siRNA c-Met腺病毒转染对人外泌汗腺上皮细胞增殖的影响(1)
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[摘要]目的:研究siRNA c-Met腺病毒转染对培养的人外泌汗腺上皮细胞(human eccrine sweat gland epithelial cells,hESGc)增殖的影响。方法:按我科已经建立的方法培养和鉴定hESGc,取第1代细胞备用。构建表达红色荧光蛋白的siRNA c-Met腺病毒, 荧光显微镜观察和Westernlot检测证实病毒的有效性。MTT法检测siRNA c-Met腺病毒对hESGc增殖的影响。结果:荧光显微镜下可见转染了siRNA c-Met腺病毒的hESGc有红色荧光表达,转染后48h,收集细胞进行westernblot检测,结果发现,siRNA c-Met腺病毒转染能抑制hESGc细胞中c-Met蛋白的表达,抑制率达70%以上,MTT法检测发现转染siRNA c-Met腺病毒能抑制hESGc增殖,抑制率分别为16.8%(2天)和34.5%(4天)。结论:siRNA c-Met腺病毒转染能抑制hESGc的增殖。
[关键词]c-Met;外泌汗腺;上皮细胞;增殖
[中图分类号]Q813.1[文献标识码]A [文章编号]1008-6455(2010)09-1324-03
Inhibitory effects of knockdown of c-Met by Adenovirus-delivered siRNA on growth of human eccrine sweat gland epithelial cells
LEI Xia1, LIU Bo2, WU Jin-jin1, LU Yuan-gang1, Yang Ya-dong1
(1. Department of Dermatology, Daping Hospital,Third Military Medical University, Chongqing, 400042,China;2. Department of Orthopadics, the First Affiliated Hospital, Chongqing Medical University)
Abstract: ObjectiveTo investigate the effect of siRNA c-Met adenovirus on proliferation of cultured hESGc.Methods The recombinant adenoviral vector containing siRNA targeting c-Met gene was successfully constructed. Adenovirus with high titer was harvested. Fluorescence microscope observation and westernblot test were used to confirm the effectiveness of adenovirus. MTT assay was used to detect the growth of the cells.ResultsRed fluorescence was observed in hESGc infected with siRNA c-Met adenovirus under fluorescence microscopy.The infected cells were harvested for westernblot test. It is observed that c-Met was inhibited in siRNA c-Met adenovirus infected cell group. The transfection of siRNA c-Met adenovirus can inhibit the growth of hESGc by MTT assay.The inhibition rate were 16.8% in 2 days and 34.5% in 4 days.ConclusionThe transfection of siRNA c-Met adenovirus can inhibit the growth of hESGc.
Key words:c-Met; eccrine sweat gland; epithelial cell; proliferation
C-Met是至今唯一被认识的肝细胞生长因子(Hepatocyte growth factor,HGF)高亲和力受体,由c-Met基因编码,HGF与c-Met结合后,可诱导其发生构像变化,进而激活磷酸化通路,逐级放大信号,从而发挥HGF的生物学效应。不少实验表明,HGF/c-Met信号通路与多种细胞增殖,器官发生,肿瘤生长和转移等生物学行为有关[1] ......
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