增强近2.5倍.划痕迁移检测发现,SNU5为-P3-SP运动能力显著增强,划痕24h愈合.体内移植瘤转移实验发现SNU5-P3-SP细胞自发性肺转移率为100%,SNU5为50%,而SNU5-P3-non-SP仅有16.7%.基因芯片分析,获得733个差异基因,其中上调基因450个,36%的基因与侵袭转移有关.结论:胃癌SP表型细胞具有干细胞的特征,在胃癌侵袭、转移中起到关键作用.
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