【摘要】目的 構(gòu)建人血栓調(diào)節(jié)蛋白（hTM）基因真核表達(dá)質(zhì)粒，并導(dǎo)入兩種真核細(xì)胞［非洲綠猴腎母細(xì)胞（COS7細(xì)胞）和臍靜脈內(nèi)皮細(xì)胞(HUVECs)］中表達(dá)，為臨床血栓病的基因治療提供實(shí)驗(yàn)依據(jù)和物質(zhì)基礎(chǔ)。 方法 采用PCR法擴(kuò)增hTM基因的表達(dá)片段，HindⅢ+EcoRⅠ酶切后與pcDNA3.1(+)/neo質(zhì)粒連接成pcDNA3.1/hTM。經(jīng)鑒定確認(rèn)后，由陽(yáng)離子脂質(zhì)體包裹轉(zhuǎn)染COS7細(xì)胞及HUVECs，RTPCR和免疫組化法分別檢測(cè)轉(zhuǎn)染后細(xì)胞hTM的mRNA及蛋白的表達(dá)。 結(jié)果 雙酶切及測(cè)序鑒定均證實(shí)hTM基因表達(dá)片段被成功克隆，pcDNA3.1/hTM經(jīng)脂質(zhì)體介導(dǎo)能有效地轉(zhuǎn)染COS7細(xì)胞和HUVECVs。 結(jié)論 成功構(gòu)建pcDNA3.1/hTM重組質(zhì)粒，并且能在兩種真核細(xì)胞中高效表達(dá)，為進(jìn)一步的基因治療和對(duì)TM抗凝機(jī)理研究提供了物質(zhì)基礎(chǔ)。
引用本文： 戴毅,陳開(kāi),鄒琳,張雪梅,喬正榮,時(shí)德. 人血栓調(diào)節(jié)蛋白基因編碼片段的克隆及在真核細(xì)胞中的表達(dá). 中國(guó)普外基礎(chǔ)與臨床雜志, 2005, 12(6): 577-580. doi: 復(fù)制
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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.薩姆布魯克，E.F.弗里奇，T.曼尼阿蒂斯．分子克隆實(shí)驗(yàn)指南 [M]. 第2版. 北京： 科學(xué)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.