应用基因芯片技术检测人脐带血CD34+细胞体外扩增的巨核细胞基因表达

目的 研究人脐带血CD34+细胞体外扩增巨核细胞的基因表达,从分子水平探讨巨核细胞的表达机制.方法 采用密度梯度离心法和免疫磁珠分选系统获取人脐带血CD34+细胞.100 ng/ml TPO诱导培养12 d后,应用抗CD+41单克隆抗体免疫磁珠法分选巨核细胞.应用基因芯片技术检测巨核细胞、非巨核细胞和meg-01细胞株...

Analysis of gene expression profiles of megakaryocytes from human cord blood CD34+ cells in vitro expanded using DNA microarray

Objective To study the gene expression profiles of megakaryocytes(MKs) from human cord blood CD34+ cells in vitro expanded and to understand megakaryopoiesis at the molecular level. Methods CD34+ cells were isolated using density gradient centrifugation and magnetic activated cell sorting. The cells were cultured and stimulated with recombinant human TPO ( 100 ng/ml). After 12 days, the MKs fraction was separated using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The gene expression profiles of MKs, non-MKs as well as meg-01 cells were studied by gene chip assay. THBSI, HOX A9,β-actin, lL-8,Annexin A6, FGF-8 were selected to validate the gene chip results by RT-PCR. Results A total of 116 genes between MKs and non-MKs cells were significantly different, 52 genes were up-regulated and 64 genes were down-regulated. In addition, 158 genes between MKs and meg-01 cells were significantly different, 71 genes were up-regulated and 87 genes were down-regulated. THBSI showed higher expression in MKs than in non-MKs. HOXA9 showed lower expression in MKs than in non-MKs. The expression of β-actin did not show any significant difference in MKs and non-MKs. IL-8 showed higher expression in MKs than in meg-01 cells, while ANXA6 showed lower expression in MKs than in meg-01 cells. The expression of FGF-8 did not show any significant difference between MKs and meg-01 cells. Conclusions MKs, non-MKs and meg-01 cells show different gene expression profiles. The regulatory genes include stress response genes,immune related genes, DNA synthesis and repair genes, metabolism genes, pro-onco genes and tumor suppressor genes.