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目的探讨乳腺癌间质成纤维细胞(BCSFs)分离、培养及鉴定的方法,为其功能的研究奠定基础。方法在无菌条件下,术中留取乳腺癌切除标本的部分癌组织块,剪切成1mm×1mm×1mm大小块后采用振荡联合Ⅰ型胶原酶和透明质酸酶消化法分离、培养癌组织间质成纤维细胞。通过观察细胞形态、免疫组织化学检测(CK、Vimentin、α-SMA以及TE-7)和流式细胞仪测定以鉴定其是否为间质成纤维细胞。结果 培养24h后有细胞贴壁生长,7～10d左右细胞近融合,可传代培养。经鉴定,BCSFs分离、培养的成功率为90%(18/20),其细胞特点为:细胞呈扁平梭形,两端有细胞质突起,有一个扁平卵圆形囊状的核,胞浆丰富;免疫组织化学检测显示CK(-)、Vimentin(+)、α-SMA(+)、TE-7(+);流式细胞仪检测CD34和CD45均为(+)。结论振荡结合双酶消化法可有效地分离、培养BCSFs。本方法为进一步研究BCSFs提供了一种新的细胞来源途径。
Objective To explore the methods of isolation, culture and identification of breast cancer mesenchymal fibroblasts (BCSFs), and lay the foundation for their function. Methods Under aseptic conditions, some cancerous tissue specimens from breast cancer specimens were collected and cut into 1mm × 1mm × 1mm size pieces and then separated by shaking combined with type Ⅰ collagenase and hyaluronidase to culture the carcinoma Tissue interstitial fibroblasts. Cell morphology, immunohistochemistry (CK, Vimentin, α-SMA, and TE-7) and flow cytometry were used to identify whether they were mesenchymal fibroblasts. Results 24h after culturing cells adherent growth, about 7 ~ 10d cells near fusion, subculture. After identification, the success rate of BCSFs separation and culture was 90% (18/20). The characteristics of the cells were as follows: the cells were flat fusiform with cytoplasmic processes at both ends, a flat oval cystic nucleus with rich cytoplasm Immunocytochemistry showed that CK (-), Vimentin (+), α-SMA (+) and TE-7 (+) were detected by flow cytometry. Conclusion Oscillation combined with double enzyme digestion method can effectively separate and culture BCSFs. The method provides a new way of cell source for further study of BCSFs.