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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 lentiviral vector miR-210 (miR-210) and transfect it into human umbilical vein endothelial cells 12 (HUVE-12) to investigate the effect of overexpression of HUVE-12 on angiogenesis, Experimental model for studying the mechanism of vascular regeneration. Methods The recombinant plasmid pGCSIL-GFP-pre-miR-210 was transfected into HUVE-12 cells. The number of GFP positive cells was detected by fluorescence microscopy and the expression of miR-210 was detected by real-time fluorescence quantitative PCR. (LV-GFP control group) and miR-210 transfection group (LV-miR-210-GFP group). The expression of ephrinA3 in each group was detected by flow cytometry. The VEGF level in cell culture supernatant was detected by ELISA. The cells were inoculated into Matrigel to observe the vascularization ability. Results The recombinant vector was identified by restriction enzyme digestion and sequencing. The expression intensity of GFP peaked at 48-72 h after transfection. The real-time PCR results showed that the expression of miR-210 in LV-miR-210-GFP group was higher than that in LV-GFP group The control group increased 9.72 times (t = -11.10, P = 0.00). The results of flow cytometry showed that the positive rate of ephrinA3 in LV-miR-210-GFP group was 12.52% ± 0.67%, significantly lower than that in LV-GFP control group (73.22% ± 1.45%) (t = -66.12, P = ). The results of ELISA showed that the content of VEGF in the supernatant of LV-miR-210-GFP group was significantly higher than that of LV-GFP control group ([305.29 ± 16.52] pg / mL vs. (42.52 ± 3.11) pg / = -27.06, P = 0.00). The number of capillary vessels in LV-miR-210-GFP group was 17.33 ± 6.33, which was significantly higher than that in LV-GFP control group (6.33 ± 2.33) (t = -2.83 , P = 0.04). Conclusion The miR-210 lentiviral vector was constructed successfully and stably expressed in HUVE-12. Overexpression of miR-210 significantly enhanced the vascularization of HUVE-12, which laid the foundation for further study of the molecular mechanism of miR-210 in regulating angiogenesis Experimental basis.