中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2013年
10期
772-777
,共6页
刘冰洁%李小平%赵丽君%王建六%魏丽惠
劉冰潔%李小平%趙麗君%王建六%魏麗惠
류빙길%리소평%조려군%왕건륙%위려혜
子宫内膜肿瘤%甲羟孕酮17-乙酸酯%抗药性,肿瘤%肿瘤干细胞
子宮內膜腫瘤%甲羥孕酮17-乙痠酯%抗藥性,腫瘤%腫瘤榦細胞
자궁내막종류%갑간잉동17-을산지%항약성,종류%종류간세포
Endometrial neoplasms%Medroxyprogesterone 17-acetate%Drug resistance,neoplasm%Neoplastic stem cells
目的 初步探讨子宫内膜癌细胞中干细胞样(SP)细胞对醋酸甲羟孕酮(MPA)的抵抗作用及其机制.方法 (1)采用流式细胞仪Hoechst33342(是一种荧光染色液)染色法分选子宫内膜癌细胞系Ishikawa细胞中的SP细胞(即Ishikawa-SP细胞)和非SP细胞(即Ishikawa-non-SP细胞),采用平板克隆形成实验、免疫组化法分别检测Ishikawa-SP和Ishikawa-non-SP细胞的集落形成率及乳腺癌耐药蛋白(BCRP)的表达.(2)不同浓度(分别为5、10、15、20 μmol/L)的MPA处理Ishikawa-SP、Ishikawa-non-SP、Ishikawa细胞不同时间(分别为24、48、72 h)后,采用甲烷硫代磺酸盐(MTS)法检测3种细胞生长的抑制率,选择Ishikawa细胞生长的抑制率最接近50％时MPA的处理浓度和时间(即为10 μmol/L的MPA处理72 h时)用于以下实验.(3)以10 μmol/L的MPA处理Ishikawa-SP、Ishikawa-non-SP、Ishikawa细胞72 h后,采用流式细胞仪检测3种细胞的凋亡率,采用免疫组化法检测3种细胞中凋亡蛋白——半胱氨酸天冬氨酸蛋白酶3(caspase-3)的表达.结果 (1)子宫内膜癌细胞系Ishikawa细胞中存在少量SP细胞,其比例为2.7％；Ishikawa-SP和Ishikawa-non-SP细胞的集落形成率分别为(6.02±1.17)％、(0.53±0.20)％,两者比较,差异有统计学意义(P=0.001)；Ishikawa-SP和Ishikawa-non-SP细胞中BCRP蛋白表达的综合得分分别为(16.3±2.0)、(13.4±1.5)分,两者比较,差异有统计学意义(P =0.001).(2)Ishikawa-SP、Ishikawa-non-SP及Ishikawa细胞生长的抑制率,随着MPA浓度的增加和处理时间的延长均增加,呈明显的浓度和时间依赖性(P＜0.05).其中,MPA对Ishikawa-SP细胞的抑制作用最弱.(3)以10 μmol/L的MPA处理72 h后,Ishikawa-SP、Ishikawa-non-SP、Ishikawa细胞的凋亡率分别为(4.01±0.43)％、(9.30±0.67)％、(4.64±0.18)％,3者间比较,差异有统计学意义(P＜0.05)；3种细胞中caspase-3蛋白表达的综合得分分别为(14.9±1.1)、(16.1±1.0)、(15.5±1.2)分,3者间比较,差异有统计学意义(P＜0.05).结论 MPA对Ishikawa、Ishikawa-SP、Ishikawa-non-SP细胞的生长均有抑制作用,但Ishikawa-SP细胞的生长抑制率及凋亡率均明显低于Ishikawa和Ishikawa-non-SP细胞,显示Ishikawa-SP细胞具有孕激素抵抗的特性,其抵抗机制可能与肿瘤干细胞的耐药性和细胞凋亡有关.
目的 初步探討子宮內膜癌細胞中榦細胞樣(SP)細胞對醋痠甲羥孕酮(MPA)的牴抗作用及其機製.方法 (1)採用流式細胞儀Hoechst33342(是一種熒光染色液)染色法分選子宮內膜癌細胞繫Ishikawa細胞中的SP細胞(即Ishikawa-SP細胞)和非SP細胞(即Ishikawa-non-SP細胞),採用平闆剋隆形成實驗、免疫組化法分彆檢測Ishikawa-SP和Ishikawa-non-SP細胞的集落形成率及乳腺癌耐藥蛋白(BCRP)的錶達.(2)不同濃度(分彆為5、10、15、20 μmol/L)的MPA處理Ishikawa-SP、Ishikawa-non-SP、Ishikawa細胞不同時間(分彆為24、48、72 h)後,採用甲烷硫代磺痠鹽(MTS)法檢測3種細胞生長的抑製率,選擇Ishikawa細胞生長的抑製率最接近50％時MPA的處理濃度和時間(即為10 μmol/L的MPA處理72 h時)用于以下實驗.(3)以10 μmol/L的MPA處理Ishikawa-SP、Ishikawa-non-SP、Ishikawa細胞72 h後,採用流式細胞儀檢測3種細胞的凋亡率,採用免疫組化法檢測3種細胞中凋亡蛋白——半胱氨痠天鼕氨痠蛋白酶3(caspase-3)的錶達.結果 (1)子宮內膜癌細胞繫Ishikawa細胞中存在少量SP細胞,其比例為2.7％；Ishikawa-SP和Ishikawa-non-SP細胞的集落形成率分彆為(6.02±1.17)％、(0.53±0.20)％,兩者比較,差異有統計學意義(P=0.001)；Ishikawa-SP和Ishikawa-non-SP細胞中BCRP蛋白錶達的綜閤得分分彆為(16.3±2.0)、(13.4±1.5)分,兩者比較,差異有統計學意義(P =0.001).(2)Ishikawa-SP、Ishikawa-non-SP及Ishikawa細胞生長的抑製率,隨著MPA濃度的增加和處理時間的延長均增加,呈明顯的濃度和時間依賴性(P＜0.05).其中,MPA對Ishikawa-SP細胞的抑製作用最弱.(3)以10 μmol/L的MPA處理72 h後,Ishikawa-SP、Ishikawa-non-SP、Ishikawa細胞的凋亡率分彆為(4.01±0.43)％、(9.30±0.67)％、(4.64±0.18)％,3者間比較,差異有統計學意義(P＜0.05)；3種細胞中caspase-3蛋白錶達的綜閤得分分彆為(14.9±1.1)、(16.1±1.0)、(15.5±1.2)分,3者間比較,差異有統計學意義(P＜0.05).結論 MPA對Ishikawa、Ishikawa-SP、Ishikawa-non-SP細胞的生長均有抑製作用,但Ishikawa-SP細胞的生長抑製率及凋亡率均明顯低于Ishikawa和Ishikawa-non-SP細胞,顯示Ishikawa-SP細胞具有孕激素牴抗的特性,其牴抗機製可能與腫瘤榦細胞的耐藥性和細胞凋亡有關.
목적 초보탐토자궁내막암세포중간세포양(SP)세포대작산갑간잉동(MPA)적저항작용급기궤제.방법 (1)채용류식세포의Hoechst33342(시일충형광염색액)염색법분선자궁내막암세포계Ishikawa세포중적SP세포(즉Ishikawa-SP세포)화비SP세포(즉Ishikawa-non-SP세포),채용평판극륭형성실험、면역조화법분별검측Ishikawa-SP화Ishikawa-non-SP세포적집락형성솔급유선암내약단백(BCRP)적표체.(2)불동농도(분별위5、10、15、20 μmol/L)적MPA처리Ishikawa-SP、Ishikawa-non-SP、Ishikawa세포불동시간(분별위24、48、72 h)후,채용갑완류대광산염(MTS)법검측3충세포생장적억제솔,선택Ishikawa세포생장적억제솔최접근50％시MPA적처리농도화시간(즉위10 μmol/L적MPA처리72 h시)용우이하실험.(3)이10 μmol/L적MPA처리Ishikawa-SP、Ishikawa-non-SP、Ishikawa세포72 h후,채용류식세포의검측3충세포적조망솔,채용면역조화법검측3충세포중조망단백——반광안산천동안산단백매3(caspase-3)적표체.결과 (1)자궁내막암세포계Ishikawa세포중존재소량SP세포,기비례위2.7％；Ishikawa-SP화Ishikawa-non-SP세포적집락형성솔분별위(6.02±1.17)％、(0.53±0.20)％,량자비교,차이유통계학의의(P=0.001)；Ishikawa-SP화Ishikawa-non-SP세포중BCRP단백표체적종합득분분별위(16.3±2.0)、(13.4±1.5)분,량자비교,차이유통계학의의(P =0.001).(2)Ishikawa-SP、Ishikawa-non-SP급Ishikawa세포생장적억제솔,수착MPA농도적증가화처리시간적연장균증가,정명현적농도화시간의뢰성(P＜0.05).기중,MPA대Ishikawa-SP세포적억제작용최약.(3)이10 μmol/L적MPA처리72 h후,Ishikawa-SP、Ishikawa-non-SP、Ishikawa세포적조망솔분별위(4.01±0.43)％、(9.30±0.67)％、(4.64±0.18)％,3자간비교,차이유통계학의의(P＜0.05)；3충세포중caspase-3단백표체적종합득분분별위(14.9±1.1)、(16.1±1.0)、(15.5±1.2)분,3자간비교,차이유통계학의의(P＜0.05).결론 MPA대Ishikawa、Ishikawa-SP、Ishikawa-non-SP세포적생장균유억제작용,단Ishikawa-SP세포적생장억제솔급조망솔균명현저우Ishikawa화Ishikawa-non-SP세포,현시Ishikawa-SP세포구유잉격소저항적특성,기저항궤제가능여종류간세포적내약성화세포조망유관.
Objective To explore the mechanism resistance of medroxyprogesterone 17-acetate (MPA) on the endometrial cancer side-population (SP) cells.Methods (1) Ishikawa-SP cells from endometrial cancer cell lines Ishikawa were be separated by Hoechst 33342 dyeing method and flow cytometry analysis.The clone formation efficiency between Ishikawa-SP cells and Ishikawa-non-SP cells were performed by clone formation assay.Breast cancer resistance protein (BCRP) was examined by immunocytochemistry method.(2)Ishikawa,Ishikawa-SP,Ishikawa-non-SP cells were treated with various concentrations of MPA at 5,10,15,20 μmoL/L.After cultured for 24,48,and 72 hours,cells growth were measured by methanethiosulfomate(MTS) assay.(3) The groups of Ishikawa,Ishikawa-SP,Ishikawa-non-SP cells incubated with MPA at the half maximal inhibitory concentration(IC50) were selected for cell apoptosis assay by using flow cytometry.After MPA treatment,the expression of caspase-3 was examined by immunocytochemistry method.Results (1)There were few proportion of Ishikawa-SP cells in Ishikawa endometrial carcinoma,which were 2.7％.There were stronger clone formation efficiency for Ishikawa-SP cells than that for Ishikawa-non-SP cells in Ishikawa [(6.02 ± 1.17)％ vs.(0.53 ±0.20)％,P =0.001].And there were higher level expression of BCRP (P =0.001)and also more resistant Taxol and radiation between Ishikawa-SP cells and Ishikawa-non-SP cells.(2)The inhibitory effect of MPA was concentrationdependent and time-dependent.(3)After MPA treatment,the apoptosis rates of Ishikawa-SP,Ishikawa-nonSP,Ishikawa were (4.01 ± 0.43) ％,(9.30 ± 0.67) ％,and (4.64 ± 0.18) ％,respectively (P ＜ 0.05).The level expression of caspase-3 in Ishikawa group after MPA treated were higher than that in Ishikawa-SP group.Conclusion MPA may be inhibit the growth of endometrial cancer,Ishikawa-SP and Ishikawa-nonSP cells,while Ishikawa-SP may be more resistant to MPA than Ishikawa-non-SP,which mechanism of resistance on MPA may be related to the properties of cancer stem-like cells and cell apoptosis.
