在妊娠合并免疫性血小板减少症动物模型中骨髓间充质干细胞的改变

Changes of bone marrow mesenchymal stem cells in animal model of immune thrombocytopenia during pregnancy

目的建立骨髓间充质干细胞的分离培养方法,探讨妊娠合并免疫性血小板减少症(immune thrombocytopenia, ITP)小鼠的骨髓间充质干细胞(bone marrow–derived mesenchymal stem cells, BM-MSCs)的改变。方法分别从正常妊娠组、ITP组及妊娠合并ITP组小鼠的骨髓中分离培养间充质干细胞,光学显微镜下观察细胞生长增殖情况;流式细胞术分析间充质干细胞的免疫表型;油红O染色及茜素红染色评估成脂成骨分化能力;CCK-8检测细胞增殖;PI试验检测细胞周期;Annexin-V/PI染色检测细胞凋亡。结果 (1)妊娠合并ITP组小鼠的BMMSCs生长缓慢,形状不规则,不同分组来源的BM-MSCs高表达干细胞相关抗原CD44、CD29及Scal-1,部分表达CD90,不表达白细胞表面抗原CD45及淋巴细胞表面抗原CDllb,能够在体外被诱导分化为成骨细胞及成脂细胞。(2)与正常妊娠组比较,ITP组及妊娠合并ITP组的BM-MSCs增殖能力显著被抑制(P <0.05),G0/G1期的细胞比例显著增高(P <0.05),凋亡率显著增加(P<0.05)。与ITP组比较,妊娠合并ITP组的BM-MSCs生长速度被抑制及凋亡率增加(P <0.05)。结论妊娠合并ITP小鼠来源的BM-MSCs表现形态异常,存在增殖能力受损及过度凋亡的现象。

Objective To establish a method for isolation and culture of bone marrow mesenchymal stem cells(BMMSCs) in mice with immune thrombocytopenia(ITP) during pregnancy. Methods Mesenchymal stem cells were isolated and cultured from mouse bone marrow of normal pregnancy, ITP and ITP pregnancy group. Cell growth and proliferation were observed under optical microscope. The immunophenotype of mesenchymal stem cells was analyzed by flow cytometry. Oil red 0 and alizarin red staining were used to evaluate the ability of lipid osteogenesis differentiation. CCK-8 test was used to detect cell proliferation. The PI test measures the cell cycle.Annexin-v/PI staining was used to detect apoptosis. Results(1) BM-MSCs derived from pregnant mice with ITP group grew slowly and were irregular in shape. BM-MSCs from different sub-groups were highly expressed in stem-cell related antigens CD44, CD29 and scal-1, partially expressing CD90, but not expressing leukocyte surface antigen CD45 and lymphocyte surface antigen CDllb. They can be induced to differentiate into osteoblasts and lipoblasts in vitro.(2) Compared with the normal pregnancy group, the proliferation of BM-MSCs in the ITP group and the ITP pregnancy group was significantly inhibited(P < 0.05), the cell proportion in G0/G1 phase was significantly increased(P < 0.05), and the apoptosis rate was significantly increased(P < 0.05). Compared with the ITP group, the growth rate of BM-MSCs in the ITP pregnancy group was inhibited and the apoptosis rate was increased(P < 0.05). Conclusions The BM-MSCs derived from mice in the ITP pregnancy group showed abnormal morphology, impaired proliferation ability and excessive apoptosis.