免疫磁珠法分选人脑胶质瘤干细胞及其培养和鉴定

Sorting of Human Brain Glioma Stem Cells through Magnetic Activated Cell Sorting, its Culture in Vitro and Identification as a Stem Cell

背景与目的:脑胶质瘤治疗效果一直不理想,这与胶质瘤细胞无限增殖能力和侵袭性生长有关,本研究旨在从人脑胶质瘤组织和细胞株中分离脑胶质瘤干细胞、进行体外培养并对其干细胞特性加以鉴定。方法:采用以CD133为标志的免疫磁珠法从人脑胶质瘤组织和细胞株中分离脑胶质瘤干细胞并进行体外培养,通过免疫荧光技术检测干细胞标志物CD133、Nestin,诱导分化后检查分化细胞标志物MAP2、GFAP、MBP以及电镜超微结构观察和移植SCID鼠致瘤实验,对其干细胞特性加以鉴定。结果:新鲜人脑胶质瘤组织和胶质瘤细胞株中存在一小部分CD133+的胶质瘤细胞,能表达干细胞的标志物CD133和Nestin,符合干细胞的超微结构特点,体外培养能连续传代;诱导分化后能产生MAP2、GFAP、MBP染色阳性的细胞;移植SCID鼠后能形成与亲本肿瘤表型一致的移植瘤。结论:新鲜人脑胶质瘤组织和胶质瘤细胞株中存在的一小部分CD133+胶质瘤细胞具有干细胞的属性,就是胶质瘤中的肿瘤干细胞,即胶质瘤干细胞。

BACKGROND＆OBJECTIVE： To sort brain glioma stem eells（BGSCs） from glioma cell lines and fresh glioma specimens, culture in vitro and identify its character as a stem cell. METHODS：BGSCs were sorted through Magnetic Activated Cell Sorting（MACS, immunomagnefie beads marking by CD133） and cultured in vitro, and character as a stem cell was identified by stem cell markers （CD133 and Nesfin）and differentiated cell markers [mierotubule-assoeiated protein 2（MAP2）, glial acidic fibrillary protein （GFAP） and myelin basic protein （MBP）], ultrastrueture observing with electron mieroseope（EM） and engrafting to severe combined immunodefieieney miee（SClD mice） for tumorigenesis test. RESULTS： Only a small subset of CD133＋ glioma cells in glioma cell lines and fresh specimens from various pathologic grade could express stem cell markers CD133 and Nestin, view ultrastrueture of a stem cell and be capacity of serial passage in culture. These CD133＋ cells possese a marked capacity for muhipotent differentiation and could differentiate into tumor cells expressing MAP2, β-Tubulin Ⅲ, GFAP and MBP; When engrafted into SCID mice, they can generate and form tumors that phenotypically resembl the tumor from the patient. CONCLUSION： The small fraction of CD133＋glioma cells in glioma cell lines and fresh specimens from various pathologic grade identified stem cells is BGSCs, so called as tumor stem cells in glioma.