骨肉瘤干细胞的分离与鉴定

Isolation and identification of osteosarcoma stem cells

背景:骨肉瘤是最常见的骨原发性恶性肿瘤,其发生可能与骨肉瘤干细胞有关。目的:评估骨肉瘤干细胞分离、培养和鉴定方法以及其相关肿瘤标记物的表达。方法:在无血清条件下以及无血清联合抗肿瘤药物的条件下应用免疫磁珠分选法对骨肉瘤干细胞进行分离、培养,分选出Stro-1阳性、CD133阳性骨肉瘤干细胞,采用免疫荧光染色、蛋白质印迹法等检测骨肉瘤肿瘤干细胞标记物CD133、Oct3/4以及Nanog等的表达水平以及致瘤性能。结果与结论:骨肉瘤干细胞在接种培养2-10d后形成悬浮细胞球,增殖潜伏期约为24h,Stro-1阳性干细胞能够形成悬浮细胞球,Stro-1阴性细胞则不能形成悬浮细胞球。此外,骨肉瘤干细胞还能高表达Oct3/4、Nanog和CD133等,CD133阳性骨肉瘤干细胞高表达CD133分子,侵袭力更强,而CD133阴性细胞则不能表达CD133分子,侵袭力相对较弱。塞来昔布对骨肉瘤干细胞的形成具有一定程度的抑制作用,能够降低肿瘤新生血管中血管内皮生长因子的表达。

BACKGROUND： Osteosarcoma is the most common bone primary malignant tumors, and the occurrence of osteosarcoma may relate with osteosarcoma stem cells. OBJECTIVE： To evaluate the isolation, culture and identification method of osteosarcoma stem cells, to investigate the expression of relative tumor markers.METHODS： The osteosarcoma stem cells were isolated and cultured with magnetic activated cell sorting method under serum-free condition and serum-free joint antineoplastic condition, in order to sort Stro-1 positive and CD133 positive osteosarcoma stem cells. The expression level and tumorigenic properties of CD133, Oct3 /4 and Nanog markers of osteosarcoma cancer stem cells were tested with immunofluorescence staining and Western blotting methods. RESULTS AND CONCLUSION： Suspended cell condensation could be seen in the osteosarcoma stem cells after culture for 2-10 days, the proliferation incubation period was about 24 hours, and the Stro-1 positive stem cells could form the suspended cell condensation, while the Stro-1 negative stem cells could not form the suspended cell condensation. In addition, osteosarcoma stem cells could highly express Oct3/4, Nanog and CD133, the CD133-positive osteosarcoma stem cells could highly express CD133 molecule with strong invasiveness, while the CD133 negative cells could not express CD133 molecule and the invasiveness was weak. Celecoxib could inhibit the formation of osteosarcoma stem cells to some extent, and could reduce the expression of tumor angiogenic blood vessels and vascular endothelial growth factor.