【Abstract】Objective To provide experimental evidence for gene therapy of thrombophilia diseases by constructing eukaryotic expression plasmid of human thrombomodulin(hTM) gene and transfecting the plasmid into COS7 cell and human umbilical vein endothelial cells(HUVECs). Methods The coding fragment of hTM gene was amplified by PCR. Both hTM gene and pcDNA3.1(+)/neo empty vector were digested with HindⅢ and EcoRⅠ. Two digested fragments were combined into pcDNA3.1/hTM with T4DNA ligase. After identification, the pcDNA3.1/hTM was transfected into COS7 cell and HUVECs using cation liposome. The expression of hTM mRNA and protein on the COS7 cell and HUVECs was detected by RTPCR and immunohistochemistry respectively. Results The hTM recombinant plasmid was confirmed by double endonuclease digesting and sequencing. It was transfected into COS7 cell and HUVECs successfully with liposome.Conclusion The pcDNA3.1/hTM plasmid can be successfully constructed and highlevel hTM can be expressed in eukaryotic cells. All of this provides us experimental evidence for gene therapy and further study of TM anticoagulant mechanism.
Citation：
DAI Y,CHEN Kai,ZOU Lin,ZHANG Xuemei,QIAO Zhengrong,SHI De. Cloning of Coding Fragment of Human Thrombomodulin Gene and Expression in Eukaryotic Cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2005, 12(6): 577-580. doi:
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1 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin
catalyzed activation of protein C [Ｊ]. Proc Natl Acad Sci USA, 1981; 78(4)22492 Jackman RW, Beeler DL, Fritze L, et al. Human thrombomodulin gene is intron depleted: nucleic acid sequences of the cDNA and gene predict protein structure and suggest sites of regulatory control [Ｊ]. Proc Natl Acad Sci USA， 1987; 84(18)64253 Wen DZ, Dittman WA, Ye RD, et al. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene [Ｊ]. Biochemistry， 1987; 26(14)43504 J.萨姆布鲁克，E.F.弗里奇，T.曼尼阿蒂斯．分子克隆实验指南 [M]. 第2版. 北京： 科学出版社， 1999463~4665 Dittman WA, Majerus PW. Structure and function of thrombomodulin: a natural anticoagulant [Ｊ]. Blood, 1990; 75(2)3296 Ohlin AK, Marlar RA. The first mutation identified in the thrombomodulin gene in a 45yearold man presenting with thromboembolic disease [Ｊ]. Blood, 1995; 85(2)3307 Norlund L, Holm J, Zoller B, et al. A common thrombomodulin amino acid dimorphism is associated with myocardial infarction [Ｊ]. Thromb Haemost， 1997; 77(2)2488 Wu KK, Aleksic N, Ahn C, et al. Thrombomodulin Ala455Val polymorphism and risk of coronary heart disease [Ｊ]. Circulation， 2001; 103(10)13869 Nan B, Lin P, Lumsden AB, et al. Effects of TNFalpha and curcumin on the expression of thrombomodulin and endothelial protein C receptor in human endothelial cells [Ｊ]. Thromb Res， 2005; 115(5)41710 Terada Y, Eguchi Y, Nosaka S, et al. Capillary endothelial thrombomodulin expression and fibrin deposition in rats with continuous and bolus lipopolysaccharide administration [Ｊ]. Lab Invest， 2003; 83(8)116511 Matsuyama T, Izumi Y, Shibatate K, et al. Expression and activity of thrombomodulin in human gingival epithelium: in vivo and in vitro studies [Ｊ]. J Periodontal Res， 2000; 35(3)14612 Waugh JM, Yuksel E, Li J, et al. Local overexpression of thrombomodulin for in vivo prevention of arterial thrombosis in a rabbit model [Ｊ]. Circ Res， 1999; 84(1)8413 Kopp CW, Grey ST, Siegel JB, et al. Expression of human thrombomodulin cofactor activity in porcine endothelial cells [Ｊ]. Transplantation， 1998; 66(2)244.
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- 1. 1 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin
catalyzed activation of protein C [Ｊ]. Proc Natl Acad Sci USA, 1981; 78(4)22492 Jackman RW, Beeler DL, Fritze L, et al. Human thrombomodulin gene is intron depleted: nucleic acid sequences of the cDNA and gene predict protein structure and suggest sites of regulatory control [Ｊ]. Proc Natl Acad Sci USA， 1987; 84(18)64253 Wen DZ, Dittman WA, Ye RD, et al. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene [Ｊ]. Biochemistry， 1987; 26(14)43504 J.萨姆布鲁克，E.F.弗里奇，T.曼尼阿蒂斯．分子克隆实验指南 [M]. 第2版. 北京： 科学出版社， 1999463~4665 Dittman WA, Majerus PW. Structure and function of thrombomodulin: a natural anticoagulant [Ｊ]. Blood, 1990; 75(2)3296 Ohlin AK, Marlar RA. The first mutation identified in the thrombomodulin gene in a 45yearold man presenting with thromboembolic disease [Ｊ]. Blood, 1995; 85(2)3307 Norlund L, Holm J, Zoller B, et al. A common thrombomodulin amino acid dimorphism is associated with myocardial infarction [Ｊ]. Thromb Haemost， 1997; 77(2)2488 Wu KK, Aleksic N, Ahn C, et al. Thrombomodulin Ala455Val polymorphism and risk of coronary heart disease [Ｊ]. Circulation， 2001; 103(10)13869 Nan B, Lin P, Lumsden AB, et al. Effects of TNFalpha and curcumin on the expression of thrombomodulin and endothelial protein C receptor in human endothelial cells [Ｊ]. Thromb Res， 2005; 115(5)41710 Terada Y, Eguchi Y, Nosaka S, et al. Capillary endothelial thrombomodulin expression and fibrin deposition in rats with continuous and bolus lipopolysaccharide administration [Ｊ]. Lab Invest， 2003; 83(8)116511 Matsuyama T, Izumi Y, Shibatate K, et al. Expression and activity of thrombomodulin in human gingival epithelium: in vivo and in vitro studies [Ｊ]. J Periodontal Res， 2000; 35(3)14612 Waugh JM, Yuksel E, Li J, et al. Local overexpression of thrombomodulin for in vivo prevention of arterial thrombosis in a rabbit model [Ｊ]. Circ Res， 1999; 84(1)8413 Kopp CW, Grey ST, Siegel JB, et al. Expression of human thrombomodulin cofactor activity in porcine endothelial cells [Ｊ]. Transplantation， 1998; 66(2)244.
