人卵巢癌H08910细胞系中肿瘤干细胞样细胞通过MMP促进血管生成拟态的形成  

Tumor stem-cell-like cells from human ovarian cancer cell line HO8910 and its relation to vasculogenic mimicry

作　　者：耿松珊 吴小华 

机构地区：[1]河北医科大学研究生学院,石家庄050000 [2]解放军白求恩国际和平医院妇产科,石家庄050082

出　　处：《现代妇产科进展》2016年第7期503-506,510,共5页Progress in Obstetrics and Gynecology

基　　金：河北省政府资助临床医学优秀人才培养和基础课题研究项目计划(No:361001)

摘　　要：目的:研究人卵巢癌HO8910细胞中肿瘤干细胞样细胞与血管生成拟态(VM)的关联。方法:(1)将亲代HO8910细胞置于无血清干细胞培养液中悬浮培养并连续传代。(2)流式细胞术检测第一、三、五、七代悬浮细胞中肿瘤干细胞标记物CD133表达情况,体外平板克隆实验、裸鼠体内成瘤实验验证第七代悬浮细胞的肿瘤干细胞样特性。(3)三维立体培养第七代悬浮细胞和亲代HO8910细胞,观察VM形成情况,并通过实时荧光定量聚合酶链式反应(RT-PCR)检测二者中基质金属蛋白酶2,9(MMP-2,MMP-9)表达情况。结果:(1)亲代HO8910细胞在无血清悬浮培养的条件下可连续传代,且传代过程中悬浮球逐渐增大。(2)流式细胞术检测第一、三、五、七代CD133阳性表达率(0.23%、31.74%、79.25%和88.18%)呈上升趋势。第七代悬浮细胞的克隆形成能力[(45.67±1.53)%]显著高于亲代细胞[(11.00±2.00)%](t=23.859,P=0.00)。裸鼠体内成瘤实验显示,第七代悬浮细胞的成瘤率(6/6)明显高于亲代细胞(0/6)。(3)三维立体培养中,第七代悬浮细胞较亲代HO8910细胞的VM形成时间更早,管状结构更密集。RT-PCR结果显示,第七代悬浮细胞中MMP-2、MMP-9表达高于亲代细胞(t=-4.544,P=0.045;t=-4.019,P=0.013)。结论:亲代HO8910细胞通过无血清悬浮培养可分选出肿瘤干细胞样细胞,其具有更强的VM形成能力,推测肿瘤干细胞样细胞可能通过影响VM的形成促进卵巢癌的生长和转移。Objective: To investigate the relationship between VM( vascularization mimicry) and tumor stem-cell-like cells from human ovarian cancer cells line HO8910. Methods:( 1) The maternal HO8910 cells were cultured in serum-free stem cell medium by continuous passage.( 2) The expression of tumor stem cells marker CD133 in the 1st,3rd,5th,7th passages suspension cells were tested by Flow cytom etry. Flat colony formation test in vitro and the tumor formation test in vivo of nude mice were applied to identify the characteristic of tumor stem-cell-like cells in the 7th suspension cells.( 3) Three-dimension cell culture was used to observe the ability of VM formation in the 7th suspension cells and maternal HO8910 cells. The expression levels of Metalloproteinases-2( MMP-2) and Metalloproteinases-9( MMP-9) in both of them were detected by real-time PCR( RT-PCR) method. Results:( 1) The maternal HO8910cells could continuous passages under the condition of serum-free medium,the suspension spheres became larger and larger with the passages.( 2) The results of flow cytom etry illustrated that CD133 expression levels in the 1st,3rd,5th and 7th generation of suspension cells increased( 0. 23%,31. 74%,79. 25% and 88. 18%). The colony-forming efficiency of the 7th generation suspension cells[( 45. 67 ±1. 53) %]were significantly higher than those of the maternal HO8910 cells[( 11. 00 ±2. 00) % ]( t = 23. 859,P<0. 00). The results of tumor formation in nude mice revealed that the rate of tumor formation in the 7th generation suspension cells( 6 /6) were significantly higher than maternal cells( 0 /6).( 3) Compared with maternal HO8910 cells,the 7th generation suspension cells took shorter time to form VM and more intensive tubular structure in the condition of tree-dimensional cultivation. RT-PCR test showed the expression levels of MMP-2 and MMP-9 gene in the 7th generation suspension cells were higher than those of the maternal cells( t =-4. 544,P = 0. 045; t =-4. 019,P = 0. 013). Conlusion: Tumor stemcell-like cells could be i

关 键 词：血管生成拟态 基质金属蛋白酶类 肿瘤干细胞 卵巢肿瘤 微循环 

分 类 号：R737.31[医药卫生—肿瘤]