CAJ | 학술논문

目的观察pEGFP-survivin对GBC-SD细胞化疗敏感性的影响.方法以MTT法检测GBC-SD、GBC-SD/EGFP和GBC-SD/survivin三种细胞的增殖活性;用RT-PCR和Western印迹、检测各组细胞中survivin mRNA和蛋白质水平的表达;以合适浓度的DDP作用相同的时间后,用MTT法检测3种细胞存活率,流式细胞仪测细胞凋亡,并用MTT法筛选IC_(50)值,TUNEL法观察细胞核改变;另外检测DDP作用后各组细胞的caspase-3蛋白酶活性的变化.结果 GBC-SD和GBC-SD/EGFP细胞的增殖活性大致相同,而GBC-SD/survivin细胞的增殖活性则明显下降;RT-PCR和Western印迹均发现GBC-SD/survivin细胞中的survivin表达水平较其余两种细胞明显下降;在给与DDP作用后,GBC-SD/survivin细胞存活率和IC_(50)明显较低,细胞凋亡率较高,而3种细胞用TUNEL法染色后均可见棕色凋亡细胞核.给与DDP作用后,各组细胞caspase-3活性均呈现先升高后下降的趋势,但GBC-SD/survivin细胞中caspase-3的活性明显高于另外两种细胞.结论 pEGFP-survivin表达的survivin shRNA能明显降低GBC-SD细胞中survivin的表达,提高对化疗药物的敏感性.
목적 관찰pEGFP-survivin대GBC-SD세포화료민감성적영향.방법 이MTT법검측GBC-SD、GBC-SD/EGFP화GBC-SD/survivin삼충세포적증식활성;용RT-PCR화Western인적、검측각조세포중survivin mRNA화단백질수평적표체;이합괄농도적DDP작용상동적시간후,용MTT법검측3충세포존활솔,류식세포의측세포조망,병용MTT법사선IC_(50)치,TUNEL법관찰세포핵개변;령외검측DDP작용후각조세포적caspase-3단백매활성적변화.결과 GBC-SD화GBC-SD/EGFP세포적증식활성대치상동,이GBC-SD/survivin세포적증식활성칙명현하강;RT-PCR화Western인적균발현GBC-SD/survivin세포중적survivin표체수평교기여량충세포명현하강;재급여DDP작용후,GBC-SD/survivin세포존활솔화IC_(50)명현교저,세포조망솔교고,이3충세포용TUNEL법염색후균가견종색조망세포핵.급여DDP작용후,각조세포caspase-3활성균정현선승고후하강적추세,단GBC-SD/survivin세포중caspase-3적활성명현고우령외량충세포.결론 pEGFP-survivin표체적survivin shRNA능명현강저GBC-SD세포중survivin적표체,제고대화료약물적민감성.
Objective To investingate the effect of survivin shRNA on chemotherapy resistance in GBC-SD cells. Methods They were divided into three groups of GBC-SD, GBC-SD/EGFP and GBC-SD/survivin. MTT assay was used to detect cell viability in the 3groups. mRNA and protein of survivin were tested by RT-PCR and Western blot. Then the cells were treated with proper construction DDP. The cell survival rate and IC_(50) were determined with MTT, cell apoptosis detected by FACS and the alteration of nucleus observed by TUNEL. Meanwhile, caspase-3 activity was determined using the colorimetric method. Results Cell viability was reduced remarkably in GBC-SD/survivin and survivin expression was decreased obviously. After being treated with DDP, cell survival rate and IC_(50) were decreased obviously in GBC-SD/survivin, apoptotic rate elevated remarkably compared with other groups. There were brownly nucleuses in three groups. Caspase-3 activity increased first and then decreased, but it exceeded in GBC-SD/survivin than that in other two groups. Conclusion The survivin shRNA can down-regulate the expression of survivin in GBC-SD cells remarkably and improve the sensibility to chemotherapy.