E2-2基因腺病毒载体的构建及其对EPCs生长、增殖及ID1表达的影响

发布时间：2018-07-10 19:37

本文选题：EPCs + E-基因　；参考：《第三军医大学学报》2014年16期

【摘要】：目的构建转录因子E2-2基因腺病毒载体,观测内皮祖细胞(endothelial progenitor cells,EPCs)过表达E2-2基因对DNA结合抑制因子-1(inhibitor of DNA binding/differentiation,ID1)表达的影响。方法分离、培养并鉴定小鼠骨髓EPCs。RT-PCR法扩增E2-2基因CDs全长DNA,克隆入载体pTG19-T后,亚克隆入腺病毒穿梭载体pAdTrack-CMV中,构建pAdTrack/E2-2重组载体,与pAdEasy-1骨架质粒同源重组形成重组病毒pAd/E2-2,经293细胞包装,获具高效感染力的重组pAd/E2-2病毒。将该病毒感染EPCs,倒置显微镜观测经感染的EPCs的GFP表达情况。CCK-8(cell count kit-8)法检测病毒pAd/E2-2对EPCs生长、增殖的影响。RT-PCR、Western blot分别检测经感染的EPCs中E2-2与ID1基因及其编码蛋白的表达情况,并予以定量分析。结果分离、培养并鉴定到小鼠骨髓EPCs。克隆到2013 bp的E2-2基因,并获得高效感染力的重组pAd/E2-2病毒。CCK-8法检测表明,与对照比较,过表达E2-2的EPCs的生长、增殖速度减慢,48h开始变得尤为明显(P0.01);RT-PCR、Western blot及定量分析结果显示,E2-2能下调ID1的表达,与对照比较,差异具统计学意义(P0.01)。结论分离、培养并鉴定小鼠骨髓EPCs,克隆出E2-2基因,证实E2-2能明显抑制EPCs的生长、增殖,并能下调ID1基因的表达。
[Abstract]:Objective to construct the adenovirus vector of transcription factor E2-2 gene and to investigate the effect of E2-2 gene overexpression on the expression of DNA-binding inhibitor 1 (inhibitor of binding / differentiation / differentiation (ID1) in endothelial progenitor cells (endothelial progenitor cells). Methods the full-length DNA of E2-2 gene CDs was amplified by RT-PCR from mouse bone marrow. After cloned into pTG19-T vector, subcloned into adenovirus shuttle vector pAdTrack-CMV, pAdTrack-E2-2 recombinant vector was constructed. The recombinant virus pAd-E2-2 was formed by homologous recombination with pAdEasy-1 skeleton plasmid. The recombinant pAd-E2-2 virus was highly infectious after being packaged in 293 cells. The EPCs were infected with the virus. The expression of EPCs was detected by inverted microscope. CCK-8 (cell count kit-8) was used to detect the effect of pAd-E2-2 on the growth and proliferation of EPCs. RT-PCRX Western blot was used to detect the expression of E2-2 and ID1 genes and their encoded proteins in infected EPCs. And quantitative analysis was made. Results the mouse bone marrow EPCs were isolated, cultured and identified. The E2-2 gene of 2013 BP was cloned, and the recombinant pAd-E2-2 virus. CCK-8 assay showed that the growth of EPCs with overexpression of E2-2 was higher than that of control. The results of Western blot and quantitative analysis showed that E2-2 could down-regulate the expression of ID1, and the difference was statistically significant (P0.01). Conclusion E2-2 gene was isolated, cultured and identified in mouse bone marrow, and E2-2 gene was cloned. It was proved that E2-2 could significantly inhibit the growth and proliferation of EPCs and down-regulate the expression of ID1 gene.
【作者单位】：成都军区昆明总医院干部病房;
【基金】：国家自然科学基金(81270224);国家自然科学基金青年基金(81000070)~~
【分类号】：R3416

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