抑制消减杂交技术筛选骨髓间充质干细胞与视网膜神经节样细胞差异表达基因

Selection of differentially expressed genes between bone mesenchymal stem cells and retinal ganglion-like cells by suppression subtractive hybridization technique

目的抑制消减杂交技术筛选和克隆视网膜神经节(RGC)样细胞与骨髓间充质干细胞的差异表达基因。方法提取RGC样细胞总RNA为检测子,骨髓间充质干细胞的总RNA为驱动子,进而用抑制消减杂交技术方法获得消减杂交产物。结果本消减文库共挑取克隆120个,经聚合酶链式反应(PCR)鉴定,其中含有插入片段的阳性克隆有20个。在这些阳性克隆中,PCR扩增片段大小分布于250~1500 bp。共得到5个差异基因片段,这些差异表达基因与视觉信号转导、神经细胞再生等功能有关。结论抑制消减杂交技术有效地克隆了骨髓间充质干细胞诱导前后差异表达的基因。

Objective To screen and clone differentially expressed genes between retinal ganglion-like cells(RGCs)and bone marrow mesenchymal stem cells by suppression subtractive hybridization(SSH)technique.Methods The total RNA of RGCs were extracted as the detector,and the total RNA of bone marrow mesenchymal stem cells were retrieved and taken as the driver.SSH technique was used to collect subtractive hybridization products.Results A total of 120 clones were selected from the subtractive library and identified using polymerase chain reaction(PCR).Among them,20 positive clones containing insertion fragments were identified.Among these positive clones,the amplified fragments of PCR were distributed in the range of 250 bp to 1500 bp.There were a total of five differentially expressed genes,and they were related to visual signal transduction,neuronal regeneration,etc.Conclusion SSH technique can effectively clone differentially expressed genes of bone marrow mesenchymal stem cells before and after induction.