【摘要】 背景和目的:多细胞球体(multicellularspheroids,MCS)对传统细胞毒化疗药物的敏感性明显低于单层细胞。本实验旨在探讨卵巢癌MCS耐药的分子机制。方法:以单层细胞为对照,以三维培养方法获得的人卵巢癌A2780MSC和CAOV3MCS为模型,采用台盼蓝拒染法比较紫杉醇对单层细胞和MCS生长的抑制作用,流式细胞仪比较单层细胞和MCS细胞周期的分布和细胞凋亡率;采用流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测单层细胞及MCS的P-糖蛋白(P-glycoprotein,P-gp)表达及亚细胞分布;采用RT-PCR法检测mdr1mRNA表达水平。结果:(1)不同浓度的紫杉醇(0.2、2.0、10.0、20.0μmol/L)作用后,MCS细胞生长抑制率明显低于单层细胞(PA2780=0.003,PCAOV3=0.015);经20.0μmol/L的紫杉醇作用后,单层细胞的细胞凋亡率明显高于MCS,差异有统计学意义(PA2780=0.034,PCAOV3=0.032)。(2)流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测提示P-gp在单层细胞中不表达,在MCS中表达明显升高(P均<0.05);RT-PCR证实MCS中有mdr1mRNA表达,而单层细胞中未检出其表达。(3)流式细胞仪检测提示将单层细胞培养成MCS时,G0/G1期细胞比率增加,S期和G2/M期细胞比率降低(P均<0.05)。结论:卵巢癌MCS对紫杉醇化疗耐药性增加,其高表达P-gp,并且与G0/G1期细更多还原

【Abstract】 BACKGROUND &OBJECTIVE: Cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than monolayer cell cultures. The aim of this study was to investigate the molecular mechanism of chemoresistance. METHODS:Ovarian cancer A2780,CAOV3 multicellular spheroids were obtained from three dimensional culture. Expression of P glycoprotein (P gp) was determined using Western blot analysis and flow cytometry (FCM).The subcellular distribution of P gp was also determined using laser confocal microscope. Reverse transcription polymerase chain reaction (RT PCR) was employed to detect the mdr1 mRNA of both monolayer cells and multicellular spheroids. Cell cycle profiles and apoptosis were also analyzed using FACS. The resistance was detected with trypan blue exclusion testing. RESULTS: Compared with control cells, no expression of P gp was detected in monolayer cells, but expression of P gp in aggregate cells was significantly elevated(P< 0 05). The mdr1 mRNA positive nodes were confirmed by RT PCR in the aggregate cells. The percentage of cells increased in G0-G1 phase and decreased in S and G2 M phase significantly in spheroids cells. Spheroids cells showed higher cell viability than monolayer cells (PA2780=0 003,PCAOV3=0 015). More apoptotic cells were induced by Taxol in MCS cells than in monolayer cells. CONCLUSION: Ovarian cancer A2780, CAOV3 multicellular spheroids cultures induced cell resistante to Taxol. High expression of P gp was induced in ovarian multicellular spheroids and the cells were arrested in G0-G1 phase.更多还原