Snail稳定表达成纤维细胞系的构建及其诱导乳腺癌细胞耐药的研究

发布时间：2018-01-25 22:23

本文关键词： 细胞遗传学 Snail 成纤维细胞乳腺癌耐药　出处：《中国科技论文》2017年18期 　论文类型：期刊论文

【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IC_(50)。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IC_(50)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。
[Abstract]:A fibroblast cell line expressing Snail stably was constructed and its effect on drug resistance of breast cancer cells was detected. Mouse fibroblasts 3T3 and L929 were transfected with pL-tdTomato-Neo and pL-tdTomato-mSnail and enriched with G418. Monoclonal selection of stable cell lines. Real-time fluorescence quantitative PCR and Western. Blotting was used to detect the expression of Snail. The culture supernatant of fibroblasts with stable expression of Snail was used as the conditioned medium. Breast cancer cells 4T1 were co-cultured in the proportion of 30% and 60%. Detection of 4T1 for cisplatin and adriamycin by MTT). The expression of cytokines in fibroblasts expressing Snail was detected by real-time fluorescence quantitative PCR. The results showed that the cell bodies of stable transfected cells emit red fluorescence. The level of intracellular Snail was up-regulated. A stable fibroblast cell line expressing Snail was successfully constructed. The expression of multiple cytokines in fibroblasts was increased by real-time fluorescence quantitative PCR assay. Snail may be involved in the resistance of breast cancer cells to chemical drugs by regulating the expression of many cytokines.
【作者单位】：中山大学药学院;
【基金】：高等学校博士学科点专项科研基金资助项目(20130171110056) 国家自然科学基金资助项目(81672943,81472643)
【分类号】：R737.9
【正文快照】： 乳腺癌是全球女性发病率最高的恶性肿瘤,也是我国发病率上升最快的恶性肿瘤之一[1]。然而,肿瘤耐药往往导致乳腺癌化疗失败[2-3],肿瘤耐药的机制及其分子调控网络并未阐明,继续探索肿瘤耐药的机制为提高化疗疗效、攻克肿瘤提供理论依据。肿瘤微环境主要由大量的异质性间质细胞

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