雄激素受体对从前列腺癌LNCaP细胞分选出的CK5+CK8+细胞的作用及其调控机制  

Role of androgen receptor in CK5+CK8+cells isolated from prostate cancer LNCaP cells and its regulating mechanism

作　　者：马志方[1] 曾胜 郐海洋 杭天昆 刘春[1] Ma Zhifang;Zeng Sheng;Kuai Haiyang;Hang Tiankun;Liu Chun(Department of Urology,First Hospital of Shanxi Medical University,Taiyuan 030001,China;Department of Urology,the First Affiliated Central Hospital of Nankai University,Tianjin 300192,China)

机构地区：[1]山西医科大学第一医院泌尿外科,太原030001 [2]南开大学附属第一中心医院泌尿外科,天津300192

出　　处：《肿瘤研究与临床》2021年第1期42-47,共6页Cancer Research and Clinic

基　　金：山西省自然科学基金面上项目(201601D011101);山西省留学回国人员科技活动择优资助项目((2017)397-10)。

摘　　要：目的探讨雄激素受体(AR)对从前列腺癌LNCaP细胞中分选出的CK5+CK8+细胞的作用及其调控机制。方法采用流式细胞术从LNCaP细胞中分选CK5+CK8+细胞,使用慢病毒载体携带AR基因转入CK5+CK8+细胞。实验分为转入AR的AR CK5+CK8+组和转入空载体的V CK5+CK8+组,在1、10 nmol/L二氢睾酮(DHT)培养条件下,采用蛋白质印迹法检测AR、p-AKT和bcl-2蛋白的表达,应用四甲基偶氮唑盐(MTT)法、细胞迁移实验和琼脂糖凝胶克隆形成实验检测AR对CK5+CK8+细胞生物学特性的影响。采用MTT法检测应用AKT信号通路活化抑制物LY 294002(LY)、γ-生育三烯酚(γ-TT)和(或)诱导AR表达的5-氮胞嘧啶(5-AZA)对CK5+CK8+细胞增殖的影响。结果AR基因转入CK5+CK8+细胞后,AR表达增强,p-AKT和bcl-2蛋白表达水平降低。在1、10 nmol/L DHT作用2、4、6 d后,AR CK5+CK8+组细胞较V CK5+CK8+组细胞增殖受抑制程度高,差异均有统计学意义(均P<0.05)。DHT 1、10 nmol/L作用3 d后,AR CK5+CK8+组细胞较V CK5+CK8+组细胞迁移能力下降[迁移细胞数:(54±9)个比(113±21)个,(13±3)个比(34±6)个],差异均有统计学意义(t=4.450,P<0.01;t=5.157,P<0.01)。DHT 1、10 nmol/L作用3周后,AR CK5+CK8+组细胞较V CK5+CK8+组细胞成瘤能力弱[克隆数:(39±7)个比(105±16)个,(41±6)个比(86±6)个],差异有统计学意义(t=6.631,P<0.01;t=8.662,P<0.01)。5 nmol/L LY+10 nmol/L 5-AZA、5 nmol/L LY+5 nmol/Lγ-TT、10 nmol/L 5-AZA+5 nmol/Lγ-TT、2.5 nmol/L LY+5 nmol/L 5-AZA+2.5 nmol/Lγ-TT联合1 nmol/L DHT或10 nmol/L DHT作用2、4、6 d均能抑制CK5+CK8+细胞增殖(均P<0.05)。结论AR可以抑制从LNCaP中分选出的CK5+CK8+细胞增殖,导致细胞迁移和成瘤能力下降;其可能通过抑制AKT-bcl-2信号通路的活化而发挥作用。Objective To investigate the role of androgen receptor(AR)in CK5+CK8+cells isolated from prostate cancer LNCaP cells and its regulating mechanism.Methods CK5+CK8+cells were isolated from LNCaP cells by using flow cytometry.Lentivirus vector carrying AR gene was transferred in CK5+CK8+cells.The experiments were divided into AR CK5+CK8+group transfering AR and V CK5+CK8+group transfering blank load.The expressions of AR,p-AKT and bcl-2 were tested by using Western blot assay under different concentrations of androgen(1 nmol/L and 10 nmol/L dihydrotestosterone).Methyl thiazolyl tetrazolium(MTT)assay,cell migration assay and soft agarose gel clone formation assay was used to detect the effect of AR on the biological property of CK5+CK8+cells.The effect of activated inhibitors such as LY 294002(LY),γ-tocotrienol(γ-TT)and/or 5-fluorocytosine inducing AR expression(5-AZA)through AKT signal pathways on CK5+CK8+cells proliferation was detected by using MTT assay.Results After AR gene was transferred into CK5+CK8+cells,the expression of AR was increased,while the expression of p-AKT and bcl-2 was decreased.After the treatment of 1 nmol/L dihydrotestosterone and 10 nmol/L dihydrotestosterone for 2,4 and 6 d,the cell proliferation inhibited degree of AR CK5+CK8+cells was higher compared with that of V CK5+CK8+cells,and the difference was statistically significant(all P<0.05).After the treatment of 1 nmol/L dihydrotestosterone and 10 nmol/L dihydrotestosterone for 3 d,the migration ability of AR CK5+CK8+cells was decreased compared with that of V CK5+CK8+cells(the number of cell migration:54±9 vs.113±21,13±3 vs.34±6),and the differences were statistically significant(t=4.450,P<0.01;t=5.157,P<0.01).After the treatment of 1 nmol/L dihydrotestosterone and 10 nmol/L dihydrotestosterone for 3 weeks,the tumorigenic ability of AR CK5+CK8+cells was reduced compared with that of V CK5+CK8+cells(the number of clone:39±7 vs.105±16,41±6 vs.86±6),and the differences were statistically significant(t=6.631,P<0.01;t=8.662,P<0.01).A

关 键 词：前列腺肿瘤 CK5+CK8+细胞 受体 雄激素 

分 类 号：R737.25[医药卫生—肿瘤]