重型再生障碍性贫血模型小鼠骨髓间充质干细胞过度衰老的机制研究

OBJECTIVE: To explore the mechanism of excessive senescence in bone marrow-derived mesenchymal stem cells (BM-MSC) of mouse model with severe aplastic anemia (SAA). METHODS: 40 BALB/c mice were randomly assigned to two groups of control (n=20) and AA (n=20). SAA mouse model was induced by intraperitoneal injection with IFN-γ and intragastric infusion with busulfan. BM-MSC were isolated and cultured from bone marrow of SAA and healthy mice. The cell morphology was observed by inverted microscope and cell cytoskeleton was stained by Rhodamine-Phalloidin; The level of proliferation was analyzed by CCK-8 method, and cell cycle was tested by flow cytometry. Senescence-associated β-galactosidase (SA-β-gal) assay was used to detect senescent BM-MSC; The expression of mTOR protein was detected by Western blot method. RESULTS: BM-MSC from normal mice presented spindle-shaped, clear boundaries and stress fibers were arranged in parallel, neat. while BM-MSCs from SAA mice presented cell volume increases, tiled, ill-shaped and the stress fiber appeared to be disordered. The decreased activity of proliferation [more cells restricted in G(0)/G(1) phase [(77.461±1.567) % vs (46.045±2.055) %, t=−34.384, P<0.001], increased percentage of SA-β-gal positive cells [(75±11) % vs (28±8) %, t=15.454, P<0.001] and notably enhanced expression of mTOR of BM-MSC from SAA mice were observed when compared with those from normal mice. CONCLUSION: This study clarified senescent BM-MSCs from SAA model mice, which could be caused by the excessive activation of mTOR pathway.