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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。
AIM: To investigate the role of T-cadherin in melanoma, a melanoma cell strain stably expressing the T-cadherin gene was first established. Methods: The recombinant plasmid pEGFP-N1 / T-cadherin was transfected into melanoma cells by lipofectamine 2000. The recombinant plasmid pEGFP-N1 / T-cadherin was transfected into melanoma cell line with gradient G418 to obtain stable clones. And immunocytochemistry identification. Results: After 14 days of G418 screening, 800μg / ml was the best screening concentration and 600μg / ml was the best culture medium. Resistant cell clones were screened and successfully transfected cells were observed under a fluorescence microscope to emit green fluorescence. The expected fragments of T-cadherin RNA were amplified by RT-PCR. Western-blot and immunocytochemistry confirmed that the transfected cells There is T-cadherin expression; untransfected cells are not expressed. Conclusion: pEGFPN1 / T-cadherin was successfully transfected into melanoma cell lines and stably expressed.