Construction of Adenovirus Vector with Human Interleukin 10 and Its Expression in Vascular Smooth Muscle Cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

WANG Shengyi ¹ , WANG Yuqi ² , FU Weiguo ² , LIU Kangda ³ , LIUYi ¹

Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China;

Corresponding author：

Keywords：

Adenovirus vector; Interleukin; Vascular intimal hyperplasia

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Objective 　To construct recombinant adenovirus vector co-expressing human interleukin (hIL)-10 and green fluorescent protein (GFP) for study of the expression of genes of interest in vascular smooth muscle cells （VSMCs）.
Methods 　hIL-10 cDNA was amplified from pUCm-T/hIL-10 cDNA using polymerase chain reaction (PCR), and cloned into shuttle plasmid pShuttle-IRES-hrGFP-1. Kanamycin resistance screeninged for recombinant plasmids, which were linealized with PmeⅠand transformed into BJ5183-AD-1 containing pAdEasy-1 by electroporation after determining the insert’s sequence correct by NotⅠ and XholⅠdigestion， sequencing and basic local alignment search tool (BLAST). Prepared recombinant adenovirus plasmids were transformed into XL10-Gold cells. Amplified plasmids were transfected to AD-293 cells for packaging after being linearized with PacⅠ. PCR was used to determine target gene; The titer of the recombinant adenovirus was measured. VSMCs were transfected by recombinant adenovirus and viewed under fluorescence microscope. hIL-10 concentration in transfected VSMCs supernant was measured by enzyme linked immune sorbent assay (ELISA).
Results 　Recombinant shuttle plasmids contained interest gene. Recombinant adenovirus had 30 kb and 3 kb fragments after digestion with PacⅠ. PCR indicated that the recombinant adenovirus contained interest gene. The titer of recombinant adenovirus was 3×10１0 efu/ml. Transfected VSMCs had GFP expression and hIL-10 concentration in supernatant was 25 ng/106 cells.
Conclusion 　The recombinant adenovirus co-expressing hIL-10 and GFP is successfully constructed and could effectively express in VSMCs, this lays the foundation for the gene therapy of vascular intimal hyperplasia.

Citation： WANG Shengyi,WANG Yuqi,FU Weiguo,LIU Kangda,LIUYi. Construction of Adenovirus Vector with Human Interleukin 10 and Its Expression in Vascular Smooth Muscle Cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2007, 14(6): 660-664. doi: Copy

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11.	吴忠均, 李昱, 时德, 等. 联合转染tPA基因和PCNA反义寡核苷酸抑制兔自体移植动脉再狭窄研究 [J]. 中国普外基础与临床杂志, 2005； 12(5)∶459.
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15.	陈吉泉，修清玉，刘涛，等. 小鼠T-bet基因重组腺病毒载体的构建及制备 [J]. 免疫学杂志， 2007； 23（1）∶84.
16.	余斌，苏秀云，汪志中，等. 应用AdEasy腺病毒载体系统构建人骨形成蛋白2基因重组腺病毒 [J]. 中华实验外科杂志， 2005； 22（5）∶538.
17.	尹冰楠，李冬田，李秋香. 一种简易、廉价、高效构建重组腺病毒载体的方法 [J]. 天津医科大学学报， 2005； 11（2）∶171.
18.	张梅，何金生，朱梅，等. 重组腺病毒方法的改建 [J]. 中国公共卫生， 2005； 21（1）∶2319　Lei P, Li WH, Liao WJ, et al. Construction and co-expression of bicistronic plasmid encoding human WEE1 and stem cell factor [J]. Acta Biochim Biophys Sin (Shanghai)， 2005; 37(2)∶107.
19.	Ngoi SM, Chien AC, Lee CG. Exploiting internal ribosome entry sites in gene therapy vector design [J]. Curr Gene Ther， 2004; 4(1)∶15.
20.	Wong ET, Ngoi SM, Lee CG. Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes [J]. Gene Ther， 2002; 9(5)∶337.

1. 　Xing Z, Ohkawara Y, Jordana M, et al. Adenoviral vector-mediated interleukin-10 expression in vivo: intramuscular gene transfer inhibits cytokine responses in endotoxemia [J]. Gene Ther, 1997; 4(2)∶140.
2. 　陈紫千，汤耀卿．白细胞介素10基因治疗急性胰腺炎的研究现状 [J]. 国外医学·外科学分册， 2004； 31(2)∶75.
3. 　汪圣毅，王玉琦，符伟国，等. 从体外培养的外周血单个核细胞克隆人白细胞介素-10 cDNA [J]. 中华实验外科杂志， 2005； 22(6)∶659.
4. 　萨姆布鲁克， D.W.拉塞尔著. 黄培堂，王嘉玺，朱厚础译. 分子克隆实验指南 [M]. 第3版．北京：科学出版社， 2002∶91～97.
5. 　江千里，王健民，江汕，等. 经典方法与LaSRT法测定绿色荧光蛋白标记重组病毒滴度的研究 [J]. 第一军医大学学报， 2003； 23（10）∶1101.
6. 　汪圣毅，王玉琦，符伟国，等. 人脐带动脉平滑肌细胞的培养及纯化 [J]. 安徽医科大学学报， 2007； 42(2)∶231.
7. 　Mitra AK, Gangahar DM, Agrawal DK. Cellular, molecular and immunological mechanisms in the pathophysiology of vein graft intimal hyperplasia [J]. Immunol Cell Biol, 2006; 84(2)∶115.
8. 　Koppensteiner R, Spring S, Amann-Vesti BR, et al. Low-molecular-weight heparin for prevention of restenosis after femoropopliteal percutaneous transluminal angioplasty: a randomized controlled trial [J]. J Vasc Surg, 2006; 44(6)∶1247.
9. 　Kibos A, Campeanu A, Tintoiu I. Pathophysiology of coronary artery in-stent restenosis [J]. Acute Card Care, 2007; 9(2)∶111.
10. 　Katayama T, Ueba H, Tsuboi K, et al. Reduction of neointimal hyperplasia after coronary stenting by pioglitazone in nondiabetic patients with metabolic syndrome [J]. Am Heart J, 2007; 153(5)∶762.
11. 　吴忠均, 李昱, 时德, 等. 联合转染tPA基因和PCNA反义寡核苷酸抑制兔自体移植动脉再狭窄研究 [J]. 中国普外基础与临床杂志, 2005； 12(5)∶459.
12. 　刘江涛, 张金山. 血管再狭窄的基因治疗 [J]. 中国介入影像与治疗学, 2006； 3(1)∶69.
13. 　Zimmerman MA, Reznikov LL, Raeburn CD, et al. Interleukin-10 attenuates the response to vascular injury [J]. J Surg Res, 2004; 121(2)∶206.
14. 　Mazighi M, Pelle A, Gonzalez W, et al. IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo [J]. Am J Physiol Heart Circ Physiol, 2004; 287(2)∶H866.
15. 　陈吉泉，修清玉，刘涛，等. 小鼠T-bet基因重组腺病毒载体的构建及制备 [J]. 免疫学杂志， 2007； 23（1）∶84.
16. 　余斌，苏秀云，汪志中，等. 应用AdEasy腺病毒载体系统构建人骨形成蛋白2基因重组腺病毒 [J]. 中华实验外科杂志， 2005； 22（5）∶538.
17. 　尹冰楠，李冬田，李秋香. 一种简易、廉价、高效构建重组腺病毒载体的方法 [J]. 天津医科大学学报， 2005； 11（2）∶171.
18. 　张梅，何金生，朱梅，等. 重组腺病毒方法的改建 [J]. 中国公共卫生， 2005； 21（1）∶2319　Lei P, Li WH, Liao WJ, et al. Construction and co-expression of bicistronic plasmid encoding human WEE1 and stem cell factor [J]. Acta Biochim Biophys Sin (Shanghai)， 2005; 37(2)∶107.
19. 　Ngoi SM, Chien AC, Lee CG. Exploiting internal ribosome entry sites in gene therapy vector design [J]. Curr Gene Ther， 2004; 4(1)∶15.
20. 　Wong ET, Ngoi SM, Lee CG. Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes [J]. Gene Ther， 2002; 9(5)∶337.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction of Adenovirus Vector with Human Interleukin 10 and Its Expression in Vascular Smooth Muscle Cells

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