乳腺癌基质成纤维细胞分离培养的优化及对乳腺癌细胞作用的初步研究

摘要

目的:分离培养乳腺癌基质成纤维细胞,初步探讨其生物学特性及对乳腺癌细胞增殖转移能力的影响.方法:取自临床手术新鲜标本,用Ⅰ型胶原酶和透明质酸酶消化法获得原代乳腺癌成纤维细胞(CAFs)及与之配对的正常乳腺成纤维细胞(NFs);显微镜观察细胞形态;免疫印迹和免疫荧光实验检测上皮细胞、间质细胞和成纤维细胞标志物表达;胶原收缩实验比较细胞收缩能力;平板克隆形成和Transwell细胞体外迁移侵袭实验,观测CAFs和NFs对乳腺癌细胞增殖、迁移和侵袭能力的影响.结果:分离得到CAFs和NFs,2次传代后可获得纯化细胞;成纤维细胞中高表达Vimentin和α-SMA,低表达E-cadherin;CAFs胶原收缩能力较NFs更强;CAFs条件培养基处理后的乳腺癌细胞体外增殖、迁移和侵袭能力增强.结论:成功获得CAFs和NFs,且与NFs相比,CAFs活化程度更高,CAFs对乳腺癌细胞体外增殖和转移能力的促进作用更强.%Objective:To isolate and culture primary breast cancer stroma fibroblasts,to explore their biological characteristics,and also to study their influence on proliferation and metastasis of breast cancer cells.Methods:The primary breast cancer tissues were obtained from fresh specimen.Primary breast cancer associated fibroblasts (CAFs)and their paired normal breast fibroblasts (NFs)were isolated by collagenase and hyaluronidase digestion.Morphology was observed by microscope.Cell markers were detected by western blot and immunofluorescence.The collagen gel contraction assay was used to compare the contractile ability of CAFs and NFs.The influence of CAFs and NFs on proliferation and metastasis of breast cancer cells was measured by colony formation assay and transwell assay.Results:The purified CAFs and NFs were maintained after passage 2.The protein levels of Vimentin and α-SMA were highly expressed,however,the protein level of E-cadherin was not detected in CAFs and NFs.The contractile ability of CAFs was stronger than NFs.CAFs-CM could promote the ability of proliferation,migration and invasion of breast cancer cells compared with NFs-CM.Conclusion:The isolation and primary culture of CAFs and NFs with digestion method could be performed successfully.The status of activation of CAFs and the ability of CAFs to promote proliferation and metastasis of breast cancer cells are higher than NFs.