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  

The mechanism of bone marrow-derived mesenchymal stem cells excessive senescence in severeaplastie anemia mouse model

作　　者：欧议清 刘海燕[1] 陆伟[1] 闻梦静 刘红[1] 

机构地区：[1]南通大学附属医院血液科,226001

出　　处：《中华血液学杂志》2017年第4期325-329,共5页Chinese Journal of Hematology

基　　金：江苏省医学创新团队与领军人才基金（LJ201136）;2013社会事业科技创新与示范基金（HS2013067）;南通市科技项目临床医学研究中心建设基金（HS2015004）

摘　　要：目的探讨重型再生障碍性贫血（SAA）模型小鼠骨髓间充质干细胞（BM-MSC）过度衰老的机制。 方法选择BALB/c小鼠40只，随机分为正常组20只、SAA组20只。应用白消安灌胃联合IFN-γ腹腔注射诱导建立SAA小鼠模型；分离培养两组小鼠的BM-MSC。观察BM-MSC细胞形态和细胞骨架；CCK-8和流式细胞术检测细胞增殖与细胞周期情况；比较衰老相关β-半乳糖苷酶（Senescence-associated β-Galactosidase, SA-β-gal）的染色阳性细胞比例；Western blot法检测mTOR蛋白的表达水平。 结果正常组小鼠的BM-MSC细胞呈纺锤状、边界清晰，应力纤维平行排列、整齐；而SAA小鼠的BM-MSC细胞体积增大，呈平铺、边界不清状，应力纤维紊乱模糊。CCK-8结果显示，SAA小鼠的BM-MSC的增殖速率较正常组缓慢，在第2天开始差异有统计学意义（P〈0.05）。SAA组小鼠BM-MSC的G0/G1期细胞比率[（77.461±1.567）%对（46.045±2.055）%，t=-34.384，P〈0.001]、SA-β-gal阳性细胞比例[（75±11）%对（28±8）%，t=15.454，P〈0.001]较正常组升高；SAA小鼠BM-MSC的mTOR表达水平高于正常小鼠。 结论SAA小鼠模型中BM-MSC是衰老的MSC，且mTOR通路激活可能是导致其衰老发生的机制。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/e 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-7 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 13-galactosidase （SA-J3-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 G0/G, phase [ （77.461 ± 1.567 ） % vs （46.045 ± 2.055）%, t=--34.384, P〈0.001 ], increased percentage of SAgal positive cells [ （75±11）% vs （28±8）%, [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.

关 键 词：贫血 再生障碍性 间质干细胞 衰老 

分 类 号：R-332[医药卫生] R556.5