体外培养前后脐血CD34^+细胞差异表达基因的筛选

Screening of Differentially Expressed Genes of Cord Blood CD34^+ Cells before and after Culture In Vitro

目的筛选扩增培养前后脐血CD34+细胞差异表达基因。方法将新鲜分离的CD34+细胞设为对照组,经静态和动态培养系统扩增的CD34+细胞设为试验组,应用基因差异显示技术(DDRT-PCR)比较两组之间的基因表达差异,对差异表达基因片段进行克隆、测序及生物信息学分析,并且通过半定量RT-PCR方法验证基因差异表达的真实性。结果应用差异显示技术筛选出5个差异表达基因,其中4个为已知功能基因,1个为未知功能基因,对其中2个基因RAN和DC24进行半定量RT-PCR检测表明,在静态和动态体系扩增培养后的CD34+细胞内,RANmRNA的表达水平分别是新鲜分离细胞的1.521和3.978倍,DC24mRNA的表达水平分别是新鲜分离CD34+细胞的14.275和2.374倍。结论发现了与CD34+细胞体外增殖相关的一些重要基因,为从基因水平调控CD34+细胞体外增殖提供依据。

Objective To screen the differentially expressed genes of cord blood CD34^＋ cells before and after culture in vitro. Methods Analyze the gene expressions of cord blood CD34^＋ cells cultured in static and dynamic systems by DDRT-PCR, using the freshly isolated CD34^＋ cells as control. The differentially expressed genes were cloned,sequenced and analyzed for bioinformatics. The authenticity of differential expression of genes was verified by semi-quantitatlve RT-PCR. Results Five differentially expressed genes were screened by DDRT-PCR,of which 4 were genes with known function and one was gene with unknown function. Two of the five genes, RAN and DC24, were analyzed by semi-quantitative RT-PCR. The result showed that, compared with that of freshly isolated cells, the expression levels of RAN mRNA of CD34^＋ cells cultured in static and dynamic systems increased 1. 521 and 3.978 folds ,and those of DC24 mRNA increased 14. 275 and 2. 374 folds, respectively. Conclusion Some important genes related to the proliferation in vitro of CD34^＋ cells were discovered, It provided a basis for the regulation of proliferation in vitro of CD34^＋ cells at molecular biological level.