The Isolation And Identification Of Esophageal Cancer Stem Cells

Object:Study the methods of separation esophageal cancer stem cells.Methods: The expression and localization of CD44, CD133, P75NTR and Oct-4 were detected using fluorescence immunocytochemistry respectively, and the cell cycles were analyzed by Flow Cytometry. And then the As2O3 toxicity to TE-1 cell were detected by MTT in different concentrations (0,0.5,1,2,4,8, and 16μmol/L) and different time (24h,48h and 72h). The cell apoptosis rate induced by As2O3 in 0,2,4 and 8μmol/L and in different time (24h,48h and 72h) were analyzed FCM with AnnexinⅤ/PI double labeling. According to MTT results, the 8μmol/L As2O3 was chose to treat the TE-1 cells when it spreaded over 80%of the bottom of the culture bottle after 12 day, TE-1 cells got the arsenic-resistant.Results:(1)The expression of CD44, CD133,P75NTR, Oct-4 were all positive in the TE-1 cell and CD44 and CD 133 fluorescence locate in the cytoplasm, but p75NTR nuclear positive rate is 44.29%, Oct-4 nuclear positive rate is 51.34%. (2) When the As2O3 concentration is greater than 1μmol/L and the poisoning of As2O3 to TE-1 cell increase depend on the As2O3 concentration and treatment time. (3) As2O3 promotes apoptosis of TE-1 cell in a concentration and time-dependent style. Apoptosis rate increase with As2O3 concentration and treatment time increase. When the As2O3 concentration is higher than 4μmol/L, promote apoptosis (p<0.05). (4) After culture with 8μmol/L As2O3 and TE-1 cells for 48h, the TE-1 cells were found forming some island-like structure surrounding by some cells in strong light refraction, which appear to form a protective barrier of the cell island. After maintain 24h, the cells in strong light refraction replaced by a layer membrane. A cycle from the strong refraction cell barriers appear to disappear alternately is two days. After 12 days culture, about 50% cells were live. When the 8μmol/L As2O3 was removed, the cell around the islands become loose, the thick membrane disappears, the space between the cell islands was recruited. (5) the cell number in G0/G1 phase after withdrawal of arsenic were significantly less than that in G0/G1 phase of the TE-1 cells (P<0.01), but S phase cells increased significantly (P<0.01). (6) The expression of CD44, CD133, P75NTR, Oct-4 were positive in the cells removing arsenic, but p75NTR and Oct-4 nuclear positive rate is obviously decreased.Conclusion:(1) P75NTR, Oct-4, CD44 and CD133 is not a specific marker of esophageal cancer stem cells, but P75NTR and Oct-4 expressed in the different position of TE-1 cells suggested that TE-1 cells are composed by the heterogeneous cells. (2)As2O3 can induce TE-1 esophageal cancer cells to acquire the arsenic resistance. Arsenic resistance cells are with self-renewal potential. (3) Whether arsenic resistance cells are esophageal cancer stem cells, need to be further confirmed.