【摘要】 构建稳定表达Snail的成纤维细胞株并检测其对乳腺癌细胞耐药的影响,运用pL-tdTomato-Neo和pL-tdTomato-mSnail转染小鼠成纤维细胞3T3和L929,G418富集,单克隆挑选稳定细胞株。采用实时荧光定量PCR和Western blotting检测细胞内Snail的表达。将Snail稳定表达的成纤维细胞的培养上清作为条件培养基,按30%和60%的比例与乳腺癌细胞4T1共培养,噻唑蓝（MTT）检测4T1对顺铂和阿霉素的IC₅₀。运用实时荧光定量PCR检测过表达Snail的成纤维中各种细胞因子的表达。结果表明:稳定转染细胞株胞体发出红色荧光,胞内Snail水平上调,成功构建了稳定表达Snail的成纤维细胞株。MTT实验发现,4T1与过表达Snail的成纤维细胞共培养后,对顺铂和阿霉素的IC₅₀均升高。实时荧光定量PCR实验发现,Snail能诱导成纤维细胞多种细胞因子的表达增加。在成纤维细胞中,Snail可能通过调控多种细胞因子的表达参与乳腺癌细胞对化学药物的抵抗。更多还原

【Abstract】 To construct stable fibroblast cell lines expressing Snail and detect its effect on resistance to breast cancer cells,3T3 and L929 cells were transfected with pL-tdTomato-Neo and pL-tdTomato-mSnail.G418 was used to select and the stable monoclonal cells were collected.The mRNA expression of Snail was detected by real-time PCR and protein was measured by Western blotting.The culture supernatant of stably Snail-expressed fibroblasts was used as conditioned medium to co-culture with mouse breast cancer cells 4T1 at 30% and 60%.MTT assay was used to detect IC₅₀ of cisplatin and doxorubicin in 4T1.The expression of various cytokines in Snail-expression fibers was detected by real-time PCR.The results showed that stable cell lines produced red fluorescence and intracellular Snail level was increased,indicating that Snail-expressing stable fibroblast cell lines were constructed successfully.The MTT method showed that the IC₅₀ of cisplatin and adriamycin increased after coculture of 4T1 with Snail overexpressing fibroblasts.Real time quantitative PCR showed that Snail could induce the expression of many cytokines in fibroblasts.Snail may mediate chemoresistance in breast cancer cell through increasing the expression of various cytokines in fibroblasts.更多还原