人脑胶质瘤干细胞扩增球体模型的建立及其蛋白质和脂质含量检测

Objective To establish a three-dimensional spheroid expansion model of human glioma stem cells with spontaneous sphere forming and multilineage differentiation potential in vitro and investigated the contents of the proteins and lipids and their secondary components, so as to lay a foundation for further study of sphere metabolism. Methods Human glioma stem cells GSC23 and SU3 were cultured in serum- free stem cell culture medium, respectively, and the cell spheres were harvested for about 2-3 weeks. After fixation in paraformaldehyde solution, dehydration, paraffin embedding, and sectioning, glioma-associated marker proteins were detected by immunohistochemical staining, and the protein and lipid and their secondary components contents in the spheroid tissues were analyzed by Raman imaging. One-way ANOVA was used to compare the protein, lipid and phenylalanine contents in the large sphere, medium sphere and small sphere groups. Results Both stem cells were able to form stem cell expansion spheres resembling solid tumors within the culture dish. Immunohistochemical staining showed that the regular marker proteins of glioblastoma multiforme, CD133, nestin, epidermal growth factor receptor (EGFR), S100, Olig2, p53, Ki-67, glial fibrillary acidic protein (GFAP), vimentin, CXC chemokine receptor 4 (CXCR4), and CD34, were all expressed. Raman imaging revealed that the constructed expanded spheres of human glioma stem cells contained protein (2 930, 1 685 and 1 586 cm^-1), lipid (2 845 and 1 444 cm^-1), phenylalanine (1 003 cm^-1) amide Ⅲ (1 250 cm^-1), while there were no significant differences of protein, lipid and phenylalanine contents among the large sphere, medium sphere and small sphere groups (P > 0. 05). Conclusions We have successfully established an expanded spheroid model of human glioma stem cells in vitro, which not only exhibits the topographical characteristics and unlimited expansion ability of three- dimensional solid tumors, but also has the ability to stably store metabolically obligatory energy sources of tumor cells such as proteins and lipids, and is expected to serve as a promising tool for human glioma research in vitro.