分离鉴定人胃癌HGC-27细胞系中的肿瘤干细胞  

Isolation and identification cancer stem cells in human gastric cancer cell line 7

作　　者：高钢龙[1] 张学利[1] 孙振亮[1] 刘文勇[2] 张萍[2] 叶冬霞[2] 

机构地区：[1]上海交通大学附属第六人民医院南院普外科,上海201400 [2]上海交通大学医学院附属第九人民医院普外科

出　　处：《上海医学》2014年第11期957-961,I0003,共6页Shanghai Medical Journal

基　　金：上海市卫生局科学基金资助项目(2010Y079)

摘　　要：目的 分析人胃癌细胞系HGC-27细胞系中肿瘤干细胞相关的侧群细胞（SP细胞）的比例,分选出相关的SP细胞和非侧群细胞（NSP细胞）,检测相关的SP细胞是否具有干细胞的生物学特性。方法 制备人胃癌细胞系HGC-27细胞中细胞悬液,实验组的人胃癌细胞株中加入Hoechst 33342至终浓度为5μg/mL,对照组的人胃癌细胞株中加入Hoechst 33342后再加入维拉帕米至终浓度为50μg/mL。应用流式细胞仪分选出人胃癌细胞系HGC-27细胞中相关的SP细胞,并进行分析。采用克隆形成实验和四甲基偶氮唑盐微量酶反应比色法（MTT法）检测相关的SP细胞和NSP细胞增殖能力差异。利用磁珠筛选实验方法测定胃癌细胞系HGC-27细胞中肿瘤干细胞表面特异性抗体。结果 实验组HGC-27细胞中SP细胞亚群所占比例为（6.00±0.05）%,显著高于对照组的（0.10±0.01）%（P〈0.05）。选用2×10^8个对数生长期细胞,经流式细胞仪分选出SP细胞7.5×10^5个和NSP细胞8.5×10^7个。分选后经低血清培养液（2%胎牛血清）培养后,SP细胞的密度较NSP细胞低,且细胞形态也不同。培养第1~7天SP细胞的光密度值分别为0.13±0.03、0.18±0.04、0.33±0.04、0.41±0.04、0.47±0.07、0.51±0.06、0.55±0.06,NSP细胞的光密度值分别为0.13±0.02、0.16±0.04、0.20±0.04、0.24±0.03、0.28±0.05、0.30±0.06、0.31±0.07,培养第3、4、5、6、7天,SP细胞的光密度值均显著高于NSP细胞（P值均〈0.05）。磁珠筛选后经流式细胞仪检测,SP细胞和NSP细胞表面抗原CD133分别为（89.94±28.19）%、（38.22±16.45）%,SP细胞和NSP细胞表面抗原CD44分别为（86.99±23.59）%、（46.09±13.60）%。结论 人胃癌细胞系HGC-27细胞中存在相关的SP细胞,应用流式细胞仪可分选出相关的SP细胞,采用克隆形成实验和MTT法检测相关的SP细胞和NSP细胞增殖能力有差异。人胃癌细胞系HGC-27细胞中相关的SP细胞可能具有肿瘤干细胞特性�Objective To detect the ratio of the side population （SP） of cancer stern cells in human gastric cancer cell line （HGC）-27, to separate SP and non side population （NSP） cells, and to initially identify whether SP cells have stem cell bionomics. Methods HGC-27 cell suspension was prepared firstly. Hoechst 33342 was added in HGC-27 cells and the final concentration was 5 μg/mL in the experimental group. Hoechst 33342 and verapamil were added in order in HGC-27 cells and the final concentration was 50 μg/mL in control group. Then SP was analyzed by flow cytometry. SP and NSP cells were separated by fluorescence activated cell sorter. The proliferation of SP and NSP cells were detected by clone formation and MTT experiment. Surface specific antibody related to cancer stem cell in HGC-27 cells was measured by magnetic activated cell sorting system. Results The percentage of SP in the experimental group was （6.00± 0.05） %, which was significantly higher than that in the control group （[0.10 - 0.01 ] %, P〈0.05）. A total of 7.5 × 10^5 SP cells and 8.5 × 10^7 NSP cells were separated by the fluorescence activated cell sorter. The density of SP cells was lower than that of NSP cells and the cell morphology were different between SP and NSP cells after culturing in the low serum culturemedium 〈2% fetal bovine serum）. The values of optical density of SP cells were 0.13±0.03, 0, 18±0.04, 0.33± 0.04, 0.41 ± 0.04, 0.47 ± 0.07, 0.51± 0.06 and 0.55 ±0. 06 on day 1 - 7 after cell culture, respectively. The values of optical density of NSP ceils were 0.13±0.02, 0.16±0.04, 0.20±0.04, 0.24±0.03, 0.28±0.05, 0. 30± 0. 06 and 0.31 ±0.07 on day 1 -7 after cell culture respectively. The values of optical density of SP cells on day 3-7 after culture were significantly higher than those of NSP cells （all P〈0. 05〉. Conclusion HGC-27 cell line contains SP cells. SP cells can be separated by flow cytometry. SP and NSP cells have disparity in clone formation and cell growth plot experiment. The

关 键 词：胃癌 肿瘤干细胞 HGC-27细胞 侧群细胞亚群 磁珠筛选 

分 类 号：R735.2[医药卫生—肿瘤]