| ObjectiveEstablish the model of CD133+/CD44+phenotype Thyroid cancer stem cells（TCSC）by culturing the 8505C people thyroid anaplastic carcinoma cell line in serum-free medium（SFM）,and analyze its biological characteristics;Test Salinomy-cin（SAL）for thyroid cancer stem cell proliferation and apoptosis regulation,prelimi-narily Salinomycin inhibition of Wnt/β-catenin signaling pathways induced thyroid cancer stem cell apoptosis mechanism of molecular biology.Methods1.Establishment of the thyroid cancer stem cell model in vitro:Culture the anaplastic thyroid carcinoma 8505C cells in serum-free medium and complete medium（CM）,and observed the changes on cell morphology and growth mode with inverted microscope.Immunofluorescence stained cell surface antigens CD133 and CD44,and observed the xpression of cell surface molecular marker CD133 and CD44 using fluorescence microscope.At the same time,analyzed the cell cycle changes and difference subsets of cells using Flow cytometry（FCM）,then detected the enrichment efficiency of the thyroid cancer stem cells.2.Mechanism of anaplastic thyroid carcinoma microsphere cells apoptosis induced by salinomycin:CCK-8 assay was used to detect the effect of salinomycin on the proliferation of anaplastic thyroid carcinoma microsphere cells.PI staining assay was used to determine the effect of salinomycin on anaplastic thyroid carcinoma microsp-here cells.Inverted microscope was used to observe apoptotic morphology and Ann-exin V/PI apoptotic detection kit was used to investigate apoptosis in the anaplastic thyroid carcinoma microsphere cells.The levels of proteins involved in cell apoptosis were evaluated by Western blotting assay.Results1.The cell line cultured by serum-free culture grew in the form of microspheres of different sizes suspending in the medium,the cell line cultured by comlete medium grew in the form of monolayer adherent growth.Serum-free media could induce CD133+/CD44+thyroid cancer cells to be the CD133+/CD44+cells.Compared to the adherent cells,the number of cells in S phase reduced and the cells in G1 phase incr-eased,that is the cell cycle progression was arrested at G1 phase.The expression of CD133 and CD44 were up-regulated in the suspension cells.The progression of CD133+/CD44+cells proportion in suspension cells and adherent cells were 18.91%±2.75%and 85.78±2.57%,respectively.2.CCK-8 assay indicated that salinomycin inhibited the proliferation of anapla-stic thyroid carcinoma microsphere cells in a dose-and time-dependent manner,and the inhibition effect was enhanced as the drug concentration increased.IC500 doses of salinomycin at 48h was 8.76±1.97%μM.After salinomycin treatment of anaplastic thyroid carcinoma microsphere cells,morphological changes of apoptosis were obsn-der the inverted microsphere,the microspheres gradually detached,the cells became edema and some of them were shrinkage into fragments.PI staining assay showed that the cells apoptosis induced by salinomycin was probably accompanied by cell cycle arrest.Annexin V/PI double-labeled assay showed that the apoptosis ratesi-nificantly increased as the concentration of salinomycin increased.Western blotting analysis increased that,as the drug concentration increased,the levels of cleaved Cas-pase3、Bax andβ-catenin were up-regulated（P<0.05）,the levels of cleaved PARP were down-regulated（P<0.05）.Conclusions1.Serum-free culture techniques make thyroid anaplastic carcinoma cell line 8505C cell morphology and transformed the cell cycle from the S phase to G0/G1 phase,then enriched CD133+/CD44+thyroid cancer stem cells.2.Salinomycin inhibited the cancer cells proliferation significantly and induced caspase-dependent apoptosis on thyroid cancer stem cells,the induction of apoptosis may be related to the action of Caspase-3,PARP,Bax,β-catenin adjustment related to each other,between multiple protein salinomycin through regulation of Wnt/beta catenin signaling pathways,ultimately forced CD133+/CD44+phenotype of thyroid cancer stem cell apoptosis. |
PDF Full Text Request