一株永生化的BRCA1突变型人卵巢表面上皮细胞系的建立及初步分析  

Establishment and initial analysis of an immortalized human ovarian surface epithelial cell line carrying a BRCA1 mutation

作　　者：任春霞 夏小艾[2] 杨树东 吕蓓[2] 童国庆[1] REN Chunxia;XIA Xiaoai;YANG Shudong;LV Bei;TONG Guoqing(Center ofReproductive Medicine,Shuguang Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200120,China;Department of Obstetrics and Gynecology,Wuxi People’s Hospital,Wuxi 214023,Jiangsu Province,China;Department of Pathology,Wuxi People’s Hospital,Wuxi 214023,Jiangsu Province,China)

机构地区：[1]上海中医药大学附属曙光医院生殖医学中心,上海200120 [2]无锡市人民医院妇产科,江苏无锡214023 [3]无锡市人民医院病理科,江苏无锡214023

出　　处：《中国癌症杂志》2019年第9期693-699,共7页China Oncology

基　　金：国家自然科学基金青年项目（81402153）

摘　　要：背景与目的:BRCA1突变女性患乳腺癌和卵巢癌的风险增加,然而,目前尚不清楚BRCA1的单个等位基因突变如何导致癌症的发生,建立BRCA1突变的永生化细胞系可能为此类肿瘤发生的研究提供一个有用的模型。方法:从无锡市人民医院收治的1例携带BRCA1单等位基因185delAG突变患者的正常卵巢组织中分离和建立了卵巢表面上皮细胞系OSE236;然后利用反转录病毒系统介导的技术,用特异的shRNA稳定沉默p53表达,并导入人端粒酶反转录酶(human telomerase reverse transcriptase,hTERT)催化亚基和致癌基因HRAS,构建了系列细胞系并测定了其生长、衰老、细胞周期及致瘤特性。结果:成功建立了永生化细胞系OSE236/p53i/hTERT,但在该永生化细胞中导入致癌基因HRAS所构建的细胞系OSE236/p53i/hTERT/HRAS并未在体外和体内诱导肿瘤的转化。对该系列细胞系进行研究发现,与原代细胞系OSE236相比,永生化细胞系OSE236/p53i/hTERT中的β-半乳糖苷酶活性明显降低,而该BRCA1突变在所有系列细胞系中都存在,细胞周期随永生化明显增强,相关细胞周期调节蛋白(如CDK4、Cyclin B1/Cyclin D1/Cyclin E)上调,该永生化细胞系目前已在体外传代超过100代。结论:尽管未能在体外和体内将该永生化细胞系转化为癌细胞,但该携带BRCA1-185delAG突变的永生化人卵巢表面上皮细胞可能为研究BRCA1突变相关卵巢癌或乳腺癌提供了一个很有价值的细胞模型。Background and purpose:Women with BRCA1 mutations have an increased risk of breast and ovarian cancer.However,it is unclear how the mutation in a single allele of BRCA1 contributes to ovarian carcinogenesis.Thus,establishment of an immortalized cell line carrying BRCA1 mutation may provide a useful model to study the tumorigenesis.Methods:We isolated and established an ovarian surface epithelial cell line OSE236 from a woman carrying an allelic 185delAG mutation of BRCA1 who treated in Wuxi People’s Hospital.Then,by using retroviral system-mediated techniques,we stably silenced p53 with a pair of specific shRNA,consecutively introduced human telomerase reverse transcriptase(hTERT)catalytic subunit and oncogenic HRAS to establish a series cell lines and tested their growth,senescence,cell cylces and tumorigenecity.Results:We successfully constructed an immortalized cell line OSE236/p53i/hTERT.But the cell line OSE236/p53i/hTERT/HRAS generated by further transfection with oncogenic HRAS into the immortalized cells failed to induce transformation in vitro and in vivo.Analysis of the cell lines showed that cellular senescence was inhibited as the activity ofβ-galactosidase was decreased in the immortalized cell line OSE236/p53i/hTERT,compared with the parental cell line OSE236,and that the BRCA1 mutation occured in all cell lines.Cell cycles were markedly increased,and the related cell cycle regulatory factors such as CDK4,Cyclin B1/Cyclin D1/Cyclin E were up-regulated in immortalized cells.The immortalized cell line was passaged over 100 times in cultured dishes.Conclusion:Although we failed to transform the immortalized cells into tumorigenic cells,this immortal cell line carrying an allelic BRCA1-185delAG mutation may provide a valuable cell model to study the BRCA1 mutation-associated ovarian or breast tumorigenesis.

关 键 词：卵巢上皮细胞 永生化 p53 人端粒酶反转录酶 BRCA1突变 

分 类 号：R737.31[医药卫生—肿瘤]