Objective       To explore whether cancer stem-like cells (CSCs) can be induced under hypoxia from glioma cells in vitro.       Methods       Glioma cell lines U87 and GL261 and primary glioma cells cultured from pathologically confirmed glioma patients were cultured in DMEM/F12+10%FBS

and

magnetic-activated cell sorting (MACS) was used to sort CD133

-

cells. The markers of CSCs

CD133

SOX-2

OCT-4

KLF-4 and Nanog

were detected by immunofluorescence staining after the CD133

-

cells exposure to hypoxia for 48 h. qRT-PCR and Western blotting were used to detect the expression of above markers at RNA and protein levels under hypoxia in 0

3

6

9

12 and 24 h or 0

12

24

48 and 72 h respectively. Flow cytometry was used to detect the proportion of CD133

+

cells

cell cycle and cell apoptosis in 1

3

5 and 7 d after hypoxia.       Results       Immunofluorescence staining demonstrated the above CSCs markers were highly expressed in 48 h after hypoxia. Both qRT-PCR and Western blotting showed obviously increase in the expression of markers (P0.05). Specifically

qRT-PCR proved that SOX-2

OCT-4 and Nanog reached the highest expression in 9 h after hypoxia

and KLF-4 and CD133 in 12 h. Western blotting displayed the highest expression of CD133 was seen in 12 h

that of KLF-4 and Nanog in 48 h

and that of SOX2 in 72 h. With the elapse of hypoxic treatment

the proportion of CD133

+

was also increased greatly (P0.05). What’s more

hypoxia treatment induced CD133

-

cells arrested in G

0

/G

1

phase

decreased the cells at G

2

/M+S phase (P0.05)

and resulted in lower apoptotic rate than the cells under normoxia (P0.05).       Conclusion       Hypoxia induces the formation of CSCs from glioma cells.