利用条件性重编程细胞技术构建前列腺癌原代细胞库  

Construction of primary prostate cancer cell bank using conditional reprogramming cell technology

作　　者：段剑礼 王自峰 杨伟娇[1] 雷汉祺 张俊夫 李永红[5] 庞俊[4] Duan Jianli;Wang Zifeng;Yang Weijiao;Lei Hanqi;Zhang Junfu;Li Yonghong;Pang Jun(Department of Urology,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China;Department of Experimental Research,Sun Yat-sen University Cancer Center,Guangzhou 510060,China;Department of Urology,The Sun Yat-sen Memorial Hospital of Sun Yat-sen University,Guangzhou 510120,China;Department of Urology,The Seventh Affiliated Hospital of Sun Yat-sen University,Shenzhen 518107,China Department of Urology,Sun Yat-sen University Cancer Center,Guangzhou 510060,China)

机构地区：[1]中山大学附属第三医院泌尿外科,广州510630 [2]中山大学附属肿瘤防治中心实验研究部,广州510060 [3]中山大学附属孙逸仙纪念医院泌尿外科,广州510120 [4]中山大学附属第七医院泌尿外科,深圳518107 [5]中山大学附属肿瘤防治中心泌尿外科,广州510060

出　　处：《中华泌尿外科杂志》2020年第5期374-381,共8页Chinese Journal of Urology

基　　金：国家自然科学基金面上项目(81772754);广东省自然科学基金重大基础研究培育项目(2017A03038009)。

摘　　要：目的利用条件性重编程细胞(CRC)技术培养人源性前列腺癌细胞,为前列腺癌体外实验研究建立个体化原代细胞库。方法 2019年1—4月共获取3例新鲜的前列腺癌病理组织标本,将标本分为2份,一份经术中快速冰冻病理检查确定为癌组织;另一份用0.25% EDTA酶消化为分散的悬浮单个的癌细胞,利用CRC技术对癌细胞进行培养。培养方法:将原代细胞与经30 Gy照射的3T3-J2小鼠成纤维细胞共培养于条件性培养基中,观察癌细胞克隆团生长情况。使用差异性消化对原代细胞进行传代:先用0.25% EDTA胰酶消化1 min去除饲养层细胞,PBS清洗后再用0.25% EDTA胰酶消化原代细胞。待癌细胞稳定后,通过免疫荧光、免疫组化染色、免疫印迹和荧光原位杂交技术等实验,验证所培养的癌细胞性质。结果体外利用CRC技术建立3例前列腺癌原代细胞株,细胞培养约15 d建系成功,并可持续稳定传代。细胞鉴定结果显示,所培养的细胞均表达AR、CK5、CK18和P504s,同时也弱表达PSA。荧光原位杂交技术显示细胞出现≥1.6%的TMPRSS2/ERG基因融合,这些现象符合前列腺癌的细胞特征。结论利用CRC技术能稳定地体外持续培养前列腺癌原代细胞。Objective To cultivate human-derived prostate cancer(PCa)cells via conditional reprogramming cell(CRC)technology,and establish individualized cell bank for PCa research in vitro.Methods We obtained three fresh PCa tissue samples from different patients between January 2019 and April 2019.Then each sample was divided into two parts.One was used for cancer nature confirmation by intraoperative biopsy.Another part was sent to the laboratory and digested into single primary cancer cells with 0.25%EDTA enzyme for CRC technology.The details were described as followed:1.The primary PCa cells were co-cultured with 3T3-J2 cells irradiated by 30 Gy(feeder cells)in conditioned medium,and observed for the growth of cell clones,2.The feeder cells were removed by 0.25%EDTA trypsin for 1 minute before primary PCa cells digested for passage.All primary PCa cells were validated by multiple experiments such as immunofluorescence,immunohistochemistry,immunoblotting and fluorescence in situ hybridization(FISH).Results Total three cases of human-derived PCa cell lines were successfully established during 15days through CRC technology.All those primary PCa cells could be steadily and continuously passaged,which also expressed AR,CK5,CK18,P504s and PSA.FISH demonstrated that each cell line harbored≥1.6%TMPRSS2/ERG fusion and conformed to the features of PCa.Conclusion CRC technology can be used for stable and continuous PCa cell culture in vitro.

关 键 词：肿瘤细胞 培养的 前列腺癌 条件性重编程技术 原代细胞培养 精准医学 

分 类 号：R737.25[医药卫生—肿瘤]