人血栓調節蛋白基因編碼片段的克隆及在真核細胞中的表達_《中國普外基礎與臨床雜志》

作者：

戴毅 ¹ , 陳開 ¹ , 鄒琳 ² , 張雪梅 ² , 喬正榮 ³ , 時德 ³

1. 川北醫學院附屬醫院普外科（南充637000）;;
2.重慶醫科大學醫學檢驗系（重慶400016）;;
3.重慶醫科大學附屬第一醫院血管外科（重慶400016）;

關鍵詞：

血栓調節蛋白真核細胞重組質粒

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【摘要】目的構建人血栓調節蛋白（hTM）基因真核表達質粒，并導入兩種真核細胞［非洲綠猴腎母細胞（COS7細胞）和臍靜脈內皮細胞(HUVECs)］中表達，為臨床血栓病的基因治療提供實驗依據和物質基礎。方法采用PCR法擴增hTM基因的表達片段，HindⅢ+EcoRⅠ酶切后與pcDNA3.1(+)/neo質粒連接成pcDNA3.1/hTM。經鑒定確認后，由陽離子脂質體包裹轉染COS7細胞及HUVECs，RTPCR和免疫組化法分別檢測轉染后細胞hTM的mRNA及蛋白的表達。結果雙酶切及測序鑒定均證實hTM基因表達片段被成功克隆，pcDNA3.1/hTM經脂質體介導能有效地轉染COS7細胞和HUVECVs。結論成功構建pcDNA3.1/hTM重組質粒，并且能在兩種真核細胞中高效表達，為進一步的基因治療和對TM抗凝機理研究提供了物質基礎。

引用本文： 戴毅,陳開,鄒琳,張雪梅,喬正榮,時德. 人血栓調節蛋白基因編碼片段的克隆及在真核細胞中的表達. 中國普外基礎與臨床雜志, 2005, 12(6): 577-580. doi: 復制

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.薩姆布魯克，E.F.弗里奇，T.曼尼阿蒂斯．分子克隆實驗指南 [M]. 第2版. 北京：科學出版社， 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 thrombomodulin gene in a 45yearold 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 Ala455Val polymorphism and risk of coronary heart disease [Ｊ]. Circulation， 2001; 103(10)13869 Nan B, Lin P, Lumsden AB, et al. Effects of TNFalpha 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.

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.薩姆布魯克，E.F.弗里奇，T.曼尼阿蒂斯．分子克隆實驗指南 [M]. 第2版. 北京：科學出版社， 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 thrombomodulin gene in a 45yearold 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 Ala455Val polymorphism and risk of coronary heart disease [Ｊ]. Circulation， 2001; 103(10)13869 Nan B, Lin P, Lumsden AB, et al. Effects of TNFalpha 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.

《中國普外基礎與臨床雜志》

人血栓調節蛋白基因編碼片段的克隆及在真核細胞中的表達

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《中國普外基礎與臨床雜志》

人血栓調節蛋白基因編碼片段的克隆及在真核細胞中的表達

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

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