乳腺癌干细胞富集及其特异性多肽的筛选  

Isolation of breast cancer stem cell and screening of specific polypeptide bonding to it

作　　者：孔冕[1,2] 戴淑琴[1] 李宝江[2] 王军业[1] 林雨[1] 臧林泉[3] 曹开源[4] 

机构地区：[1]中山大学肿瘤防治中心.华南肿瘤学国家重点实验室,广东广州510060 [2]泰安市中心医院乳腺外科,山东泰安271000 [3]广东药学院药科学院药理教研室,广东广州510006 [4]中山大学微生物教研室,广东广州510080

出　　处：《中华肿瘤防治杂志》2013年第24期1892-1895,1899,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省科技计划(2010B031600066)

摘　　要：目的:无血清培养法富集乳腺癌干细胞(breast cancer stem cell,BCSC)并采用噬菌体展示技术,筛选能特异性结合乳腺癌干细胞的噬菌体多肽。方法:无血清培养法富集乳腺癌MDA-MB-231细胞株中干细胞并以此为靶标,以hs578bst人正常乳腺细胞及普通培养的MDA-MB-231细胞为减性筛选细胞,对噬菌体随机肽库进行双重减性筛选,选取富集后的阳性噬菌体单克隆,ELISA及DAB染色鉴定阳性噬菌体特异性并测序。结果:经过3轮筛选,噬菌体得到约500倍的富集,随机挑选10株单克隆噬菌体。ELISA显示,6号噬菌体单克隆对乳腺癌干细胞的亲和力是对照的6.14倍;DAB鉴定亦显示,其对乳腺癌干细胞的特异性及亲和力最高,对阳性噬菌体DNA测序翻译得到十二肽氨基酸为GYSASRSTIPGK。结论:通过干细胞富集及噬菌体展示技术,成功筛选出能够特异性结合乳腺癌干细胞的特异性噬菌体多肽,为乳腺癌的干细胞靶向治疗和深入研究奠定基础。OBJECTIVE： To isolate breast cancer stem cells using serum-free culture technique,and screen peptides bonding to it by phage display technology. METHODS： A phage display random 12 peptide library was screened using breast cancer stem cells as target isolated from MDA-MB-231 cell line by using serum-free culture technique, hs578bst cells and MDA-MB-231 cells as subtract screening cells. The specificity of the positive clones was identified by ELISA and DAB staining,positive phages were sent to sequencing company for DNA sequencing. RESULTS： Phages were gathered a- bout 500 times after 3 round of biopanning. ELISA showed the affinity to breast cancer stem cells of NO. 6 phage was 6.14 times higher than that in the control group. Immunohistoehemistry also showed NO. 6 phage had a high specificity bonding to breast cancer stem cells. The peptide sequence of the positive phage was GYSASRSTIPGK after translation. CONCLUSION： We isolated a phage display peptide bonding to breast cancer stem cells by phage display technology, and laid the foundation of the further study of the stem cell targeted therapy of breast cancer.

关 键 词：乳腺肿瘤 肿瘤干细胞 噬菌体展示 噬菌体随机十二肽库 筛选 

分 类 号：R737.9[医药卫生—肿瘤]