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

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

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。100ng／mlTPO诱导培养12d后，应用抗CD41^＋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细胞株的基因差异表达。选择THBSl、HOXA9、B-actin、IL-8、ANXA6、FGF-8对检测结果进行RT—PCR验证。结果 巨核细胞样本与非巨核细胞样本比较，呈显著差异表达的基因有116个，其中52个上调，64个下调；巨核细胞样本与meg-01细胞株样本比较呈显著差异表达的基因有158个，其中71个上调，87个下调。THBSl在巨核细胞表达高于非巨核细胞，HOXA9在巨核细胞表达低于非巨核细胞；13一actin在巨核细胞与非巨核细胞的表达无差别。IL-8在巨核细胞表达高于meg-01细胞株；ANXA6在巨核细胞表达低于meg-01细胞株；FGF一8在巨核细胞与meg-01细胞株的表达无差别。结论 巨核细胞、非巨核细胞和meg—01细胞株基因表达存在显著差异，差异表达中的调控基因包括应激反应基因、免疫相关基因、DNA合成和修复基因、新陈代谢基凶、肿瘤基因和肿瘤抑制基因等。

Objective To study the gene expression profiles of megakaryocytes（MKs） from human cord blood CD34^＋ ceils 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 immunomagnctic sorting. The gene expression profiles of MKs, non-MKs as well as meg-01 cells were studied by gene chip assay. THBS1, HOX A9,β-actin,IL-8,Annexin A6,FGF-8 were selected to validate the genc 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. THBS1 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 ceils, 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.