【摘要】 目的构建人microRNA-210(miR-210)慢病毒重组载体并转染人脐静脉内皮细胞株(human umbilical vein endothelial cells 12,HUVE-12),探讨其过表达对HUVE-12成血管的影响,为研究血管再生机制提供实验模型。方法构建pGCSIL-GFP-pre-miR-210重组质粒表达载体并转染HUVE-12,荧光显微镜观察GFP阳性表达细胞数及实时荧光定量PCR法检测miR-210表达变化;细胞分为空病毒对照组(LV-GFP对照组)和miR-210转染组(LV-miR-210-GFP组),流式细胞仪检测各组细胞ephrinA3表达变化;ELISA检测细胞培养上清中VEGF含量;将两组细胞分别接种于Matrigel观察血管形成能力。结果重组载体经酶切、测序鉴定正确,GFP表达强度在转染后48～72 h达峰值;实时荧光定量PCR检测结果显示:LV-miR-210-GFP组miR-210表达水平较LV-GFP对照组增加9.72倍(t=—11.10,P=0.00)。流式细胞仪检测结果显示LV-miR-210-GFP组ephrinA3阳性细胞率为12.52%±0.67%,明显较LV-GFP对照组(73.22%±1.45%)降低(t=—66.12,P=0.00);ELISA检测结果显示LV-miR-210-GFP组细胞上清中VEGF含量显著高于LV-GFP对照组([305.29±16.52)pg/mL vs.(42.52±3.11)pg/mL](t=—27.06,P=0.00);血管形成能力实验显示LV-miR-210-GFP组毛细血管管腔数为17.33±6.33,较LV-GFP对照组(6.33±2.33)显著增加(t=—2.83,P=0.04)。结论成功构建miR-210慢病毒重组载体,并能在HUVE-12中稳定表达,过表达miR-210能明显增强HUVE-12血管形成能力,为进一步研究miR-210调控血管新生的分子机制奠定了实验基础。更多还原

【Abstract】 Objective To construct human recombinant lentiviral expression vector of microRNA-210(miR-210) and to explore the over-expression of miR-210 on the capillary formation in human umbilical vein endothelial cells 12(HUVE-12). Methods The recombinant lentiviral expression vector of pGCSIL-green fluorescent protein(GFP)-pre-miR-210 was constructed by molecular cloning and transfected to HUVE-12(LV-miR-210-GFP group),only pGCSIL-GFP was transfected as control group(LV-GFP group).The miR-210 expression activity was evaluated by GFP reporter through fluorescence detection and real-time fluorescent quantitative PCR.The ephrinA3 protein expression was measured by flow cytometry.The concentration of vascular endothelial growth factor(VEGF) in culture supernatant was determined by ELISA.The cells were cultured in 96-well culture plate coated with Matrigel to assess the ability of capillary formation. Results The recombinant plasmid pGCSIL-GFP-pre-miR-210 was confirmed by restriction endonuclease analysis and DNA sequencing.Fluorescence detection showed that the fluorescence intensity of GFP was highest between 48 and 72 hours after transfection.Real-time fluorescent quantitative PCR showed that the miR-210 expression of LV-miR-210-GFP group was 9.72 times higher than that in LV-GFP group(t= —11.10,P=0.00).Flow cytometry analysis showed that the positive cell rate of enphrinA3 in LV-miR-210-GFP group(12.52% ± 0.67%) was significantly lower than that in LV-GFP group(73.22% ± 1.45%)(t= —66.12,P=0.00).The concentration of VEGF in supernatant in LV-miR-210-GFP group was significantly higher than that in LV-GFP group [(305.29 ± 16.52) pg/mL vs.(42.52 ± 3.11) pg/mL,t= —27.06,P=0.00].In vitro capillary-like formation assay showed that the number of capillaries was significantly larger in LV-miR-210-GFP group than in LV-GFP group(17.33 ± 6.33 vs.6.33 ± 2.33,t= —2.83,P=0.04).Conclusion The recombinant lentiviral expression vector of miR-210 is constructed successfully and HUVE-12 over-expressing miR-210 can significantly increase the capillary formation,which facilitates further study on the molecular functions of miR-210 in angiogenesis.更多还原