目的:胃癌干细胞的制备困难一直是阻碍胃癌研究进展的主要因素。文中研究无血清培养基悬浮培养胃癌MKN-45细胞系技术,并根据胃癌干细胞(cancer stem cells,CSCs)的生物学标记筛选出该细胞系中的干细胞。方法 MKN-45细胞系在无血清培养基中培养8周后,取对数生长期的MKN-45细胞以Hoechst33342孵育后流式细胞仪( FACS)检测侧群细胞。当侧群细胞达约25%时,收集全部细胞微球体,加荧光标记的CD133和CD44单抗孵育后 FACS 筛选,选CDl33+CD44+细胞即为胃癌干细胞。结果胃癌MKN-45细胞系中约40%的肿瘤细胞在无血清培养基中能够存活、增殖并形成自由飘浮的细胞球。双苯酰胺33342染色检测提示MKN-45细胞中侧群细胞含量为3.4%,而细胞球中侧群细胞细胞含量则明显增高,可达到26.9%;收集的侧群细胞通过FACS检测表面标记,MKN-45细胞CDl33+CD44+细胞含量为11.2%,而细胞球中CD133+CD44+细胞含量为90.3%。结论应用无血清培养基悬浮培养法能成功获得CSCs富集的肿瘤球,Hoechst33342及表面标记双重检测提取法可筛选出其中的CSCs,并可作为今后胃癌干细胞研究的实验基础。
[Abstract ] Objective Difficulties with the preparation of gastric cancer stem cells (CSCs) have been a main obstacle to the studies of gastric cancer .This article addresses the technology of the serum-free medium suspension cultivation of the MKN-45 gastric cancer cell line and screening of stem cells from the cell line based on the biomarkers of gastric CSCs . Methods MKN-45 cells were cultured in serum-free medium for 8 weeks and those in the logarithmic phase cultivated with hoechst 33342 followed by detection of the side cells by flow cytometry .When the side population cells reached 25%, all the cell microspheres were collected , hatched with CD133 and CD44, and subjected to fluorescence-activated cell sorting.The CDl33 +and CD44 +cells were selected as gastric CSCs . Results About 40%of the MKN-45 gastric CSCs were alive , prolif-erated, and formed floating cell balls .Side population cells constitu-ted 3.4% of the MKN-45 cells and 26.9% of the cell balls.The CDl33 +and CD44 +cells made up 11.2% of the MKN-45 cells and 90.3%of the cell balls. Conclusion Cell balls rich in CSCs can be successfully obtained by serum-free medium suspension culti-vation and CSCs can be screened out with hoechst 33342 and surface markers , which may serve as an experimental ground for the stud-ies of gastric CSCs .