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

目的 从中枢神经系统原发性神经外胚层肿瘤(Central nervous system primitive neu-roectodermal 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细胞在于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Nes-tin.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,并能表达干细胞的标记抗体,可以作为理想的CSCs进行一系列的后续研究.

Isolation and identification of cancer stem cells from central nervous system primitivetumors

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-free NSC medium was used to promote the formation of cells spheres to enrich CSCs subpopulation. CSCs were identified by second-ary 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 in-to multiple morphologic cell types with negative Sox2 and Nestin expression. All nude mice that im-planted 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-free NSC medi-um,CSCs with the ability to self-renew,initiate brain tumors upon implantation and multipotency can be isolated and enriched from CNS-PNET.