免疫磁珠法分离、培养人脑胶质瘤干细胞

目的建立免疫磁珠法分离，并培养人脑胶质瘤干细胞的方法。方法将术中取得的脑胶质瘤标本，通过剪切、消化和吹打成单细胞悬液，筛网过滤，免疫磁珠分选试剂盒分选出CD133^＋细胞，用神经干细胞无血清培养法培养出具有单细胞克隆能力的细胞球，取第3代进行诱导分化，分化前后用免疫细胞荧光化学方法鉴定肿瘤干细胞及分化后细胞。结果免疫磁珠分选出的CD133^＋细胞，可悬浮生长并形成神经干细胞样细胞球，有较强的增殖能力，干细胞标志物巢蛋白（nestin）阳性，分化后细胞表达神经元小管相关蛋白β-3（β-tubulin3）和星形胶质细胞胶质纤维酸性蛋白质（GFAP）特异性抗原，而巢蛋白、CD133^＋阴性，并具有肿瘤的核型。结论免疫磁珠分选法可避免原代培养中众多细胞混杂生长的发生，能够从大量肿瘤细胞中分离出只占极少比例的肿瘤干细胞，细胞结合磁珠后在体外可以长期培养和传代，进一步证实了肿瘤干细胞的存在，并为胶质瘤干细胞的研究奠定基础。

Isolation and incubation of the glioma stem cells by immunomagnetic bead techniques

Objective To isolate and culture the glioma stem ceils by immunomagnetic bead techniques. Methods Glioma tissues obtained surgically were fully chopped and filtered into single cell suspension. Then CD133 positive cells selected by immunomagnetie bead techniques from the suspension were cultured in serumfree medium with epidermal growth factor （EGF）, basic fibroblast growth factor （bFGF）, leukemia inhibitory factor （LIF）, b27 to obtain tumor cell spheres. Tumor stem cells of the third passage were induced to differentiation. Specific markers such as nestin, glial fibriUary acidic portein （GFAP） and β-tubulin3 were employed before and after differentiation to recognize tumor stem cells and differentiated glioma cells respectively by immunofluorescence cytochemistry techniques. Results CD133 positive cells selected by immunomagnetic bead techniques showed the characteristics of stem cells such as suspended and sphere-like growth, self-renewal, and proliferation. The cultured sphere-like cells which had tumor karyotype and strong positive nestin protein could conditionally be differentiated into β-tubulin 3 positive neurons or GFAP positive astrocytes. Conclusion Primary cells cultured with irnmunomagnetic bead techniques can avoid the growth of other unwanted cell in the tissue and separate the tumor stem cells efficiently from the tumor cells. These cells have the capacity of proliferation, self-renewal, multi-directional differentiation and can be cultured and passaged in vitro for a long term. The experiment not only confirms the existence of tumor stem cell as previous study, but also provides a new efficient method for studying cellular and molecular biology of glioma.