五指山小型猪近交系主动脉内皮细胞体外培养模型的建立和TLR2介导炎症相关因子的表达

doi:10.3864/j.issn.0578-1752.2012.02.017

Abstract

Abstract: 【Objective】 This study aimed to explore a protocol of isolation, culture and identification of Wuzhishan Inbred Miniature Pig aortic endothelial cells (EC), and to study the regulation of Toll-like receptor 2 (TLR2) expression by LPS in EC with the expression of in?ammatory cytokines mediated by TLR2.【Method】Thoracoabdominal aortic were isolated from 3-4 month-old Wuzhishan Inbred Miniature Pig, disassociated with 0.1% collagenase I. Aortic EC were identified with the methods of uptake of fluorescence-labeled acetylated low density lipoproteins (DiI-Ac-LDL) and detecting of CD31 expression in the cells by flow cytometry. After stimulation of aortic derived EC with 1 μg•mL-1 lipopolysaccharide (LPS) for 0, 6, 8 and 12 h, the level of TLR2 and TLR4 mRNA was detected by real-time PCR. Furthermore, we detected the influence of lipoteichoic acid (LTA) on IL-6, IL-8 and ICAM-1 expression in aortic EC pretreated for 12 h with 1 μg•mL-1 LPS. 【Result】 Obtained Wuzhishan Inbred Miniature Pig aortic EC displayed paving stone-like pattern with high vitality. The cultured cells expressed CD31 and Ac-LDL Receptor in membrane, demonstrating that they were indeed EC. Stimulation of aortic derived EC with LPS induced a strong increase in TLR2 mRNA but not in TLR4 mRNA. LTA potentiated the increase of the in?ammatory cytokines IL-6, IL-8 and ICAM-1 expression in EC pretreated with LPS, but not in EC without LTA. 【Conclusion】The present study successfully isolated and identified Wuzhishan Inbred Miniature Pig aortic EC in vitro culture system. LPS stimuli induced a high TLR2 expression in EC, which mediated the expression of inflammatory cyokines.

Key words: Wuzhishan Inbred Miniature Pig, aortic endothelial cells, TLR2

WANG Pan, TAO Xiao-Li, SHEN Zu-Nan, LI He-Gang, PAN Jie, MOU Yu-Lian, LI Kui. Establishment of Aortic Endothelial Cells Model in Vitro from Wuzhishan of Inbred Miniature Pig and Expression of In?ammatory Cytokines Mediated by TLR2[J].Scientia Agricultura Sinica, 2012, 45(2): 346-352.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2012.02.017

https://www.chinaagrisci.com/EN/Y2012/V45/I2/346

References

[1]冯书堂, 褚武军, 王雅春. 中国五指山猪. 北京: 中国农业科学技术出版社, 1999.

Feng S T, Chu W J, Wang Y C. China Wuzhishan Pig. Beijing: China Agriclure Scinece Press, 1999. (in Chinese)

[2]山丽梅, 张锦超, 汪海. 血管内皮细胞功能与动脉粥样硬化关系的研究进展. 解放军药学学报, 2002, 18(5): 294-297.

Shan L M, Zhang J C, Wang H. The review of the relationship between Vascula endothelial cell function and atherosclerosis. Pharmaceutical Journal of Chinese People’s Liberation Army, 2002, 18(5): 294-297. (in Chinese)

[3]Yang X F, Yin Y, Wang H. Vascular inflammation and atherogenesis are activated via receptors for PAMPs and suppressed by regulatory T cells. Drug Discovery Today. Therapeutic Strategies, 2008, 5(2): 125-142.

[4]Jaffe E A, Nachman R L, Becker C G. Culture of human endothelial cells derived from umbilical veins. Indentificafion by morphologic and immunologic criteria. Journal of Clinical Investigation, 1973, 52: 2745-2756.

[5]胡野荣, 邓又华, 姜志斌, 吴忠仕. 人脐静脉内皮细胞体外分离培养的改进及其鉴定. 医学临床研究, 2005, 22(5): 587-589.

Hu Y R, Deng Y H, Jiang Z B, Wu Z S. Improved cultivation of HUVECs in vitro and their identification. Journal of Clinical Research, 2005, 22(5): 587-589. (in Chinese)

[6]Bian C, Zhao K, Tong G X, Zhu Y L, Chen P. Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen. Journal of Zhejiang University Science, 2005, 6(7): 631-636.

[7]Kobayuashi M, Inoue K, Warabi E, Minami T, Kodama T. A simple method of isolating mouse aortic endothelial cells. Journal of Atherosclerosis and Thrombosis, 2005, 12: 138-142.

[8]周晓彤, 沈振亚, 滕小梅, 夏漫辉. 小鼠动脉血管内皮细胞的原代培养和鉴定. 徐州医学院学报, 2007, 27(5): 21-25.

Zhou X T, Shen Z Y, Teng X M, Xia M H. Culture of vascular endothelial cells from mouse artery. Acta academiae Medicinae Xuzhou, 2007, 27(5): 21-25. (in Chinese)

[9]Carrillo A, Chamorro S, Rodríguez-Gago M, Alvarez B, Molina M J. Isolation and characterization of immortalized porcine aortic endothelial cell lines. Veterinary Immunology and Immunopathology, 2002, 89(1-2): 91-98.

[10]马泉荔, 洪海霞, 张彦明, 徐浩, 杨蒙, 苏正元. 猪脐静脉血管内皮细胞永生化的初步研究. 西北农林科技大学学报, 2007, 35(7): 43-47.

Ma Q L, Hong H X, Zhang Y M, Xu H, Yang M, Su Z Y. Study on immortalization of swine umbilicus veins endothelial cells. Journal of Northwest Sci-Tech University of Agriculture and Forestry, 2007, 35(7): 43-47. (in Chinese)

[11]Ross R. Atherosclerosis: an inflammatory disease. The New England Journal of Medicine, 1999, 340: 115-126.

[12]Bjorkbacka H. Multiple roles of toll-like receptor signaling in atherosclerosis. Current Opinion in Lipidology, 2006, 17(5): 527-533.

[13]Blander J M, Medzhitov R, Regulation of phagosome maturation by signals from toll-like receptors. Science, 2004, 304: 1014-1018.

[14]Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell, 2006, 124: 783-801.

[15]Kathrin S. Michelsen M A. Toll-like receptor signaling and atherosclerosis. Current Opinion in Hematology, 2006, 13: 163-168.

[16]Dunzendorfer S, Lee H K, Tobias P S. Flow-sependent regulation of endothelial toll-like receptor 2 expression through inhibition of SP1 activity. Circulation Research, 2004, 95: 684-691.

[17]Mullick A E, Soldau K, Kiosses W B, Bell T A, Tobias P S, Curtiss L K. Increased endothelial expression of toll-like receptor 2 at sites of disturbed blood ?ow exacerbates early atherogenic events. The Journal of Experimental Medicine, 2008, 3: 373-383.

[18]Adam E. Mullick, Peter S. Tobias, Linda K. Curtiss. Modulation of atherosclerosis in mice by toll-like receptor 2. The Journal of Clinical Investigation, 2005, 115: 3149-3156.

[19]Man G T, Feng Z H, Zhang T L. Improvement in the techniques of culturing and purifying pig, human, and rat endothelial cells and smooth muscle cells. Chinese Journal of Histochemistry and Cytochemistry, 2001, 10(2): 123-128.

[20]Satta N, Kruithof E K, Reber G. de Moerloose P. Induction of TLR2 expression by in?ammatory stimuli is required for endothelial cell responses to lipopeptides. Molecular Immunology, 2008, 46: 145-157.

[21]穆杨, 张彦明. 猪血管内皮细胞体外培养方法的建立. 畜牧兽医学报, 2004, 35(1): 46-49.

Mu Y, Zhang Y M. Establish the cultivation of swine vascular endothelial cell in vitro. Acta Veterinaria et Zootechnica Sinica, 2004, 35(1): 46-49. (in Chinese)

[22]Hiromichi Y, Hsin-Hua C, Yusuke T. Sensitization of human aortic endothelial cells to lipopolysaccharide via regulation of toll-like receptor 4 by bacterial fimbria-dependent invasion. Infection and Immunity, 2005, 12: 8050-8059.

[23]Trianta?lou M, Gamper F G, Lepper P M, Mouratis M A, Schumann C, Harokopakis E, Schifferle R E, Hajishengallis G, Trianta?lou K. Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced in?ammatory responses in human vascular endothelial cells. Cellular Microbiology, 2007, 9: 2030-2039.

[24]Devaraj S, Dasu M R, Rockwood J, Winter W, Griffen S C, Jialal I. Increased toll-like receptor(TLR) 2 and TLR4 expression in monocytes from patients with type 1 diabetes: further evidence of a proin?ammatory state. The Journal of Clinical Endocrinology and Metabolism, 2008, 93: 578-583.

[25]Erridge C, Spickett C M, Webb D J. Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via toll-like receptor 2. Cardiovascular Research, 2007, 73: 181-189.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Establishment of Aortic Endothelial Cells Model in Vitro from Wuzhishan of Inbred Miniature Pig and Expression of In?ammatory Cytokines Mediated by TLR2

PDF

Abstract

Cite this article

share this article

References

Related Articles 0

Metrics

Comments

Recommended 0