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国际病理 2009, 29(3) 196-201 DOI:
ISSN: 0412-1961 CN: 21-1139/TG |
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| 论著 |
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小鼠Wnt-1基因真核表达载体构建及其在大鼠成肌细胞中的表达 |
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王秋艳1，杜文娟2， 李继科3，杨艳波4， 于波2 |
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1.河北省秦皇岛港口医院心内科CCU，河北 秦皇岛 066000； 2. 哈尔滨医科大学附属第二临床医院心内科，哈尔滨 150086;
3.哈尔滨医科大学附属第二临床医院心外科, 哈尔滨 150086； 4.哈尔滨市第二医院心内科, 哈尔滨 150056 |
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摘要：
目的：构建p-EGFP-C3-Wnt-1真核表达载体并转染体外培养大鼠成肌细胞，观察Wnt-1在其中的表达。方法：利用RT-PCR方法从小鼠胚脑中扩增获得Wnt-1基因编码区全长序列，克隆到含有增强型绿色荧光蛋白报告基因的真核表达载体p-EGFP-C3上，形成重组载体p-EGFP-C3-Wnt-1。大鼠成肌细胞原代培养及desmin免疫荧光染色鉴定。利用Lipofectamine TM2000脂质体将重组质粒p-EGFP-C3-Wnt-1转染成肌细胞。结果：重组质粒p-EGFP-C3-Wnt-1构建成功；将其转染成肌细胞72 h后，观察到EGFP报告基因和目的基因Wnt-1表达。结论：成功构建了小鼠胚脑Wnt-1基因的真核表达载体p-EGFP-C3-Wnt-1，并可转染成肌细胞。 |
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关键词：
Wnt-1
载体构建
成肌细胞
转染
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Construction of mouse Wnt-1 gene eukaryotic expression vector and
its expression in skeletal myoblasts of rats |
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WANG Qiuyan1,DU Wenjuan2, LI Jike3, YANG Yanbo4, YU Bo2
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1.Department of Cardiology, Qinhuangdao Port Hosipital,Qinhuangdao Hebei 066000;
2. Department of Cardiology,Second Clinical Hospital, Harbin Medical University,Harbin 150086;
3.Department of Cardiology Surgery, Second Clinical Hospital, Harbin Medical University,Harbin 150086;
4.Department of Cardiology,Second Hosipital of Harbin, Harbin 150056, China
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Abstract:
ObjectiveTo construct mouse Wnt-1 gene eukaryotic expression vector and to detect its expression in rat skeletal myoblasts in vitro. MethodsMouse Wnt-1 gene was amplified from the brain tissue of fetal mouses by RT-PCR, and was inserted into an eukaryotic expression vector p-EGFP-C3 carrying the enhanced green fluorescent protein, to generate a new plasmid p-EGFP-C3-Wnt-1.Skeletal myoblasts were cultured and identified by desmin immunofluorescence staining. The identified recombinate p-EGFP-C3-Wnt-1 eukaryotic expression vector was transfected into rat skeletal myoblasts with Lipofectamine TM2000 liposome. After transfection for 72h, p-EGFP-C3-Wnt-1 expression was examined in skeletal myoblasts. Results The eukaryotic expression vector containing mouse Wnt-1 gene was constructed successfully. EGFP report gene and Wnt-1 gene were expressed in myoblasts transfected with the recombinate plasmid p-EGFP-C3-Wnt-1. Conclusion The recombinant vector p-EGFP-C3-Wnt-1 is constructed and mouse Wnt-1 gene can be normally expressed in skeletal myoblasts. |
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Keywords:
Wnt-1
vector construction
skeletal myoblasts
transfectione
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收稿日期 2009-02-18 修回日期 2009-06-29 网络版发布日期 |
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DOI: |
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基金项目:
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通讯作者: 于波 |
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