| Objective:To establish stable regulated brain tumor mouse model with sternness coexpressing c-myc and SV40Tag induced by Tet-on system and lay the ground work for future research on the special property of the brain tumor stem cells (BTSCs). To isolate and identify normal adult neural stem cells (NSCs), induced adult neural stem cells (NSC's) and mouse brain tumor stem cells from subventricular zone (SVZ) tissue of non-induced genetically engineered mouse models, SVZ tissue and tumor tissue of induced genetically engineered brain cancer mouse models respectively. To learn the similarities and differences among normal adult neural stem cells, induced adult neural stem cells and mouse brain tumor stem cells, and define the differential molecular mechanisms. To provide evidences for the hypothesis that brain tumor stem cells originate from neural stem cells.Methods:Transgenic mouse coexpressing pTRE2-c-myc, Tet-on and pTRE2-SV40Tag were generated by crossing the pTRE2-c-myc transgenic mouse with transgenic mouse coexpressing both Tet-on and pTRE2-SV40Tag, and induced by doxycycline (DOX) to generate brain tumor mouse model. Magnetic resonance image, macroscopy and histopathology of the tumor specimen and immunohistochemistry were used to investigate the characteristics of the model. Cells from SVZ tissue of non-induced genetically engineered mouse models and SVZ tissue of induced genetically engineered brain cancer mouse models were cultured in DMEM/F12 mediume containing FGF2, epidermal growth factor and B27 minus retinyl acetate. Differentiated cells of the two group cell spheres were cultured in DMEM/F12 containing 10% FCS. Brain tumor tissues were obtained from genetically engineered mouse which symptoms appear. Cells from tumor tissue were cultured in DMEM/F12 mediume containing fibroblast grwoth factor-2 (bFGF), epidermal growth factor (EGF) and B27 minus retinyl acetate. Differentiated cells of mouse brain tumor spheres were cultured in DMEM/F12 containing 10% FCS. The morphous and proliferation of normal adult neural stem cells, induced adult neural stem cells and mouse brain tumor stem cells were obeserved under phase contrast microscope. The ultrastructures of the three group cells spheres were obeserved under transmission electron microscope (TEM). To determine whether the three group cells spheres contain stem cells, Hoechst 33342 was used to sort for the side population (SP) phenotype. Cell cycle and chromosome ploidy of the three group cells spheres were performed on Flow cytometry. Multiple immunofluorescence was used to assays the cell markers of the three group cells spheres and differentiated cells. Immunofluorescence was used to define the differential expression in c-myc and p53 pathway among normal adult neural stem cells, induced adult neural stem cells and mouse brain tumor stem cells.Results:(1) Thirty of Seventy-three transgenic mouse coexpressing pTRE2-c-myc, Tet-on and pTRE2-SV40Tag generate brain tumor induced by DOX. The incidence of brain tumor was 41.1%. Beside of one brain stem tumor, all of the other tumors were located on the pineal region. On magnetic resonance imaging (MRI), these lesions appear isointense to hyperintense on TI-weighted images and and isointense to hypointense on T2 weighted images. These lesions were enhanced and rather homogeneous to moderately heterogeneous with contrast images. The brain tumors were diagnosed as undifferentiated medulloblastoma. The brain tumors were highly invasive. And immunohistochemistry indicated that the tumors overexpressed c-myc, SV40Tag and stem/progenitor cells mark nestin.(2) Cells from SVZ of uninduced and induced genetically engineered adult mouse formed cell spheres when cultured in DMEM/F12 mediume containing FGF2, epidermal growth factor and B27 minus retinyl acetate. Uninduced adult neural stem cells spheres differentiated into neuronal and glial cells when cultured in DMEM/F12 containing 10% FCS. Induced adult neural stem cell spheres exhibited enhanced proliferating capacity and impaired differentiation. The ultrastructures of uninduced and induced adult neural stem cell spheres indicated stem cells characteristics of undevelopped organelles, high nuclear-cytoplasmic ratio. Compared with uninduced adult neural stem cell spheres, cellular organs associated with differentiation and degradation, for example lysosome, autophagosome, were significant reduced in induced adult neural stem cell spheres. The average first sort percentage of SP cells was 10.43% and 16.89% in uninduced and induced adult neural stem cell spheres respectively. All uninduced and induced adult neural stem cell spheres were diploid. A majority of cells in uninduced adult neural stem cell spheres were at S phase. A majority of cells in uninduced adult neural stem cell spheres were nestin positive. The differentiated cells of uninduced adult neural stem cell spheres were GFAP and MAP2 positive respectively. A majority of cells in induced adult neural stem cell spheres were nestin positive, and small fraction were GFAP and NSE positive. But partially differentiated cells still coexpressed stem cells marker nestin and differentiation markers GFAP and MAP2, and even coexpressed differentiation markers GFAP and MAP2.(3) Cells from genetically engineered mouse brain cancer tissue formed stem-like sphere when cultured in DMEM/F12 mediume containing FGF2, epidermal growth factor and B27 minus retinyl acetate, and differentiated into neuronal and glial like cells when cultured in DMEM/F12 containing 10% FCS. The ultrastructures of brain tumor sphere indicated stem cells characteristics of undevelopped organelles, high nuclear-cytoplasmic ratio. Cellular organs associated with differentiation and degradation were seldom seen. The average first sort percentage of SP cells was 17.27%. Heteroploid in mouse brain cancer spheres was 26.77%. A majority of cells were at S phase. A majority of cells in mouse brain cancer spheres were nestin positive and small fraction of cells were NSE and GFAP positive. Most differentiated cells coexpress nestin with MAP2 or GFAP, and even coexpressed differentiation markers GFAP and MAP2. Brain tumor stem cells and induced neural stem cells overexpressed c-myc and MDM2 (negative regulator of p53).Conclusion:(1) Stable regulated brain tumor mouse model coexpressing c-myc and SV40Tag induced by Tet-on system was established. The ground work was laied for future research on the special property of the brain tumor stem cells (BTSCs).(2) Adult neural stem cells were isolated from SVZ of uninduced and induced Tet-on genetically engineered mouse models. Induced neural stem cells exhibited enhanced proliferating capacity and impaired differentiation. Our data indicated adult neural stem cells acquired early malignant transformation.(3) Tet-on genetically engineered mouse brain cancer contains brain tumor stem cells which have the capacity to self-renew and muti-differential potentiality. Brain tumor stem cells exhibited enhanced proliferating capacity and impaired differentiation as same as induced neural stem cells. Activation of c-myc and inactivation of p53 pathway play critical roles in substance of brain tumor stem cells and malignant transformation of neural stem cells.(4) Adult neural stem cells and brain tumor stem cells were isolated from one Tet-on genetically engineered brain cancer mouse models. Tet-on genetically engineered brain cancer mouse models become powerful tool for brain tumor stem cells research.
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