Study On The Characteristics Of Stem Cell On Human Esophageal Cancer Cell Line Eca109Mammospheres By Serum-free Culture Method

Objective: To enrich the cancer stem cells in esophageal carcinoma cell line Eca109using method ofmammospheres formation in serum-free environment.To investigate the relationship between theexpression characteristics of p75NTRand Eca109cancer stem cells.Methods: Adjust the density of esophageal carcinoma cell line Eca109to l×105cell/ml. Eca109cells werecultivated in serum-free culture medium for14days to get mammospheres. Then observing the cell stationby the HE staining;The expression of p75NTR、p75ICDby western-blot in Eca109mammospheres;Theexpression and localization of p75NTR、p75ICD、Oct-4、Sox2、Lin28、Nanog were detected byImmunofluorescence technique in mammospheres,;Applicating MTT method and tableting cloningexperiment to detect the proliferation of mammospheres and Eca109cells in vitro; Using differentconcentrations of chemotherapeutic drugs to affect on mammospheres and Eca109cells48hours then testthe proportion of cell viability by MTT assay; Using transwell chamber assay to detect invasive ability ofmammospheres and Eca109cells.Results: Esophageal carcinoma cell Eca109form suspended mammosphere in serum-free medium for3days. Cultured for14days can be passaged, the second generation of mammospheres foemed faster thanthe first generation. The cells were seeded prepared by mammospheres and HE staining show that Eca109mammospheres Round or short spindle and a large nucleus central but cytoplasm below normal.Western-blot experiments showed that protein of p75NTRin mammospheres are less than Eca109cells; thenucle-oprotein of75ICDin mammospheres are significantly higher than plasma proteins,but thenucle-oprotein of p75ICDEca109cells are lower than the plasma proteins.Stem cell markers of p75NTR、p75ICD、Oct-4、Sox2、Lin28、Nanog are positive expression in mammospheres and Eca109cells.Todetectthe expression and localization of p75NTR、 p75ICD、 Oct-4、 Sox2、 Lin28、 Nanog byImmunofluorescence technique in mammospheres and Eca109cells, The expression of p75NTR、p75ICD、Oct-4and Lin28were positive in nuclear of t mammospheres,but Eca109cells most localized in thecytoplasm, only a small part localized in the nucleus,.The expression of Sox-2and Nanog were positive incytoplasm of mammospheres and Eca109cells were not significant difference. MTT assay showed thatmammosphere proliferation was significantly higher than the Eca109cells, the difference was statisticallysignificant (P <0.001). mammospheres were seeded in culture plates were cultured for14days after theformation of the cell count greater than50cells, and the colony forming rate was significantly higher thanEca109cells (P <0.05).Using different concentrations of carboplatin, paclitaxel and mitomycin were actingon the mammosphere cells and Eca109cells were cultured cells48hours the number of surviving cellswere more than Eca109cells and the difference was statistically significant.Transwell chamber assayresults were shown that the invasion ability of mammospheres were more stronger than Eca109cells, thedifference was statistically significant (P <0.01).Conclusion:(1) Stem cell markers p75NTR、p75ICD、Oct-4、Lin28may be sorting esophageal cancer stemcells important marker, p75NTRmay be specific markers.(2) p75NTR、p75ICD、Oct-4and Lin28nuclear-positive cells may be esophageal cancer stemcells.