中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
4期
632-635,封4
,共5页
纪影畅%李宇%胡志奇%蔡湘娜%黄铿%高建华
紀影暢%李宇%鬍誌奇%蔡湘娜%黃鏗%高建華
기영창%리우%호지기%채상나%황갱%고건화
Wnt10b%COS细胞%基因表达
Wnt10b%COS細胞%基因錶達
Wnt10b%COS세포%기인표체
Wnt10b%COS cell%Gene expression
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-Wnt10b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-Wnt10b真核表达载体,酶切和测序鉴定该载体；LpofectamineTM 2000将该载体转染入COS-7细胞,计算转染率.Western blot和免疫细胞化学法检测COS-7细胞Wnt10b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,Western blot法检测皮肤Wnt10b和β-连环蛋白的表达.结果 PCR扩增出的人Wnt10b基因cDNA正确克隆到pEGFP-N1载体,成功构建pEGFP-N1-Wnt10b；pEGFP-N1-Wnt10b,成功转染COS-7细胞,转染率为20％ ～ 40％,转染后COS-7细胞Wnt10b蛋白表达较对照组增加(P＜0.05),COS-7细胞分泌的Wnt10b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,Wnt10b蛋白促进子鼠皮肤β-连环蛋白的表达增加.
目的 基因工程製備重組人Wnt10b蛋白,觀察其促進子鼠皮膚β-連環蛋白的錶達.方法 以pUC19-Wnt10b為模闆,聚閤酶鏈反應(PCR)擴增cDNA,擴增產物和pEGFP-N1載體雙酶切,連接酶連接構建pEGFP-N1-Wnt10b真覈錶達載體,酶切和測序鑒定該載體；LpofectamineTM 2000將該載體轉染入COS-7細胞,計算轉染率.Western blot和免疫細胞化學法檢測COS-7細胞Wnt10b蛋白錶達；將子鼠揹部皮膚置于含Wnt10b蛋白的培養液中培養2d,Western blot法檢測皮膚Wnt10b和β-連環蛋白的錶達.結果 PCR擴增齣的人Wnt10b基因cDNA正確剋隆到pEGFP-N1載體,成功構建pEGFP-N1-Wnt10b；pEGFP-N1-Wnt10b,成功轉染COS-7細胞,轉染率為20％ ～ 40％,轉染後COS-7細胞Wnt10b蛋白錶達較對照組增加(P＜0.05),COS-7細胞分泌的Wnt10b蛋白能促進子鼠皮膚β-連環蛋白的錶達(P＜0.05).結論 成功製備具有生物活性的重組人Wnt10b蛋白,Wnt10b蛋白促進子鼠皮膚β-連環蛋白的錶達增加.
목적 기인공정제비중조인Wnt10b단백,관찰기촉진자서피부β-련배단백적표체.방법 이pUC19-Wnt10b위모판,취합매련반응(PCR)확증cDNA,확증산물화pEGFP-N1재체쌍매절,련접매련접구건pEGFP-N1-Wnt10b진핵표체재체,매절화측서감정해재체；LpofectamineTM 2000장해재체전염입COS-7세포,계산전염솔.Western blot화면역세포화학법검측COS-7세포Wnt10b단백표체；장자서배부피부치우함Wnt10b단백적배양액중배양2d,Western blot법검측피부Wnt10b화β-련배단백적표체.결과 PCR확증출적인Wnt10b기인cDNA정학극륭도pEGFP-N1재체,성공구건pEGFP-N1-Wnt10b；pEGFP-N1-Wnt10b,성공전염COS-7세포,전염솔위20％ ～ 40％,전염후COS-7세포Wnt10b단백표체교대조조증가(P＜0.05),COS-7세포분비적Wnt10b단백능촉진자서피부β-련배단백적표체(P＜0.05).결론 성공제비구유생물활성적중조인Wnt10b단백,Wnt10b단백촉진자서피부β-련배단백적표체증가.
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-Wnt10b by polymerase chain reaction (PCR) and cloned into eukaryotic expression vector pEGFP-N1.The positive clone was confirmed by enzyme digestion and sequencing.The recombinant 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 Wnt10b gene was constructed.COS-7 cells transfected with the recombinant plasmid expressed high level of Wnt10b 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.
