| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The following steps have been performed for the study. (1) preparation of 18 primary glioblastoma (GBM) stem-like cell cultures from patients with primary GBM; among which 14 with high O6-methylguanine DNA methyltransferase (MGMT) expression and resistant to temozolomide were used in experiments;
(2) 5 primary cultures of bone marrow mesenchymal stem cells (MSCs) and a primary neural stem cell (NSC) culture were isolated from C57BL mice;
(3) 7 primary MSC cultures were isolated from human adipose tissue samples with confirmed phenotype (CD14, CD29, CD31, CD34, CD44, CD45, CD90, CD105);
(4) I have investigated the effects of TMZ, GSK3β inhibition and co-culture of primary GBM stem-like cells and MSCs on GBM stemness phenotype by proliferation assay, time-lapse microscopy and by evaluation of MGMT promoter methylation status and gene expression;
(5) I have investigated the effects of GSK3β inhibition and intra-tumor transplantation of MSCs on neurological state, cognitive functions and survival of C57BL mice with GBM model. This model is generated by using GBM line EPNT-5(Institute for Cytology, Saint-Petersburg) it was induced in С57BL mouse by 7,12-dimethylbenzanthracene and adequately represents human GBM; I have investigated with fluorescent microscopy the effects on mice EPNT-5 GBM cells by co-culture with mice MSCs.
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| 今後の研究の推進方策 |
For in vitro study, we isolate glioblastoma (GBM) stem-like cell culture from patients’ GBM tumors, mesenchymal stem cells (MSCs) from human adipose tissues and C57BL mice bone marrow, and primary neural stem cell cultures from C57BL mice. Molecular characteristics of these cells are examined to confirm the phenotypes of the respective cells. MSCs are labeled with a fluorescent linker and MSCs and GBM stem-like cells are transfected with GSK3β-specific short hairpin (sh)RNA expression vector. We then examine effects of MSCs, neural stem cells, GSK3β inhibition, TMZ and various combination of them against GBM stem-like cells. To address our working hypothesis of interaction between GBM stem-like cells and MSCs via GSK3β-mediated signaling, we investigate (a) c-Myc and O6-methylguanine DNA methyltransferase (MGMT) expression and MGMT promoter methylation; and (b) c-Myc and DNA (cytosine-5)-methyltransferase (DNMT) 3A binding to MGMT promoter according to our previous study (Carcinoenesis 2013).
For animal model study, we examine effects and underlying mechanism of intracranial transplantation of fluorescence-labeled MSCs, neural-transdifferentiated MSCs and neural stem cells against GBM in C57BL mice bearing mouse GBM. We observe survival as well as consequence of neurological state and cognitive functions in mice following transplantation of the respective cells.
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