再生障碍性贫血患儿骨髓间充质干细胞体外造血支持作用的研究

Hematopoiesis support of mesenchymal stem cells in children with aplastic anemia

摘要:

目的：骨髓间充质干细胞（mesenchymal stem cell, MSC）具有造血支持作用，再生障碍性贫血（aplastic anemia, AA）的发病机制涉及到造血微环境的异常，将两方面联系起来，体外研究再障患儿骨髓MSC的造血支持作用与再障的造血微环境异常的关系。方法：采集24例再障患儿和19例对照儿童的骨髓标本，分离、培养和扩增MSC；观察细胞形态及骨髓MSC成纤维细胞集落形成单位（CFU-F）计数分析；应用MTT法检测骨髓MSC粘附造血细胞的能力；应用ELISA检测骨髓MSC分泌干细胞生长因子（SCF）的浓度；对骨髓MSC进行贴壁培养，接种骨髓单个核细胞，计数扩增细胞数和红系爆式集落生成单位（BFU-E），粒细胞巨噬细胞集落生成单位（CFU-GM），混合细胞集落生成单位（CFU-GMME）。结果：①再障患儿骨髓MSC传代时间延长，CFU-F计数15.70±5.78显著低于对照组21.73±5.74，P<0.05；②再障患儿骨髓MSC培养上清中SCF的浓度30.69±16.82 pg/mL显著低于对照组50.74±14.83 pg/mL，P<0.01；③再障患儿骨髓MSC支持下的单个核细胞（MNC）细胞扩增总数和红系爆式集落生成单位（BFU-E），粒细胞巨噬细胞集落生成单位（CFU-GM），混合细胞集落生成单位（CFU-GMME）计数显著低于对照组，P<0.01。结论： 再障患儿骨髓MSC体外造血支持作用较对照儿童骨髓MSC显著降低，其发生机制可能与再障患儿骨髓MSC增殖能力减低及分泌SCF减少有关。

Abstract:

ObjectiveThe abnormality of hemopoietic inductive microenvironment (HIM) is involved in the pathophysiology of aplastic anemia (AA). Mesenchymal stem cells (MSC) are main source of bone marrow stromal cells which constitute the bone marrow HIM. Thus, the bone marrow failure in AA may be related to the function of MSC. The aim of the study was to investigate the hematopoiesis support function of MSC in children with AA in vitro. MethodsBone marrow samples were collected from 24 children with AA at diagnosis and 19 children with idiopathic thrombocytopenic purpura (ITP), infectious mononucleosis or lymphadenitis (controls). MSCs from bone marrow samples were isolated, cultured and expanded. Morphology, proliferation activity and colony forming unit-fibroblast (CFU-F) were measured. The ability of bone marrow MSC to adhere hemopoietic cells was assayed by MTT. The concentration of stem cell factor (SCF) released from MSC was tested using ELISA. Mononuclear cells (MNC) of bone marrow were plated onto a feeder layer formed by MSC. Cells count and BFU-E, CFU-GM, CFU-GMME productions were measured.ResultsThe first and third passage time of MSC in children with AA was longer than that in the controls. The number of CFU-F in children with AA (15.70±5.78) was less than that in the controls (21.73±5.74) (P<0.05). The concentration of SCF in MSC supernatants in children with AA (30.69±16.82 pg/mL) was significantly lower than the controls (50.74±14.83 pg/mL) (P<0.01). The total MNC count and the number of BFU-E, CFU-GM and CFU-GMME colonies in the support of MSC in children with AA were significantly lower than those in the controls (P<0.01). ConclusionsThe hematopoiesis support function of MSC was significantly reduced in children with AA in vitro. The decreased hematopoiesis support function of MSC may be related its decreased proliferation capacity and SCF release activity.