microRNA-210 基因修飾人臍靜脈內皮細胞誘導血管形成_《中國修復重建外科雜志》

作者：

婁遠蕾 ¹ , 高法梁 ² , 謝安 ¹ , 郭菲 ¹ , 鄧志鋒 ² , 汪泱 ¹

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院神經外科;

關鍵詞：

microRNA-210 人臍靜脈內皮細胞血管再生慢病毒載體

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目的構建人microRNA-210（miR-210）慢病毒重組載體并轉染人臍靜脈內皮細胞株（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。方法構建pGCSIL-GFP-pre-miR-210 重組質粒表達載體并轉染HUVE-12，熒光顯微鏡觀察GFP 陽性表達細胞數及實時熒光定量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。結果重組載體經酶切、測序鑒定正確，GFP 表達強度在轉染后48 ～ 72 h 達峰值；實時熒光定量PCR 檢測結果顯示：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）。結論成功構建miR-210 慢病毒重組載體，并能在HUVE-12 中穩定表達，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

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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. 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.

《中國修復重建外科雜志》

microRNA-210 基因修飾人臍靜脈內皮細胞誘導血管形成

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《中國修復重建外科雜志》

microRNA-210 基因修飾人臍靜脈內皮細胞誘導血管形成

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