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目的:探讨靶向葡萄糖神经酰胺合成酶(glucosylceramide synthase,GCS)对乳腺癌多药耐药的逆转作用及机制。方法:GCS反义寡核苷酸(GCSASODN)转染人耐药乳腺癌细胞MCF-7/AdrR,RT-PCR检测细胞GCS和多药耐药基因1(MDR1)mRNA的表达,免疫细胞化学染色检测细胞GCS蛋白和P-糖蛋白(P-gp)的表达,流式细胞仪分析细胞周期和凋亡。Caspase-3活性测定法检测Caspase-3活性改变。结果:GCSASODN转染人耐药乳腺癌细胞MCF-7/AdrR后,GCS和MDR1mRNA的表达水平分别为0.3、0.7,GCS蛋白和P-gp表达阳性率分别为23%、33%,GCS和MDR1的mRNA和蛋白水平较对照组有显著性差异(P<0.05)。转染后细胞周期改变不明显,但细胞凋亡增加为13.7±4.1,Caspase-3活性升高为0.0725±0.0003,与对照组差异有显著性(P<0.01)。结论:靶向GCS通过直接抑制GCS活性,并间接下调MDR1mRNA和蛋白表达,激活Caspase-3活性,诱导耐药细胞凋亡增加,有效逆转乳腺癌多药耐药。
Objective: To investigate the reversal effect and mechanism of targeting glucosylceramide synthase (GCS) on multidrug resistance in breast cancer. METHODS: GCSASODN was used to transfect human breast cancer cell line MCF-7 / AdrR. The expression of GCS and multidrug resistance gene 1 (MDR1) mRNA was detected by RT-PCR. Immunocytochemistry The expression of GCS and P-glycoprotein (P-gp) was detected by flow cytometry. Cell cycle and apoptosis were analyzed by flow cytometry. Caspase-3 activity assay to detect changes in Caspase-3 activity. Results: The expression of GCS and MDR1 mRNA in MCS-7 / AdrR cells transfected with GCSASODN were 0.3, 0.7, respectively. The positive rates of GCS and P-gp were 23%, 33% MRNA and protein levels were significantly different from the control group (P <0.05). The changes of cell cycle were not obvious after transfection, but the apoptosis increased 13.7 ± 4.1 and the activity of Caspase-3 increased 0.0725 ± 0.0003, which was significantly different from the control group (P <0.01). CONCLUSIONS: Targeting GCS can effectively reverse the multidrug resistance of breast cancer by directly inhibiting GCS activity and indirectly down-regulating MDR1 mRNA and protein expression, activating Caspase-3 activity, and inducing apoptosis of drug-resistant cells.