分别应用基因芯片和solexa技术研究脐血干细胞体外诱导的巨核细胞基因表达谱

摘要

Objective To study the gene expression profile of megakaryocytes from human cord blood CD34 + cells ex vivo expanded using DNA microarray and solexa sequencing. Methods CD34 + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (MACS). Cultures were stimulated with recombinant human TPO (100ng/ml). After 12 days, the MK fraction was separated from the non - MK fraction by immunomagnetic sorting. The gene expression of MKs and non - MKs was detected by DNA microarray and solexa sequencing. Results A total of 38000 genes exhibited 1834 differential expression genes between MKs and non -MKs cells by DNA microarray. The expression of 711 genes was up - regulated and that of 1123 genes was down - regulated. Using solexa sequencing, we obtained 3773147 and 3533805 Tags from MKs and non - MKs. Unambiguous Tags were 3291132 and 2967947,and distinct Tags were 197769 and 245318. The expression of 1161 genes was up - regulated and that of 902 genes was down - regulated. The expression of 2717 Tags was up - regulated and that of 1519 Tags was down - regulated. Conclusion MKs, non - MKs showed different mRNA expression profiles. The regulation of genes including signal transduction genes, metabolism genes, development genes, apoptosis genes, adhesion genes, and immune related genes, may be involved in the differential gene expression. The findings presented herein may have clinical significance for the development of molecular pathogenesis. Some results of DNA microarray and solexa sequencing are identical, and some are different. It suggestes that more complete gene expression profile will be obtained when both technologies are applicated.%目的 分别应用基因芯片和solexa技术研究人脐带血CD34+细胞来源的巨核细胞的基因表达谱.方法 采用密度梯度离心法和免疫磁珠法分选人脐带血CD34+细胞.100 ng/ml TPO诱导培养12天后,应用免疫磁珠法分选巨核细胞(MKs)和非巨核细胞(非MKs).分别应用基因芯片和solexa技术检测MKs和非MKs的差异表达基因.结果 基因芯片技术在38000多个基因表达谱的筛选中,检测出呈现显著差异表达的基因有1834个,其中711个上调基因,1123个下调基因.应用solexa技术获取的MK和非MK细胞Tag初始数量分别为3773147和3533805个,整个清晰的Tag数量分别为3291132和2967947,明显独特的tag数量分别为197769和245318个.其中上调基因表达数量为1161个,下调为902个;上调Tag表达数量为2717个,下调为1519个.结论 MKs和非MKs mRNA表达存在显著差异,差异基因编码产物与细胞内信号转导、新陈代谢、细胞发育、运动、细胞凋亡、细胞黏连和免疫应答等功能相关,进一步深入研究将有助于探讨巨核细胞的表达机制、信号传导及调控机制.基因芯片和solexa技术的检测结果存在重叠,同时又相互补充,两种方法联合应用能得到更完整的巨核细胞的基因表达谱.