造血干细胞相关microRNAs的筛选及其促分化研究

为了获得人脐血造血干细胞(HSCs)的microRNAs(miRNAs)表达谱,并对相关miRNAs功能进行初步鉴定.利用免疫磁珠(MACS)和流式细胞仪(FACS)细胞分选技术分离人脐血造血干细胞(HSCs),分别提取细胞总RNA并分离小分子RNA,经荧光标记后与miRNAs基因芯片杂交,获得HSCs的miRNAs表达谱,集落形成实验(CFC)研究在HSC中高表达miR-520h对HSC的促分化作用.成功分离人脐血CD34 细胞和HSC,经基因芯片杂交获得31个造血干细胞相关miRNAs,其中22个为低表达,9个为高表达;经实时定量RT-PCR验证miR-520h显著升高,CFC实验表明其可增加多种集落形成,具有促进HSC向祖细胞分化的作用.上述结果表明,人脐血HSC具有自身特征性miRNAs,参与并调控HSC生物学功能,为深入探讨miRNAs在造血系统发育中的作用打下基础.

Screening of Hematopoietic Stem Cell-correlative MicroRNAs and Analysis of Their Differentiation-promoting Function

In order to obtain MicroRNA (miRNAs) expression profiles of human hematopoietic stem cells (HSCs), and to preliminarily investigate functions of HSC-correlative miRNAs, by using miniMACS magnetic beads and fluorescence-activated cell sorting (FACS), isolated hematopoietic stem cells (HSCs) were isolated from human umbilical cord blood and performed total RNA extraction. Next, using a microarray, miRNA gene expression profiles of HSCs were obtained. CFC assays were performed to research on differentiation-promoting function of miR-520h, enriched in HSCs. Results showed that CD34 hematopoietic cells and HSCs were successfully isolated from human umbilical cord blood, and 31 HSCs-correlative miRNAs were screened by microarrray. Among them, 22 were low in HSCs, and 9 were high. The result of real time RT-PCR confirmed high expression of miR-520h in HSCs. CFC assays showed that miR-520h promotes differentiation of HSCs into progenitor cells. In conclusion, human HSCs have a set of specific miRNAs that contribute to regulation of HSCs functions, which pave the way for exploring the roles of miRNAs in development of hematopoiectic system.