中枢神经系统原发性神经外胚层肿瘤干细胞的分离与鉴定

Isolation and identification of cancer stem cells from central nervous system primitive neuroectodermai tumors

目的从中枢神经系统原发性神经外胚层肿瘤（Central nervous system primitive neuroectodermal tumor, CNS-PNET）中分离并鉴定肿瘤干细胞（cancer stem cells，CSCs）。方法应用无血清的干细胞培养基，分离CNS-PENT的细胞球细胞，应用二代细胞球形成分析、免疫细胞化学染色以及不同亚群肿瘤细胞裸鼠颅内接种等方法，进行CSCs鉴定。结果2种CNS-PNET细胞在干细胞培养条件下均形成了细胞克隆球，部分细胞球细胞均表达了神经干细胞标记抗体Sox2和Nestin。原代细胞球在连续传代培养中均可形成2代细胞球，且也能表达Sox2和Nestin。在血清的刺激下细胞球细胞可分化为多种形态的细胞，分化细胞亚群免疫染色均为阳性。10。个CNS-PNET细胞球细胞在颅内接种后几乎全部裸鼠形成肿瘤，相反，同等数量的原代肿瘤细胞接种后未见肿瘤生长。结论使用无血清的干细胞培养条件可以简便快捷的培养并分离出CNS-PNET的悬浮细胞克隆球体，具备自我更新、多向分化、体内成瘤的基本特征，并能表达于细胞的标记抗体，可以作为理想的CSCS进行一系列的后续研究。

Objective To isolate and identify the cancer stem cells （CSCs） from central nervous system primitive neuroectodermal tumors （CNS-PNET）. Methods Primary cultured tumor cells were derived from PNET/MB and sPNET specimens. The defined serum-flee NSC medium was used to promote the formation of cells spheres to enrich CSCs subpopulation. CSCs were identified by secondary sphere forming assay, fluorescent immunocytostaining and intracranial implantation in nude mice. Results Two types of CNS-PNET gave rise to clonal subsphere cells, some of which expressed neural stem cell markers including Sox2 and Nestin. Primary tumor cells could grow for 2 passages with the expression of Sox2 and Nestin. In the culture medium containing serum, sphere cells differentiated into multiple morphologic cell types with negative Sox2 and Nestin expression. All nude mice that implanted with 105 CNS-PNET cells developed intracranial tumors, but none of the nude mice developed tumors when primary tumor cells were implanted. Conclusions Using defined serum-flee NSC medium, CSCs with the ability to self-renew, initiate brain tumors upon implantation and multipotency can be isolated and enriched from CNS-PNET.