三维无血清条件培养结合抗癌药物分离人骨肉瘤肿瘤干细胞

目的探讨无血清三维条件培养结合抗癌药物分离及鉴定人骨肉瘤肿瘤干细胞的可行性。方法将来源于人体的骨肉瘤细胞种植于1．2％藻酸盐凝胶中，并置于添加有阿霉素（Epirubicin，0．8μg／ml）的无血清DMEM／F12条件培养基中培养。培养7～10天后，可见凝胶内出现由单细胞增殖形成的单克隆球。取出该单克隆球，并通过细胞免疫荧光（Oct3／4和Nanog）、体内致瘤实验，检测该单克隆球细胞的生物特性。结果单克隆球主要由Oct3／4和Nanog阳性细胞组成，这些阳性细胞具有明显的致瘤作用：结论分离所得的单克隆细胞既能表达部分干细胞基因（Oct3／4和Nanog）。又表现出明显的抗肿瘤药物性和体内致瘤性，三维无血清条件培养结合抗癌药物分离所得的单克隆骨肉瘤细胞可能为人骨肉瘤干细胞。

Isolation and Identification of Human Osteosarcom Cancer Stem Cells by Serum-free three-dimensional Culture Combined with Anticancer Drug

Objective To isolate and identify cancer stem cells （CSCs） from human osteosarcoma by serum-free three- dimensional culture combined with anticancer drug. Methods The primary cells from human osteosarcoma were digested by trypsin to prepare a single-cell suspension,and mixed homogeneously into 1.2% alginate gel. Single-cell alginate gel was cultured with serum-free DMEM/F12 medium. Epirubicin （0.8 μg/ml） was added to the medium to enrich CSCs. After been cultured conventionally for 7 to 10 days,most of cells suspended in alginate gel were killed by epirubicin. But there were a few cells survived and formed some single-cell cloning spheres. Immunofluorescent staining for Oct3/4 and Nanog was implemented to find cells provided with properties of self-renew and multi-potential differentiation. Cells from cloning spheres were transplanted into BALB/c mice to detect the tumorigenicity in vivo. Results Some positive cells stained by Oct3/4 （TRITC） and Nanog （TRITC） were found in single-cell cloning spheres,and most of positive cells were concentrated in the core of sphere. Cells from spheres could form osteosarcoma in the body of mice. Conclusion Cells from single-cell cloning spheres have the properties of the expression of parts of stem cell gene （Oct3 / 4 and Nanog）, resisting anti-cancer drugs, and tumorigenicity in vivo. Summary of above,we can believe that cells we have gotten from osteosarcoma by serum-free three-dimensional culture combined with anticancer drug are cancer stem cells.