乳腺癌前病变组织原代成纤维细胞生物学特征初步研究

Biological characteristics of fibroblasts primarily cultured form breast precancerosis

目的比较乳腺导管不典型增生成纤维细胞(atypical intraductal hyperplasia fibroblasts,AFs)与正常乳腺成纤维细胞(normal fibroblasts,NFs)、乳腺癌肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)在生物学特征上的差异。方法采用Ⅰ型胶原酶法分离并培养AFs、NFs和CAFs,免疫细胞荧光化学检测其粘连蛋白(fibronectin,FN)表达以鉴定细胞纯度。比较3种细胞的细胞生长曲线、细胞周期和周期蛋白cyclin D1、p21waf1及p53的变化。结果获得并鉴定了NFs、AFs、CAFs;其生长速度依次增加(P<0.05),细胞周期S期百分比(8.24±2.47)、(16.94±4.77)、(33.31±7.90)依次增大(P<0.05);与NFs相比,AFs周期蛋白cyclin D1表达增加(P<0.05),p21waf1和p53蛋白表达降低(P<0.05);与CAFs相比,AFs的周期蛋白cyclin D1没有升高(P<0.05),p21waf1蛋白和p53蛋白表达更强(P<0.05)。结论 AFs相对于NFs和CAFs,细胞生长增殖具有特异性,这种变化提示乳腺肿瘤的癌前病变阶段AFs可能是NFs逐步向CAFs转变的过渡阶段。

Objective To investigate hyperplasia fibroblasts （AFs） in tumorigenesis of biological characteristics of breast breast cancer, normal mammary primary atypical intraductal fibroblasts （NFs） and the breast primary cancer-associated fibroblasts （CAFs）. Methods The primary human breast fibroblasts were isolated by collagenase I digestion from normal mammary tissue, atypical intraductal hyperplasia and mammary tumor. The purity of these fibroblasts was identified by immunofluorescent staining using the specific first anti- body to fibronectin, the fibroblast biomarker. The proliferation and cell cycle for each kind of fibroblasts was detected by MTY assay and flow cytometry. The expression of cyclin D1, p53 and p21wafl in the fibroblasts was detected by Western blotting. Results More than 95% of the isolated fibroblasts were positive to fibronectin. Comparing with NFs （8.24 ＋ 2.47 ）, AFs （16.94 ＋ 4.77 ） had higher growth rate and more cells at S-phase of cell cycle （P 〈 0.05 ）. However, there were lower growth rate and lesser S-phase cells （ P 〈 0.05 ） when compared these parameters in AFs with that in CAFs （ 33.31 ＋ 7.90）. Similar changes were found on the expression of cyclin D1, p21 wall and p53 among AFs, NFs and CAFs. Conclusion Different biological characteristics of AFs with NFs and CAFs indicate that AFs are on the way of transformation of NFs to CAFs in the precancerous lesion of breast cancer.