microRNA-210基因修饰人脐静脉内皮细胞诱导血管形成

microRNA-210 MODIFIED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS INDUCE CAPILLARY FORMATION

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。

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. Theconcentration 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.