稳定表达T-钙粘蛋白黑素瘤细胞株建立及鉴定  

Establishment and Identification of Melanoma Cell Lines Stably Transfected T-cadherin

作　　者：曹娜[1,2] 牛艳东[1,2] 白秀会[1,2] 李娜[3] 邬新春 陆洁[1,2] 孙立新[3] 胡晓梅[1,2] 段昕所[1,2] 

机构地区：[1]承德医学院 [2]承德医学院附属医院皮肤科,河北承德067000 [3]承德医学院附属医院中心实验室,河北承德067000 [4]迁西县医院内分泌科,河北唐山064300

出　　处：《生物技术》2013年第5期63-66,共4页Biotechnology

基　　金：河北省科技支撑计划项目("T-钙粘蛋白在恶性黑素瘤中表达及基因转染研究";09276101D-14)资助

摘　　要：目的:研究T-钙粘蛋白在黑素瘤中的作用,因此首先建立稳定表达T-钙粘蛋白基因的黑素瘤细胞株。方法:应用脂质体将重组质粒pEGFP-N1/T-cadherin转染到黑素瘤细胞株中,以梯度浓度的G418进行筛选,获得稳定表达克隆;采用荧光显微镜、RT-PCR、Western-blot和免疫细胞化学鉴定。结果:G418筛选14d后,800μg/ml为最佳筛选浓度,600μg/ml为最佳维持培养浓度。筛选后形成抗性细胞克隆,成功转染的细胞在荧光显微镜下观察发出绿色荧光;RT-PCR扩增出T-钙粘蛋白RNA预期的片段;Western-blot、免疫细胞化学证实转染的细胞有T-钙粘蛋白的表达;未转染的细胞则不表达。结论:pEGFPN1/T-cadherin成功转染至黑素瘤细胞株并稳定表达。Objective： To investigate the functions of T - cadherin gene in melanoma cells, so first, it is necessary for us to establish the melanoma cells that stably transfected T-cadherin. Method： The recombinant plasmid, pEGFP-N1/T-cadherin, was transfected into human melanoma cells by liposome and positive clones were chosen by five concentrations of G418. The transfection and the expression of T-cadherin were observed to green fluorescent protein（ pEGFP-NI ）by fluorescent microscope and tested by RT-PCR, Western-blot and immunocytochemistry. Result：Though G418 screening 14 days,800μg/ml is the best screening concentration,600μg/ml is the best to maintain concentration. Screening can form resistant cells cloning. After transfection, green fluorescent was observed in cells which trans-fected with pEGFP - N1/T - cadherin under fluorescent microscope. RT - PCR can show the fragment which is the cDNA of T - cadherin mRNA in melanoma cells transfected with pEGFP - N1/T - cadherin. Western - blot and Immunocytochemistry confirm that melanoma cells with T - cadherin gene have also expressed T - cadherin. Conclusion：The recombinant of pEGFP - N1/T - cadherin transcript stab-ly express in the transfected melanoma cells.

关 键 词：T-钙粘蛋白 黑素瘤细胞 pEGFP—N1 脂质体2000 G418 

分 类 号：Q812[生物学—生物工程]