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目的 :观察从卵巢癌SKOV3细胞中筛选获得的全克隆(holoclone)细胞的特性及其形成血管生成拟态(vasculogenic mimicry,VM)的能力。方法 :通过有限稀释法对SKOV3细胞进行单细胞培养,形成3种单细胞克隆[全克隆、部分克隆(meroclone)和亚克隆(paraclone)];通过裸鼠体内成瘤实验和软琼脂克隆形成实验比较3种单克隆的生物学特性,并通过三维细胞培养模型观察其形成VM的能力。蛋白质印迹法检测全克隆细胞中CD133、基质金属蛋白酶2(matrix metalloproteinases-2,MMP-2)及MMP-9的表达水平。结果 :SKOV3细胞单细胞培养10~14 d后,全克隆、部分克隆和亚克隆细胞所占的比例分别为(17.4±0.8)%、(30.1±0.4)%和(52.5±1.1)%。裸鼠体内成瘤率结果显示,全克隆细胞的成瘤率为80%,部分克隆和亚克隆细胞在裸鼠中均未有肿瘤形成。软琼脂克隆形成实验结果提示,全克隆细胞的克隆形成率为(16.7±3.8)%,显著高于部分克隆及亚克隆细胞的(6.5±1.5)%和(1.2±0.7)%(F=4.085,P<0.05)。三维培养中,全克隆组可以形成VM,部分克隆和亚克隆细胞不能形成VM。全克隆细胞中CD133、MMP-2和MMP-9蛋白的表达水平均高于亲本SKOV3细胞(P值均<0.05),并高于部分克隆和亚克隆细胞(P值均<0.05)。结论 :SKOV3细胞中单细胞克隆在形态和生物学特性上存在异质性,其中全克隆细胞亚群具有肿瘤干细胞样细胞特性,具有形成VM的能力。全克隆是SKOV3细胞中具有较高复发能力的细胞亚群。
Objective: To observe the characteristics of holoclone cells screened from ovarian cancer SKOV3 cells and their ability to form vasculogenic mimicry (VM). METHODS: SKOV3 cells were cultured in a single cell by limiting dilution to form three kinds of single cell clones [full, meroclone, and paraclone]; by in vivo tumor formation in nude mice and formation of soft agar The biological characteristics of three kinds of monoclonal were compared and their ability to form VM was observed by three-dimensional cell culture model. Western blotting was used to detect the expression of CD133, matrix metalloproteinases-2 (MMP-2) and MMP-9 in all the cloned cells. Results: After 10-14 days of single cell culture, the percentage of fully cloned, partially cloned and subcloned SKOV3 cells was (17.4 ± 0.8)%, (30.1 ± 0.4)% and (52.5 ± 1.1)%, respectively. The rate of tumorigenesis in nude mice showed that the rate of tumorigenesis of all the cloned cells was 80%, and some of the cloned and subcloned cells had no tumor formation in nude mice. The results of soft agar colony formation assay showed that the rate of clonogenicity of all the cloned cells was (16.7 ± 3.8)%, which was significantly higher than that of the partial clones and subclones (6.5 ± 1.5) and (1.2 ± 0.7)% (F = 4.085 , P <0.05). In the three-dimensional culture, the full clone group can form the VM, and some of the cloned and subcloned cells can not form the VM. The expression levels of CD133, MMP-2 and MMP-9 in all the cloned cells were higher than that of the parental SKOV3 cells (P <0.05), and higher than that of some cloned and subclone cells (P <0.05). CONCLUSION: The single cell clone in SKOV3 cells is heterogeneous in morphology and biological characteristics. The full clone cell subpopulation has the characteristics of tumor stem cell like cells and has the ability of forming VM. Full cloning is a subset of cells that have a high ability to recurrent in SKOV3 cells.