羟基喜树碱对高危浅表性膀胱癌模型5637细胞的凋亡作用研究

Hydroxycamptothecin Induced Apoptosis in 5637 Cells Line:An In-vitro Model for High-risk Superficial Bladder Cancer

目的:观察羟基喜树碱(HCPT)对高危浅表性膀胱癌模型5637细胞的凋亡作用,研究HCPT不同作用时间和不同浓度对5637细胞的影响,进一步讨论HCPT在膀胱灌注化疗中的作用。方法:采用四甲基偶氮唑盐(MIT)检测不同浓度、不同作用时间下HCPT对5637细胞增殖的影响;采用流式细胞术测定低浓度(5μmol/L)、不同作用时间(12、24 h)HCPT诱导5637细胞的凋亡率;用Hochest 33258染色观察细胞凋亡情况;采用荧光定量PCR检测5μmol/L HCPT作用5637细胞12 h相较未加药5637细胞中凋亡相关基因的变化。结果:5μmol/L HCPT作用5637细胞12 h后,早期凋亡细胞占23.5%。HCPT随着作用时间的延长,凋亡率并不会随之增加,HCPT诱导凋亡的作用在肿瘤细胞与药物作用后的某一时间点达到高峰。Hochest 33258染色和荧光定量PCR检测凋亡相关基因也证明HCPT这一浓度在这一时间点有诱导癌细胞凋亡的作用。结论:羟基喜树碱具有诱导高危浅表性膀胱癌细胞凋亡的作用。适当延长药物作用时间,可以促进其药效的发挥。

Objective：To observe whether hydroxycamptothecin （HCPT） can induce apoptosis in 5637 cells of High-risk Superficial bladder cancer model, to study the influence on 5637 cells of different time and concentrations of HCPT, and to discus {urtherly the role of HCPT in bladder infusion chemotherapy. Methods： The influence on 5637 cells proliferation of different time and concentrations of HCPT was detected by MTT. Apoptosis rate of 5637 cells were induced by low concentration（5μmol/L）, various lengths of time（12,24 h） of HCPT was determined by Flow cytometry. Hochest 33258 were used to determine the induction of apoptosis after use of HCPT. The changes of apoptosis-related genes in 5637 cells induced by 5μmol/L HCPT for 12h and in 5637 cells with no HCPT was detected by fluorescent quantitative polymerase chain reaction （PCR）. Results：Cell shrinage, nuclear fragmentation and condensed chromosomes showed that apoptosis can be induced by HCPT within the concentration of 5μmol/L. The flow cytometry analysis showed that the percentage of apoptotic cells were 23.5% after 12 hours treatment. There was no significant change about the percentage of apoptotic cells with higher drug concentration and the time of induction. Hochest 33258 and fluorescence quantitative PCR also confirmed the same findings. Conclusions：The induction of apoptosis exposed to HCPT in 5637 human urinary bladder cancer cells is a good model for further studying urinary bladder cancer. Appropriately extend time of the drug can exert to promote its efficacy.