卵巢癌细胞中CD117^+细胞群分离及其意义

Isolation of CD117^+ cell subsets from the ovarian cancer cells and its significance

目的从人卵巢癌细胞株HEY中筛选出CD117^+细胞亚群,探讨其体内、外增殖、呈瘤和分化能力。方法卵巢癌细胞株HEY采用流式细胞仪分选,筛选出CD117^+细胞亚群,分为CD117^+细胞组和CD117^-细胞组,2组分别取100个细胞接种在6孔板中培养2周,采用平板克隆实验检测克隆形成率;分别取100个细胞在无血清DMEM培养基中培养1周,显微镜下观察细胞成球情况;分别取2×10~4个细胞接种在6孔板中培养2周,采用流式分选法检测细胞中CD117^+细胞比例,检测细胞体外分化能力。BALB/c裸鼠16只,根据接种细胞密度分为10~3组和10~4组,每组8只。10~3组将1×10~3个CD117^+细胞和CD117^-细胞分别接种于裸鼠背部的左侧和右侧皮下,10~4组将1×10~4个CD117^+细胞和CD117^-细胞分别接种于裸鼠背部的左侧和右侧皮下,比较2组肿瘤形成情况和肿瘤体积。结果 HEY细胞中CD117^+细胞占1.23%;培养2周后,CD117^+组细胞克隆形成率[(44±4)%]和CD117^+细胞比例(31.4%)高于CD117^-组[(25±3)%、0](P<0.05);培养1周后,CD117^+组细胞可形成状态良好、连接紧密的细胞球,生长速度较快,每个细胞球由40~50个细胞组成,CD117^-组细胞呈悬浮、球形生长,不能增殖成细胞球;接种CD117^+细胞4周后,103组裸鼠皮下移植瘤肿瘤体积[(0.300±0.042)cm^3]小于10~4组[(0.490±0.031)cm^3](P<0.05),接种CD117^-细胞后,10~3组和10~4组裸鼠均未成瘤。结论卵巢癌细胞株HEY中仅小部分表达CD117^+抗原标记,但该亚群细胞具有较强增殖和分化能力,可在无血清悬浮培养中形成细胞球。

Objective To screen the CD117^＋ cell subsets from human ovarian cancer cell line HEY to compare the ability of in vivo and in vitro proliferation,tumor formation and differentiation capacity.Methods The ovarian cancer HEY cells were selected the CD117^＋ subsets by flow cytometry,and were divided into CD117^＋ group and CD117^- group.One hundred cells from each group were seeded into 6-well plates for 2 weeks,and the cell colony-forming ability was detected by the plate cloning experiment.Another 100 cells from each group were cultured in the serum-free DMEM medium.After one week,the formation of cell spheres was observed under the microscope.To test the in vitro differentiation ability,2×10^4 cells were seeded into 6-well plates and cultured for 2 weeks,and the proportion of CD117^＋ was detected by flow cytometry analysis.Sixteen nude mice were averagely divided into 10^3 group and 10^4 group according to the density of injected cells.To compare the ability of tumor formation and the tumor volume,1×10^3 CD117^＋ cells and CD117^- cells were inoculated subcutaneously in the left and right sides of the back in 10^3 group,and 1×10^4 CD117^＋ cells and CD117^- cells were inoculated subcutaneously in the left and right sides of the back in 10^4 group.Results CD117^＋ accounted for 1.23% of the ovarian cancer HEY cells.After two-week culturing,the cells colony-forming rate and percentage of CD117^＋ cells were higher in CD117^＋ group（（44±4）%,31.4%）than those in CD117^- group（（25±3）%,0）（P〈0.05）.After one-week culturing in the serum-free medium,CD117^＋ cells formed finely a closely-connected cell spheres which grew fast.Each sphere consisted of 40 to 50cells.CD117^-cells suspension emerged and grew spherically,with no proliferating into cell spheres.After inoculating the CD117^＋ cells into the nude mice for 4 weeks,the average xenograft tumor volume was significantly smaller in 10^3 group（（0.300±0.042）cm^3）than that in 10^4 group（（0.490±0.031）cm^3）（P〈0.05）.After inoculating the CD117^- cells,no tumor formation was found in nude mice in either groups.Conclusion CD117^＋ marker is expressed in only a small amount of the ovarian cancer HEY cells,which has a strong ability of proliferation and differentiation to form cell spheres in serum-free medium.