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X. Jin et al.
normal hematopoietic stem and progenitor cells (HSPCs), especially in erythroid cells, which may result from growth differentiation factor 11(GDF11)-induced reactive oxygen species (ROS) and shortens survival in MDS mice. Here we are the first to utilize RUNX1-S291fs-induced MDS mice to successfully construct an iron overload model. This can provide the experimental basis for further exploring the influence and mechanism of iron overload on MDS.
Methods
Ethical considerations
Our experiment has been approved by the Ethics Committee of Tianjin First Central Hospital. All animal experiments were con- ducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (n. 1204).
Vector and retrovirus construct
The Empty control vector (pMYs-Empty control-IRES-GFP) and the RUNX1-S291fs mutant vector (pMYs-RUNX1 S291fs-IRES- GFP) were kindly provided by Dr Atsushi Iwam (Kumamoto University, Japan). Production of retrovirus construct has been described previously.8
Mice and treatment
C57BL/6 mice were purchased from Vital River (Beijing, China) at 6-8 weeks of age. The animals were quarantined and allowed to acclimatize for one week. They were maintained in a room at 22±2°C, with a relative humidity of 50%±10%. The mice were housed 5 individuals per cage and used at a weight of approxi- mately 20.0-22.0g. Retroviral construct transduced into bone mar- row mononuclear cells (BMMNCs) was performed as previously described.9 Briefly, retrovirus-infected BMMNCs were cultured in αMEM medium containing 10 ng/mL of human IL-6, 10 ng/mL of mouse IL-3, and 100 ng/mL of mouse SCF for three days, and then these cells were intravenously injected into 9Gy lethally irradiated recipient mice together with radioprotective dose of 1×106 BM cells to generate MDS mice or control mice. Eight weeks post transplantation, the recipient mice were administered an intraperi- toneal injection of 0.2 mL iron dextran at a concentration of 25 mg/mL once every other day for a total of 8 times to establish an iron overload model. At the same time, normal saline was given to the control group.
Flow cytometric analysis
Flow cytometry was performed by using the following mono- clonal mouse antibodies: CD45.2, CD45.1, Gr1, Mac1/CD11b, Ter119, CD71, B220, CD19, CD3, CD5, CD41, CD117/c-Kit, Sca1 and CD34. All Flow cytometric analysis of the stained cells was performed with Coulter Altra (Beckman Coulter) equipped with Cytexpert software (Beckman Coulter).
Western blot analysis
To detect the expression of RUNX1S291fs protein, BM cells and spleen cells were lysed and blotted as previously described.10 Polyclonal anti-RUNX1 and anti-α-Tubulin antibodies were used to detect RUNX1S291fs protein and α-Tubulin, respectively.
Diagnosis
The diagnosis was made according to the Bethesda proposals for classification of MDS in mice.11
Statistical analysis
Results were analyzed with the GraphPad Prism program (GraphPad Software, Inc., San Diego, CA, USA). Data obeyed nor- mal distribution were presented as means±Standard Deviation (SD) and multiple group comparisons were performed by using one-way analysis of variance (ANOVA), whereas data with non- normal distribution were showed by median and quartiles, and compared by Kruskal-Wallis test. The survival curves were ana- lyzed using the Kaplan-Meier method with the log-rank test. P<0.05 was considered statistically significant.
Results
Iron overload model can be established in RUNX1S291fs-induced MDS mice
In order to explore the role of iron overload in MDS, we first transduced a pMYs-RUNX1S291fs-IRES-GFP or a pMYs-Empty control-IRES-GFP retroviral construct into BMMNCs (Figure 1A). After five days culture in vitro, the transduced cells were transplanted into lethally irradiated recipient mice together with radioprotective BM cells to generate MDS mice or control mice (Figure 1A). Eight weeks after transplantation, the recipient mice were administered iron dextran or normal saline by intraperi- toneal injection. Hereafter, we refer to the Empty control mice with normal saline, the Empty control mice with iron dextran, the RUNX1S291fs mice with normal saline, and the RUNX1S291fs mice with iron dextran as Empty/NS, Empty/FE, RX291/NS and RX291/FE mice, respectively. The ratio of GFP+ cells gradually increased over 24 weeks after the transplantation in RX291/NS and RX291/FE mice, but not in mice who received transplants of the Empty control-transduced cells (Figure 1B), which indicat- ed that the RUNX1S291fs-transduced cells successfully engrafted and established chimerism in the peripheral blood (PB).
To verify the establishment of an MDS model, several mice were sacrificed at 24 weeks post transplantation. The RUNX1S291fs protein was detected high expression in spleen and low expression in BM in RX291/NS and RX291/FE groups, but not in Empty/NS and Empty/FE mice (Figure 1C), suggesting that RUNX1S291fs was suc- cessfully transduced and expressed. We then examined whether RUNX1S291fs contributes to the development of MDS in recipient mice. The mice received RUNX1S291fs- transduced cells showed significantly reduced white blood cell (WBC) counts (Figure 1D), hemoglobin (HG) levels (Figure 1E), platelet counts (PLT) (Figure 1F), and increased mean corpuscular volume (MCV) (Figure 1G) compared with Empty control recipient mice. The RUNX1S291fs mutant mice also showed dysplastic cells, such as dot-like and polychromatic erythrocytes, giant platelets, pseudo- Pelger-Huet granulocytes in the PB, together with dual- nucleated granulocytes, increased immature blasts (less than 20%) in the BM, characteristic of human MDS (Figure 1H). Then we examined the pathological changes in the femur, liver and spleen. The femur showed extremely active myeloid hyperplasia, mainly in blast cells, with the increased ratio of granulocyte/red cells and abnormal localization of blast precursor cells in RUNX1S291fs mice but not Empty control mice (Figure 1I). Moreover, the RUNX1S291fs mice showed many nucleated cells around the central vein and the portal area of the liver, and exhibited enlargement and irregular shape
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