| Objective: The issue was first introduced the separation method to isolate the brain tumor stem cells (BTSC) from human glioblastoma tumor cell line U251, which combines suspension culture and magnetic activated cell sorting (MACS). Then, based on U251 cell lines tumor stem cell apoptosis mechanism will be discussed. Accordingly provide new strategies for the therapy in glioma.Methods: First through suspension culture of U251 cells inoculated into serum - free medium with growth factor to obtain brain tumor sphere (BTS) which contains a lot of BTSC. Then separate and collect the BTS for further separation and purification through immunomagnetic beads sorting and verification for the positive test rate by flow cytometry. Then, approach the BTS mechanism of apoptosis by using flow cytometry cycle, AO/EB double staining and RT-PCR methods and from different levels to clarify U251 cell lines BTSC special apoptosis model.Results: With the. method combining suspension culture with MACS the positive rate of BTSC can be raised over 60%. Through flow automatic cytometry for cycle detection and AO/EB double staining and RT-PCR, the apoptosis of BTSC in the U251 cell line were found decreased in which the apoptosis promoting genes were down-regulated and the apoptosis suppressing genes were up-regulated.Conclusion: 1. In U251 cell line there is a certain proportion of stem cell characteristics of the cells, with the use of growth factor in serum - free medium suspension culture technology, it can effectively isolated and cultured BTS. When it was combind with MACS a higher positive rate of BTSC population can be isolated, so this thesis provide a new way for effective isolation. 2. The apoptosis of BTSC in the U251 cell line were decreased, it may be one of the important molecular mechanisms about unlimited growth. 3. Both from the technical platform or molecular mechanism it provides new strategy for glioma treatment.
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