重组人Wnt10b蛋白的制备及其促进子鼠皮肤β-连环蛋白的表达  

Preparation of rWntlOb protein and upregulation of β-catenin expression in cultured rat embryonic skin

作　　者：纪影畅[1] 李宇[1] 胡志奇[2] 蔡湘娜[1] 黄铿[3] 高建华[2] 

机构地区：[1]汕头大学医学院第一附属医院整形外科,515041 [2]南方医科大学南方医院整形外科 [3]汕头大学医学院第二附属医院

出　　处：《中华实验外科杂志》2012年第4期632-635,F0004,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81071577）;广东省科技计划资助项目（20098030801320）;广东省自然科学基金资助项目（9151008.901000195、9151503102000037）;广东省医学科研基金资助项目（A2009433）

摘　　要：目的基因工程制备重组人Wnt10b蛋白，观察其促进子鼠皮肤β-连环蛋白的表达。方法以pUC19-Wntlob为模板，聚合酶链反应（PCR）扩增cDNA，扩增产物和pEGFP—N1载体双酶切，连接酶连接构建pEGFP-N1-Wntlob真核表达载体，酶切和测序鉴定该载体；Lipofectamine^TM 2000将该载体转染入COS-7细胞，计算转染率。Westernblot和免疫细胞化学法检测COS-7细胞Wntlob蛋白表达；将子鼠背部皮肤置于含Wnt10b蛋白的培养液中培养2d，Westernblot法检测皮肤Wntlob和B．连环蛋白的表达。结果PCR扩增出的人Wntlob基因cDNA正确克隆到pEGFP-N1载体，成功构建pEGFP—N1一Wntlob；pEGFP—N1-Wntlob，成功转染COS-7细胞，转染率为20％-40％，转染后COS-7细胞Wntlob蛋白表达较对照组增加（P〈0．05），COS-7细胞分泌的WntlOb蛋白能促进子鼠皮肤β-连环蛋白的表达（P〈0．05）。结论成功制备具有生物活性的重组人Wnt10b蛋白，Wntlob蛋白促进子鼠皮肤β-连环蛋白的表达增加。Objective To prepare rWnt10b protein through genetic engineering and observe the upregulation of β-catenin expression in cultured rat embryonic skin. Methods 1170 bp cDNA fragment was amplified from pUC19-Wntl0b by polymerase chain reaction （PCR） and cloned into eukaryotic expression vector pEGFP-N1. The positive clone was confirmed by enzyme digestion and sequencing. The recom-binant plasmid was transiently transfected into COS-7 cell line with LipofectamineTM 2000. The expression of Wnt10b gene was detected by Western blotting and immunocytochemistry. The dorsal skins of SD rat at embryos 14. 5-15.0 were cultured in DMEM in the presence or absence of rWnt10b protein, and two days later, Wnt10b/β-catenin expression was analyzed by Western blotting. Results Eukaryotic expression vectors containing human WntlOb gene was constructed. COS-7 cells transfected with the recombinant plas-mid expressed high level of Wntl0b protein. The transfection efficiency was about 20% -40%. Compared with controls, Wnt10b could upregulate β-catenin expression in cultured rat embryonic skin （P 〈 0. 05 ）. Conclusion We prepared rWnt10b protein successfully, rWnt10b protein can upregulate β-catenin expression in cultured rat embryonic skin.

关 键 词：Wnt10b COS细胞 基因表达 

分 类 号：R33[医药卫生—人体生理学]