CAJ | 학술논문

目的建立子宫内膜癌顺铂耐药细胞株ISH/DDP,鉴定其生物学特性,并探讨其顺铂耐药的机制.方法采用顺铂浓度逐步递增联合大剂量顺铂冲击方法,在体外连续诱导、培养子宫内膜癌细胞株Ishikawa细胞,建立子宫内膜癌顺铂耐药细胞株ISH/DDP.非放射性细胞增殖法(MTS法)测定ISH/DDP细胞对顺铂的耐药指数;绘制细胞生长曲线,计算细胞群体倍增时间;流式细胞术检测ISH/DDP细胞的细胞周期比例;免疫细胞化学法检测ISH/DDP细胞中耐药蛋白--乳腺癌耐药蛋白(BCRP)、P糖蛋白(P-gp)以及谷胱甘肽S转移酶π(GST-π)蛋白的表达水平,以综合积分表示.结果 Ishikawa细胞在体外采用顺铂浓度逐步递增联合大剂量顺铂冲击方法连续诱导、培养,历时10个月,成功建立了子宫内膜癌顺铂耐药细胞株ISH/DDP,其耐药指数为3.77.Ishikawa细胞和ISH/DDP细胞的群体倍增时间分别为34.1 h和40.4 b,两者比较,差异有统计学意义(P<0.05).与Ishikawa细胞相比,ISH/DDP细胞的S期细胞比例[分别为(44.2±6.1)%和(55.3±8.4)%]增加,G2/M期细胞比例[分别为(11.9±0.7)%和(5.7±2.4)%]减少,差异均有统计学意义(P<0.05);G0/G1期细胞比例[分别为(44.3±5.7)%和(39.0±10.7)%]减少,但差异无统计学意义(P>0.05).ISH/DDP细胞中BCRP蛋白的表达水平为(16.3±2.0)分,高于Ishikawa细胞的(13.4±1.5)分,差异有统计学意义(P<0.05);而Ishikawa和ISH/DDP细胞中P-gp蛋白的表达水平分别为(16.1±1.0)和(15.5±1.2)分,GST-π蛋白的表达水平分别为(14.9±1.1)和(15.2±1.9)分,分别比较,差异均无统计学意义(P>0.05).结论 ISH/DDP细胞具有耐药表型及耐药细胞的生物学特性;其耐药机制可能与细胞中BCRP蛋白的过度表达相关.
목적 건립자궁내막암순박내약세포주ISH/DDP,감정기생물학특성,병탐토기순박내약적궤제.방법 채용순박농도축보체증연합대제량순박충격방법,재체외련속유도、배양자궁내막암세포주Ishikawa세포,건립자궁내막암순박내약세포주ISH/DDP.비방사성세포증식법(MTS법)측정ISH/DDP세포대순박적내약지수;회제세포생장곡선,계산세포군체배증시간;류식세포술검측ISH/DDP세포적세포주기비례;면역세포화학법검측ISH/DDP세포중내약단백--유선암내약단백(BCRP)、P당단백(P-gp)이급곡광감태S전이매π(GST-π)단백적표체수평,이종합적분표시.결과 Ishikawa세포재체외채용순박농도축보체증연합대제량순박충격방법 련속유도、배양,력시10개월,성공건립료자궁내막암순박내약세포주ISH/DDP,기내약지수위3.77.Ishikawa세포화ISH/DDP세포적군체배증시간분별위34.1 h화40.4 b,량자비교,차이유통계학의의(P<0.05).여Ishikawa세포상비,ISH/DDP세포적S기세포비례[분별위(44.2±6.1)%화(55.3±8.4)%]증가,G2/M기세포비례[분별위(11.9±0.7)%화(5.7±2.4)%]감소,차이균유통계학의의(P<0.05);G0/G1기세포비례[분별위(44.3±5.7)%화(39.0±10.7)%]감소,단차이무통계학의의(P>0.05).ISH/DDP세포중BCRP단백적표체수평위(16.3±2.0)분,고우Ishikawa세포적(13.4±1.5)분,차이유통계학의의(P<0.05);이Ishikawa화ISH/DDP세포중P-gp단백적표체수평분별위(16.1±1.0)화(15.5±1.2)분,GST-π단백적표체수평분별위(14.9±1.1)화(15.2±1.9)분,분별비교,차이균무통계학의의(P>0.05).결론 ISH/DDP세포구유내약표형급내약세포적생물학특성;기내약궤제가능여세포중BCRP단백적과도표체상관.
Objective To establish the cisplatin(DDP)-resistant cell line from human endometrial cancer cell line Ishikawa and to investigate its resistant mechanism to DDP. Methods A resistant endometrial cancer cell line ISH/DDP was established by gradually increasing dose of cisplatin and high-dose stimulation. The resistant index was estimated by 3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay. Cell growth curve, doubling time and cell cycle phase distribution were measured; drug-resistant protein of breast cancer resistance protein (BCRP),P-glycoprotein (P-gp) and glutathione-S-transferase-π (GST-π) were examined by immuocytochemistry. Results The DDP-resistant cell line ISH/DDP was established with the resistant index of 3. 77. The proliferation of ISH/DDP got slow, doubling time prolonged, which were 40. 1 hours, while it was 34. 1 hours in Ishikawa (P<0.05); and the cell number of G0/G1 phase [(44. 3 ±5. 7)% and (39. 0 ± 10. 7)% ,P >0. 05] and G2/M phase [(11.9 ±0.7)% and (5. 7 ±2. 4)% ,P <0. 05] decreased, while S-phase [(44. 2 ±6.1)% and (55. 3 ± 8. 4) % , P < 0. 05] increased compared with parent cells. The comprehensive score of the expression of BCRP in ISH/DDP was 16. 3 ± 2. 0, while it was 13.4 ± 1. 5 in Ishikawa (P < 0. 05). The score of the expression of P-gp in ISH/DDP and Ishikawa were 15. 5 ± 1. 2 and 16. 1 ± 1. 0 (P > 0. 05) ,respectively. The score of the expression of GST-π in ISH/DDP and Ishikawa were 15. 2 ± 1. 9 and 14. 9 ± 1.1 (P > 0. 05) . Conclusion ISH/DDP cell line showed a typical resistant phenotype and biological characteristics, which may be accounted for high BCRP expression.