The Expression And Relationship Of NANOG ,CD133 And GFAP In Gliomas

Objective The co-expression of NANOG,CD133 and GFAP are detectted in the organization of the glioma,discussing the relationship between NANOG and CD133,GFAP;culturing and identificating stem cells in U87,discussing the relationship between NANOG and CD133 in the cellular levels and demonstrate its significance in gliomas formation.Methods (1) The double-labeled of NANOG/CD133 and NANOG/GFAP were detected by immunohistochemical double staining method in 60 cases of human gliomas respectively. The percentage of NANOG~+cells, CD133+ cells, GFAP+ cells, NANOG+/CD133+ cells and NANOG+/GFAP+ cells was calculated. All the results were carried on statistics analysis. (2) Culture glioma cell lines U87 with the method of serum deprivation .The formation of tumor spheres were observed when they were inoculated onto serum-containing medium; The immunofluorescence staining of cells was employed to identify the surviving cells cultured in serum deprivation medium,tumor spheres and differentiated cells.(3) The double-labeled of NANOG/CD133 were detected by immunohistochemical double staining method in glioma stem cells, analysis of the relationship between them.Results (1) The percentage of the NANOG+, CD133~+, and GFAP~+ cells was significantly different in different pathological levels of the human glioma ,and the NANOG~+expression and the CD133+ expression were positively correlated (r = 0.904, P<0.001), the NANOG~+ expression and the GFAP~+ expression were negative related (r =-0.907, P<0.001). With the pathological level rise,the percentage difference of NANOG+/CD133~+ cells was significant (F = 560.805, P<0.001), the percentage difference of NANOG+/GFAP~+ cells was not statistically significant(F=0.355,P>0.1), and NANOG~+/CD133~+ expression was positively correlated with CD133~+ expression (r=0.823,P<0.001). (2) Using of the serum deprivation method selected tumor stem cells successfully which expressed CD133 and could form tumor spheres.The tumor spheres had a ability of multi-differentiations,the daughter cells expressed GFAP and NSE; The NANOG and CD133 has a significant correlation,they are co-expression in tumor sphere cells,the cells around the stem cells with a polarization of the state express CD133 only without express NANOG.Conclusion (1) NANOG presented at different levels of a brain tumor, there was an obvious correlation between CD133 ,CD133~+/ NANOG~+ cells and the pathological were positively correlated, the tumor with CD133~+ cells would also express NANOG, and they are co-expression in the cell level. In different pathological levels of the human glioma, the NANOG~+ expression and the GFAP~+ expression were negative related. The hight expression of NANOG in glioma would be relatively low level expression of GFAP, and in different levels of pathological tumor, NANOG~+/GFAP~+ cells are not statistical significance. (2) The U87 cell lines exist glioma stem cells which have the capacities of self-renewal and multi-differentiation.It is the first time to applicate serum deprivation to isolate cancer stem cells from glioma cell lines U87 successfully and they could form tumor spheres also; the NANOG and CD133 are co-expression significant in the cellular levels, NANOG was high expressed in tumor stem cells, could not express in undifferent cells, Maybe played an important role in maintaining a state of stem cell state.