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|本期目录/Table of Contents|

腺病毒介导hHO-1基因体外转染骨髓间充质干细胞

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2009年第2期
页码:
174
栏目:
基础研究
出版日期:
2009-03-30

文章信息/Info

Title:
Experimental study on bone marrow mesenchymal stem cell transfected by Adv-HO-1
作者:
马粉娜; 林国生; 曾彬
武汉大学人民医院心血管内科,湖北 武汉 430060
Author(s):
MA Fen-na;  LIN Guo-sheng;  ZENG Bin
Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei, China
关键词:
腺病毒; 血红素加氧酶-1; 基因转染; 骨髓间充质干细胞
Keywords:
adenovirus;  hemo-oxygenase-1;  gene transfection;  mesenchymal stem cells
分类号:
Q343.1
DOI:
-
文献标识码:
A
摘要:
目的 以腺病毒（adenovirus，Adv）为载体将人血红素加氧酶(human hemo-oxygenase,hHO)-1基因转染至骨髓间充质干细胞（MSC）中，为干细胞移植联合hHO-1基因治疗心血管疾病奠定实验基础。方法 以密度梯度离心结合贴壁筛选法分离培养骨髓MSC。将携带有hHO-1基因与绿色荧光蛋白（GFP）报告基因的腺病毒体外扩增与鉴定后，以不同感染复数(multiplicity of infection，MOI)转染MSC。在荧光显微镜下观察转染后GFP的表达，以反转录(RT)-聚合酶链式反应(PCR)法检测目的基因hHO-1 mRNA在MSC中的表达，4’6’-二乙酰基-2-苯基吲哚(DAPI)染色观察hHO-1基因转染对MSC的影响。结果 将Adv-hHO-1体外扩增、纯化后,经PCR鉴定可得到555 bp的目的条带。转染后24 h，MSC中即可见GFP的表达，荧光强度随MOI的增加而增强。RT-PCR法检测显示，不同MOI的转染组均有hHO-1 mRNA表达，随MOI的增加其表达强度也逐渐增强（P<0.05）。DAPI染色显示，hHO-1基因转染后,细胞的生长及形态无明显变化。结论 以Adv为载体可成功地将hHO-1基因转染至骨髓MSC中，转染效率随转染滴度的增加而增加，转染后对细胞的生物学活性无明显影响。
Abstract:
AIM To transfect human hemo-oxygenase-1(hHO-1) gene into bone marrow Mesenchymal stem cell(MSC) via adenovirus (Adv) vector, so as to lay the experimental foundation for the therapy of cardiovascular disease. METHODS Bone marrow-derived MSC were separated and purified in vitro by Percoll density gradient centrifugation and screened by cell adhered to the bottom. MSC were transfected with different multiplicity of infection (MOI) of Adv-hHO-1-green fluorescent protein(GFP) after amplification, purification and identification. GFP expression was examined with fluorescence microscope and hHO-1 mRNA expression was observed with reverse transcription(RT)-polymerase chain reaction(PCR). 4’,6’-amidino-2’-phenylindol(DAPI) staining was used to observe the morphology of MSC after transfection. RESULTS PCR result showed a band of 555 bp DNA after Adv-GFP-hHO-1 amplification and purification. GFP was observed after 24 h transfection and the intensity of GFP expression enhanced in parallel with MOI. RT-PCR showed that the HO-1 mRNA expressed in different transfection groups and the expression also elevated in parallel with MOI. DAPI staining showed no obvious changes in the morphology of MSC. CONCLUSION hHO-1 gene is successfully transfected into MSC via Adv vector, which exerts no obvious adverse influence on the growth and proliferation of MSC. The transfection efficiency increases along with the increase of MOI.

参考文献/References

[1］ Tang JM, Xie QY, Pan GD, et al. Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion［J］. Eur J Cardiothorac Surg, 2006, 30(2):353-361.

［2］ 李培建,李兵仓. 一种重组腺病毒载体的扩增、纯化和病毒滴度检测方法［J］. 中华神经医学杂志， 2002, 1(1):52-55.

［3］ Juan SH, Lee TS, Tseng KW, et al. Adenovirus-mediated heme oxygenase-1 gene transfer inhibits the development of atherosclerosis in apolipoprotein E-deficient mice［J］. Circulation, 2000, 104(13): 1519-1525.

［4］ Price MJ, Chou CC, Frantzen M, et al. Intravenous mesenchymal stem cell therapy early after reperfused acute myocardial infarction improves left ventricular function and alters electro- physiologic properties［J］. Int J Cardiol, 2006, 111(2):231-239.

［5］ Grauss RW, Winter EM, van Tuyn J, et al. Mesenchymal stem cells from ischemic heart disease patients improve left ventricular function after acute myocardial infarction［J］. Am J Physiol Heart Circ Physiol, 2007, 293 (4):H2438-H2447.

［6］ Baron F, Gothot A. Mesenchymal stem cells: a new versatile therapeutic option［J］. Rev Med Liege, 2007, 62(Spec No):9-14.

［7］ 杨胜利,何作云. 干细胞移植治疗心肌梗死［J］. 心脏杂志, 2003, 15(4):372-374.

［8］ Potier E, Ferreira E, Meunier A, et al. Prolonged hypoxia concomitant with serum deprivation induces massive human mesenchymal stem cell death［J］. Tissue Eng, 2007, 13(6):1325-1331.

［9］ Angermayr B, Mejias M, Gracia-Sancho J, et al. Heme oxygenase attenuates oxidative stress and inflammation, and increases VEGF expression in portal hypertensive rats［J］. J Hepatol, 2006, 44(6):1033-1039.

［10］李锦,蒋晖,毛建斌，等. 血红素氧合酶-1在胰岛素对乳鼠心肌细胞保护机制中的作用［J］. 心脏杂志, 2005, 17(2):111-114.

［11］Tang YL, Tang Y, Zhang YC, et al. Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated heme oxygenase-1 vector［J］. J Am Coll Cardiol, 2005, 46(7):1339-1350.

［12］Barbagallo I, Tibullo D, Di Rosa M, et al. A cytoprotective role for the heme oxygenase-1/CO pathway during neural differentiation of human mesenchymal stem cells［J］. J Neurosci Res, 2008, 86(9):1927-1935.

［13］Vorburger SA, Hunt KK. Adenoviral gene therapy［J］. Oncologist, 2002, 7(1):46-59.

［14］Liu X, Pachori AS, Ward CA, et al. Heme oxygenase-1 (HO-1) inhibits postmyocardial infarct remodeling and restores ventricular function［J］. FASEB J, 2006, 20(2):207-216.

备注/Memo

备注/Memo:
收稿日期：2008-7-2.通讯作者：林国生，主任医师，主要从事冠心病基础与临床研究Email：lguosheng1027@yahoo.com.cn 作者简介：马粉娜,硕士生Email：mfn1983@126.com
更新日期/Last Update: 2009-04-16