荧光定量聚合酶链反应检测GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在再生障碍性贫血患者及正常人骨髓间充质干细胞中的表达

背景:GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在骨髓造血及成脂调控机制中起重要作用。目的:观察GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在再生障碍性贫血患者及正常人间充质干细胞中的表达。方法:收集6例再生障碍性贫血患者及6位正常人骨髓液各10mL,Ficoll贴壁法分离培养骨髓单个核细胞,达70%~80%融合后扩增传代。取传至第3代的间充质干细胞,应用流式细胞仪进行鉴定。提取总RNA,比较各组基因与内参基因之间的差别。荧光定量聚合酶链反应检测GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在再生障碍性贫血患者及正常人间充质干细胞中的表达量。结果与结论:与正常人间充质干细胞相比,再生障碍性贫血患者间充质干细胞的GATA-2和干细胞因子基因表达量降低(P=0.012,0.039),过氧化物酶体增殖物激活受体γ基因表达量升高(P=0.035);两组GATA-1基因表达量差异无显著性意义。提示GATA-2、干细胞因子基因的异常表达可能影响AA患者骨髓微环境的造血调控作用;过氧化物酶体增殖物激活受体γ基因的异常表达可能解释再生障碍性贫血患者骨髓易成脂的原因。

Real-time PCR detection of GATA-1, GATA-2, peroxisome proliferator-activated receptor gamma, SCF gene in bone marrow mesenchymal stem cells of aplastic anemia patients and normal people

BACKGROUND: GATA-1,GATA-2,peroxisome proliferator-activated receptor gamma (PPAR-γ),and SCF gene play an important role in bone marrow hematopoietic and concomitant regulation mechanism.OBJECTIVE: To observe the expressions of GATA-1,GATA-2,PPAR-γ,SCF gene in bone marrow mesenchymal stem cells of aplastic anemia patients and normal people.METHODS: we collected bone marrow samples from six aplastic anemia patients and six normal persons.Ficoll stick wall method was used to isolate and culture bone marrow mononuclear cells that began to passage at 70%-80% confluence.The 3rd generation of mesenchymal stem cells were detected using flow cytometry.Total RNA was extracted to synthesis and design primer sequence,analysis standard curve,amplification curve,dissolved curve,to be compared with NaCan each gene genetic differences.Fluorescent quantitative PCR detection was employed for GATA-1,GATA-2,PPAR-γ,SCF gene in bone marrow mesenchymal stem cells of aplastic anemia patients and normal people.RESULTS AND CONCLUSION: Expression of GATA-2 and SCF gene was lower (P=0.012,0.039) and PPAR-γ expression was higher (P=0.035) in the aplastic anemia patients as compared with normal persons.There was no difference in GATA-1 expression.The findings indicated that abnormal expression of GATA-2 and SCF gene could affect macro-control of bone marrow hematopoietic microenvironment,and abnormal expression of PPAR-γ could explain why bone marrow adipogenesis easily formed in aplastic anemia patients.