乳腺癌间质成纤维细胞的分离、培养及鉴定  

Separation,Culture,and Identification of Breast Cancer Stromal Fibroblasts

作　　者：张晓耀[1] 康骅[1] 孙海晨[2] 刘爽[2] 崔叶青[2] 张琳[1] 蔡伟[1] 

机构地区：[1]首都医科大学宣武医院普外科,北京100053 [2]首都医科大学宣武医院普外科实验室,北京100053

出　　处：《中国普外基础与临床杂志》2011年第11期1160-1164,共5页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金资助项目(项目编号:30872516)~~

摘　　要：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 separation,culture,and identification of breast cancer stromal fibroblasts（BCSFs）,which could build up a good basis for the further research of function.Methods Breast cancer tissues were obtained during breast cancer operation,and were cut into pieces with size of 1 mm×1 mm×1 mm under aseptic conditions,then the pieces of the tissues were digested by collagenase Ⅰ and hyaluronidase.Finally the cells separated from the tissues incubated at 37 ℃ with 5% CO2 and 95% air humidified incubator.Morphological characteristics of the fibroblasts were observed under light microscope.The certain proteins were examined by immunohistochemistry（using CK,Vimentin,α-SMA,and TE-7 antibody） and flow cytometric analysis（CD34 and CD45）.Results The separated cells begin to attach to the wall of flask within 24 h and reached almost confluency in about 7 d to 10 d.According to identification,the successful rate of separation and culture of BCSFs was 90%（18/20）,and the characteristics of cells showed that morphological characteristics of the fibroblasts was flat spindle,rich cytoplasm,and a flat ovoid cystic nuclear.The fibroblasts in breast cancer tissues showed negative staining for cytokeratin,positive staining for vimentin,alpha-smooth muscle actin,and TE-7,and negative for CD34 and CD45 by flow cytometric analysis.Conclusions The fibroblasts in breast cancer tissues could be easily obtained by tissues cuting combined enzyme digestion and rocking technology in vitro.The present study provide an experimental foundation for further studies on fibroblasts in breast cancer.

关 键 词：乳腺癌 间质 成纤维细胞 培养 鉴定 

分 类 号：R737.9[医药卫生—肿瘤]