雄激素非依赖性前列腺癌细胞系LNCaP-ADR的建立与鉴定  

Establishment and Characterization of Androgen-independent Prostate Cancer Cell Line LNCaP-ADR

作　　者：史昕艺 芦晓红[1] 吴晓婷 罗明秀 陈捷珲 王芳[2] 赵薇[1] 高玉婧[1] SHI Xinyi;LU Xiaohong;WU Xiaoting;LUO Mingxiu;CHEN Jiehui;WANG Fang;ZHAO Wei;GAO Yujing(Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education,School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;Department of Gastroenterology,the General Hospital of Ningxia Medical University,Yinchuan 750004,China;School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学生育力保持教育部重点实验室基础医学院,银川750004 [2]宁夏医科大学总医院消化内科,银川750004 [3]宁夏医科大学临床医学院,银川750004

出　　处：《宁夏医科大学学报》2021年第10期989-995,共7页Journal of Ningxia Medical University

基　　金：国家自然科学基金项目(81660486);中国科学院“西部之光”人才培养引进计划(2017);宁夏回族自治区大学生创新创业训练计划(NXCX2016119);宁夏医科大学校级科研项目(XZ2020006)。

摘　　要：目的建立雄激素非依赖性前列腺癌细胞系LNCaP-ADR,为进一步研究前列腺癌的去势抵抗治疗机制奠定实验基础。方法将雄激素依赖性的前列腺癌细胞LNCaP作为亲本细胞,用含有10%木炭-右旋糖酐剥离的胎牛血清无酚红的RPMI 1640培养基进行雄激素剥夺培养,建立雄激素非依赖性的前列腺癌细胞系LNCaP-ADR。通过MTS实验和平板克隆形成实验,比较LNCaP亲本细胞和LNCaP-ADR细胞在雄激素剥夺培养条件下的增殖能力及对雄激素受体(AR)拮抗剂比卡鲁胺的敏感性;采用流式细胞术观察两种细胞在雄激素剥夺培养或比卡鲁胺处理下的细胞凋亡率,采用RT-qPCR检测AR信号通路靶基因PSA、TMPRSS2的表达变化。结果MTS实验和平板克隆形成实验结果表明,在雄激素剥夺培养条件下,雄激素非依赖性前列腺癌细胞系LNCaP-ADR的增殖能力和克隆形成能力较LNCaP亲本细胞增高(P均<0.01);细胞凋亡结果显示,LNCaP亲本细胞在雄激素剥夺培养条件下的细胞凋亡率高于LNCaP-ADR细胞(P<0.05);细胞毒性实验及凋亡检测结果表明,与LNCaP亲本细胞相比,LNCaP-ADR细胞对比卡鲁胺的敏感性降低(LNCaP IC_(50)=49.18μmol·L^(-1)、LNCaP-ADR IC_(50)=94.21μmol·L^(-1)),相同浓度比卡鲁胺诱导LNCaP-ADR细胞发生的凋亡率较LNCaP亲本细胞减少;RT-qPCR结果显示,在雄激素剥夺培养条件下,LNCaP-ADR中AR下游靶基因PSA、TMPRSS2的表达水平较亲本细胞增高(P<0.01)。结论本实验成功构建了雄激素非依赖性前列腺癌细胞系LNCaP-ADR,该细胞系对雄激素剥夺及雄激素受体拮抗剂比卡鲁胺具有较强的抵抗特性。Objective To establish and characterize androgen-independent prostate cancer cell line LNCaP-ADR from parental LNCaP cell line,so as to lay an experimental foundation for further study on the mechanism of castration resistance in prostate cancer.Methods An androgen-independent prostate cancer cell line LNCaP-ADR was established by culturing the LNCaP parental cells in androgen deprivation condition(maintained in phenol red-free RPMI 1640 with 10%charcoal-dextran stripped FBS)for about one month.MTS assay and plate colony formation assay were then used to compare the cell proliferation and colony formation abilities between LNCaP parental cells and LNCaP-ADR cells under androgen deprivation condition.Flow cytometry and MTS assay were performed to compare the effect of androgen receptor(AR)antagonist bicalutamide on apoptosis and cell viability between LNCaP parental cells and LNCaP-ADR cells.RT-qPCR was used to compare the expression levels of AR target genes(PSA and TMPRSS2)in LNCaP parental cells and LNCaP-ADR cells.Results Cell proliferation assay and colony formation assay showed that the proliferation ability and colony formation ability of androgen-independent LNCaP-ADR cells were significantly higher than parental cells in androgen deprivation condition(P all<0.01).Result of flow cytometry revealed that the apoptosis rate of LNCaP-ADR cells was significantly lower than that of parental LNCaP cells under androgen deprivationcondition(P<0.05).Cytotoxicity assay and flow cytometry showed that LNCaP-ADR cells were less sensitive to the androgen receptor antagonist bicalutamide(LNCaP IC_(50)=49.18μmol·L^(-1),LNCaP-ADR IC_(50)=94.21μmol·L^(-1)).In addition,expression levels of androgen receptor target genes PSA and TMPRSS2 were higher in LNCaP-ADR than in parental cells under androgen deprivation condition(P<0.01).Conclusion In this study,androgen-independent cell line LNCaP-ADR was successfully established from parental LNCaP cells.LNCaP-ADR cell line has strong androgen-independent growth ability and resist

关 键 词：前列腺癌 LNCAP细胞 雄激素依赖性 去势抵抗 

分 类 号：R737.25[医药卫生—肿瘤]