胃癌MKN-45细胞系的无血清培养技术及干细胞的制备  

Serum-free culture of the MKN-45 gastric cancer cell line and preparation of stem cells

作　　者：姜贻乾[1] 管晓翔[2] 郭庆敏[1] 徐小平[1] 何一扬 梁军才[1] 安素红[1] 韩丽娟[1] 

机构地区：[1]杭州师范大学医学院附属萧山第一医院放疗科,杭州医学硕士研究生311201 [2]南京医科大学金陵医院(南京军区南京总医院)肿瘤内科,南京210002

出　　处：《医学研究生学报》2016年第4期380-383,共4页Journal of Medical Postgraduates

基　　金：杭州市科技发展计划项目(20130733Q45)

摘　　要：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。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 hoechst33342 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,proliferated,and formed floating cell balls. Side population cells constituted 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 and90. 3% of the cell balls. Conclusion Cell balls rich in CSCs can be successfully obtained by serum-free medium suspension cultivation and CSCs can be screened out with hoechst33342 and surface markers,which may serve as an experimental ground for the studies of gastric CSCs.

关 键 词：胃癌干细胞 无血清培养 侧群细胞 CDl33+ CD44+ 

分 类 号：R735.2[医药卫生—肿瘤]