• 1 南昌大學泌尿外科研究所（南昌，330006）;;
  • 2 南昌大學第二附屬醫(yī)院神經(jīng)外科;

目的 構(gòu)建人microRNA-210（miR-210）慢病毒重組載體并轉(zhuǎn)染人臍靜脈內(nèi)皮細胞株（human umbilical vein endothelial cells 12，HUVE-12），探討其過表達對HUVE-12 成血管的影響，為研究血管再生機制提供實驗模型。方法 構(gòu)建pGCSIL-GFP-pre-miR-210 重組質(zhì)粒表達載體并轉(zhuǎn)染HUVE-12，熒光顯微鏡觀察GFP 陽性表達細胞數(shù)及實時熒光定量PCR 法檢測miR-210 表達變化；細胞分為空病毒對照組（LV-GFP 對照組）和miR-210 轉(zhuǎn)染組（LV-miR-210-GFP 組），流式細胞儀檢測各組細胞ephrinA3 表達變化；ELISA 檢測細胞培養(yǎng)上清中VEGF 含量；將兩組細胞分別接種于Matrigel 觀察血管形成能力。 結(jié)果 重組載體經(jīng)酶切、測序鑒定正確，GFP 表達強度在轉(zhuǎn)染后48 ～ 72 h 達峰值；實時熒光定量PCR 檢測結(jié)果顯示：LV-miR-210-GFP 組miR-210 表達水平較LV-GFP 對照組增加9.72 倍（t= —11.10，P=0.00）。流式細胞儀檢測結(jié)果顯示LV-miR-210-GFP 組ephrinA3 陽性細胞率為12.52% ± 0.67%，明顯較LV-GFP 對照組（73.22% ± 1.45%）降低（t= —66.12，P=0.00）；ELISA 檢測結(jié)果顯示LV-miR-210-GFP 組細胞上清中VEGF 含量顯著高于LV--GFP 對照組（[ 305.29 ± 16.52）pg/mL vs.（42.52 ± 3.11）pg/mL]（t= —27.06，P=0.00）；血管形成能力實驗顯示LV-miR-210-GFP 組毛細血管管腔數(shù)為17.33 ± 6.33，較LV-GFP 對照組（6.33 ± 2.33）顯著增加（t= —2.83，P=0.04）。 結(jié)論 成功構(gòu)建miR-210 慢病毒重組載體，并能在HUVE-12 中穩(wěn)定表達，過表達miR-210 能明顯增強HUVE-12 血管形成能力，為進一步研究miR-210 調(diào)控血管新生的分子機制奠定了實驗基礎。

引用本文： 婁遠蕾,高法梁,謝安,郭菲,鄧志鋒,汪泱. microRNA-210 基因修飾人臍靜脈內(nèi)皮細胞誘導血管形成. 中國修復重建外科雜志, 2012, 26(5): 587-591. doi: 復制

1. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature, 2011, 473(7347): 298-307.
2. Ribatti D. Novel angiogenesis inhibitors: addressing the issue of. redundancy in the angiogenic signaling pathway. Cancer Treat Rev, 2011, 37(5): 344-352.
3. Liu D, Krueger J, Le Noble F. The role of blood flow and microRNAs in blood vessel development. Int J Dev Biol, 2011, 55(4-5): 419-429.
4. van Solingen C, de Boer HC, Bijkerk R, et al. MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1 (+) /Lin (-) progenitor cells in ischaemia. Cardiovasc Res, 2011, 92(3): 449-455.
5. Caporali A, Emanueli C. MicroRNA regulation in angiogenesis. Vascul Pharmacol, 2011, 55(4): 79-86.
6. Anand S, Cheresh DA. MicroRNA-mediated regulation of the angiogenic Switch. Curr Opin Hematol, 2011, 18(3): 171-176.
7. Tie J, Fan D. Big roles of microRNAs in tumorigenesis and tumor development. Histol Histopathol, 2011, 26(10): 1353-1361.
8. Sato F, Tsuchiya S, Meltzer SJ, et al. MicroRNAs and epigenetics. FEBS J, 2011, 278(10): 1598-1609.
9. Fasanaro P, D’alessandra Y, Di Stefano V, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem, 2008, 283(23): 15878-15883.
10. Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol, 2007, 17(3): 118-126.
11. Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of-target mRNAs. Nature, 2005, 433(7027): 769-773.
12. Stark A, Brennecke J, Bushati N, et al. Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3’UTR evolution. Cell, 2005, 123(6): 1133-1146.
13. Ivan M, Harris AL, Martelli F, et al. Hypoxia response and microRNAs: no longer two separate worlds. J Cell Mol Med, 2008, 12(5A): 1426-1431.
14. Kelly TJ, Souza AL, Clish CB, et al. A hypoxia-induced positive feedback loop promotes hypoxia-inducible factor 1alpha stability through miR-210 suppression of glycerol-3-phosphate dehydrogenase 1-like. Mol Cell Biol, 2011, 31(13): 2696-2706.
15. Mutharasan RK, Nagpal V, Ichikawa Y, et al. microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53-dependent pathways and exerts cytoprotective effects. Am J Physiol Heart Circ Physiol, 2011, 301(4): H1519-H1530.
16. Shibuya M. Vascular endothelial growth factor-dependent and -independent regulation of angiogenesis. BMB Rep, 2008, 41(4): 278-286.
17. Kuijper S, Turner CJ, Adams RH. Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc Med, 2007, 17(5): 145-151.
18. Byrne AM, Bouchier-Hayes DJ, Harmey JH. Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J cell Mol Med, 2005, 9(4): 777-794.
19. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol, 2005, 23(5): 1011-1027.
20. Kume T. Specification of arterial, venous, and lymphatic endothelial cells during embryonic development. Histol Histopathol, 2010, 25(5): 637-646.
  1. 1. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature, 2011, 473(7347): 298-307.
  2. 2. Ribatti D. Novel angiogenesis inhibitors: addressing the issue of. redundancy in the angiogenic signaling pathway. Cancer Treat Rev, 2011, 37(5): 344-352.
  3. 3. Liu D, Krueger J, Le Noble F. The role of blood flow and microRNAs in blood vessel development. Int J Dev Biol, 2011, 55(4-5): 419-429.
  4. 4. van Solingen C, de Boer HC, Bijkerk R, et al. MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1 (+) /Lin (-) progenitor cells in ischaemia. Cardiovasc Res, 2011, 92(3): 449-455.
  5. 5. Caporali A, Emanueli C. MicroRNA regulation in angiogenesis. Vascul Pharmacol, 2011, 55(4): 79-86.
  6. 6. Anand S, Cheresh DA. MicroRNA-mediated regulation of the angiogenic Switch. Curr Opin Hematol, 2011, 18(3): 171-176.
  7. 7. Tie J, Fan D. Big roles of microRNAs in tumorigenesis and tumor development. Histol Histopathol, 2011, 26(10): 1353-1361.
  8. 8. Sato F, Tsuchiya S, Meltzer SJ, et al. MicroRNAs and epigenetics. FEBS J, 2011, 278(10): 1598-1609.
  9. 9. Fasanaro P, D’alessandra Y, Di Stefano V, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem, 2008, 283(23): 15878-15883.
  10. 10. Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol, 2007, 17(3): 118-126.
  11. 11. Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of-target mRNAs. Nature, 2005, 433(7027): 769-773.
  12. 12. Stark A, Brennecke J, Bushati N, et al. Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3’UTR evolution. Cell, 2005, 123(6): 1133-1146.
  13. 13. Ivan M, Harris AL, Martelli F, et al. Hypoxia response and microRNAs: no longer two separate worlds. J Cell Mol Med, 2008, 12(5A): 1426-1431.
  14. 14. Kelly TJ, Souza AL, Clish CB, et al. A hypoxia-induced positive feedback loop promotes hypoxia-inducible factor 1alpha stability through miR-210 suppression of glycerol-3-phosphate dehydrogenase 1-like. Mol Cell Biol, 2011, 31(13): 2696-2706.
  15. 15. Mutharasan RK, Nagpal V, Ichikawa Y, et al. microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53-dependent pathways and exerts cytoprotective effects. Am J Physiol Heart Circ Physiol, 2011, 301(4): H1519-H1530.
  16. 16. Shibuya M. Vascular endothelial growth factor-dependent and -independent regulation of angiogenesis. BMB Rep, 2008, 41(4): 278-286.
  17. 17. Kuijper S, Turner CJ, Adams RH. Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc Med, 2007, 17(5): 145-151.
  18. 18. Byrne AM, Bouchier-Hayes DJ, Harmey JH. Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J cell Mol Med, 2005, 9(4): 777-794.
  19. 19. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol, 2005, 23(5): 1011-1027.
  20. 20. Kume T. Specification of arterial, venous, and lymphatic endothelial cells during embryonic development. Histol Histopathol, 2010, 25(5): 637-646.