| Objectives1.Based on the resistance of cancer stem cells(CSC),exploring the efficiency of enriched adrenocortical carcinoma(ACC)stem cells with the intervention of cyclophosphamide(CTX)in vivo by consecutively passages,provide more alternativer ways to find the ACC stem cells.2.To detect the expression levels of CD 133,the expression of ABCG2 and CXCR4 in the enriched cells and control group cells was evaluated to find the surface marker of ACC stem cell,which provide an experimental basis for the separation of cancer stem cells in the future.Methods:1.The enrichment of ACC stem cells was performed by consecutively passaging ACC cell line SW-13 for 3 times in nude mice with the trearment of CTX in vivo.2.To study the efficiency of enriched cells:the capacity of self-renewal of enriched cells was determined by observing sphere formation;The differentiation and proliferation of enriched cells Was analyzed by MTT;Single cell and flat colony formation were used to evaluate the clonogenicity of enfiched cells.3.The expression levels of CD133?CXCR4 and ABCG2 in enriched cells were analyzed by flow cytometry.Results:1.Among the three generations of enriched cells and control group cells,the single cell colony formation rate were(58.67±2.31)%?(75.00±3.61)%?(79.00±2.00)%and(43.33±3.06)%,flat colony formation rate were(349.67±12.99)‰?(449.33±42.70)‰?(618.67±24.58)‰ and(196.33±24.46)‰,sphere formation rate were(1.52±0.29)%?(4.17±0.41)%?(5.41 ±0.90)%and(0.37±0.11)%;The single cell colony formation rate,flat colony formation rate and sphere formation rate of the CTX enriched cells were higher than the control group cells(P<0.05).The growth curve of the cells in four groups showed proliferative activities of CTX enriched cells were higher than the control group cells(P<0.05).2.Among the three generations of enriched cells and control group cells analyzed by flow cytometry,the expression of CD133 were negative;The expression rate of ABCG2 were(98.03±1.35)%?(92.86±5.33)%?(89.40±5.27)%and(87.09±12.34)%,among the 4 groups(P>0.05);CXCR4 were(22.77±1.11)%.(40.93±2.33)%?(87.41±3.78)%and(27.85±12.71)%,and the expression of 3rd-generation cells was higher than control group(P<0.05).Conclusions:1.Cyclophosphamide intervention can increase the relative proportion of cancer stem cells in the adrenocortical carcinoma.2.CXCR4 was highly expressed in the the 3rd-generation cells and it could be used as a sorted markers for adrenocortical carcinoma stem cells. |
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