人乳腺癌相关成纤维细胞的原代培养及其生物学特性

目的原代培养人乳腺癌相关成纤维细胞(Cancer-associated fibroblast,CAF),并检测其生物学特性。方法采用胶原酶消化培养法对26份乳腺癌患者标本进行原代细胞分离培养,倒置显微镜下观察人乳腺癌CAF的形态学特性,免疫荧光染色法鉴定所分离培养的人乳腺癌CAF纤维连结蛋白(Fibronectin,FN)和成纤维细胞活化蛋白(Fibroblast activated protein,FAP)的表达,并通过MTT法和细胞计数法绘制细胞生长曲线。结果胶原酶消化法的最适酶浓度为0.12%,最适消化时间为10 h。原代培养23份成功,3份失败。培养成功的细胞贴壁生长,形态完整,生长状态良好,背景清晰,有明显的对数生长期,并可传代培养。培养的细胞FN和FAP表达阳性,表明原代培养的CAF为乳腺癌CAF。结论胶原酶消化培养法适合人乳腺癌CAF的原代培养,通过该方法可建立理想的人乳腺癌CAF体外实验模型。

Primary culture and biologic characteristics of human breast cancer-associated fibroblasts

Objective To culture primary human breast cancer-associated fibroblasts(CAFs)and determine their biologic characteristics.Methods The specimens of CAFs from 26 patients with breast cancer were subjected to primary culture by collagenase digestion method,then observed for morphology under inverted microscope,and for expressions of fibronection(FN)and fibroblast activated protein(FAP)by IFA,based on which the growth curve was plotted by MTT and cell counting methods.Results The optimal collagenase concentration and time for digestion were 0.12% and 10 h respectively.The specimens from 23 patients were cultured successfully,while those from 3 patients failed to culture.The successfully cultured cells were adherent,which grew well and showed integrate shape,clear background,and could be subcultured.Obvious logarithmic growth phase was observed.The cultured cells were positive for expression of FN and FAP,which were identified as CAFs.Conclusions Collagenase digestion method is suitable for the primary culture of human breast CAFs,by which an ideal in vitro experimental model of CAFs may be established.