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目的长期应用多柔比星(doxorubicin,ADM)能够增加药物耐受性及毒副作用,单壁碳纳米管(single-walled carbon nanotubes,SWCNTs)作为药物载体可提高载药率。本研究通过制备携载ADM的碳纳米管复合物然后作用于人卵巢癌ADM耐药细胞株,探讨研究碳纳米管复合物对细胞的杀伤作用。方法制备ADM/PEG/SWCNTs复合物,傅里叶变换红外光谱扫描仪表征检测PEG/SWCNTs表面官能团的变化;紫外分光光度计测定载药量;WST-1法检测ADM/PEG/SWCNTs对SKOV3/ADM的增殖抑制作用;流式细胞术和Hoechst33258荧光染色观察ADM/PEG/SWCNTs对细胞凋亡的影响;PCR及蛋白质印迹法检测ADM/PEG/SWCNTs对MDR-1 mRNA及P-gp糖蛋白的表达影响。结果成功制备ADM/PEG/SWCNTs复合药物,其中ADM在PEG/SWCNTs的载药率为(79.5±10.6)%,包封率(32.0±4.2)%。WST-1实验显示,ADM/PEG/SWCNTs及ADM作用细胞平均IC50为(18.0±1.2)和(37.5±1.9)μg/mL。ADM/PEG/SWCNTs对SKOV3/ADM细胞24、48和72h细胞增殖抑制率分别为(43.27±3.60)%、(53.40±8.12)%和(68.00±4.26)%,组间比较,差异有统计学意义,P<0.001。流式细胞术检测ADM/PEG/SWCNTs组及ADM组SKOV3/ADM细胞凋亡率为(14.83±1.86)%和(5.28±0.45)%,F=137.292,P=0.001。Hoechst33258染色ADM/PEG/SWCNTs组细胞呈明显凋亡形态改变。ADM/PEG/SWCNTs组可下调MDR-1mRNA及P-gp蛋白的表达,经灰度扫描分析,组间比较,差异有统计学意义,F=18.634,P=0.003。结论体外制备的ADM/PEG/SWCNTs纳米复合物具有高载药率,可抑制人卵巢癌细胞多柔比星耐药细胞增殖,促进人卵巢癌ADM耐药细胞凋亡,可下调MDR-1mRNA及P-gp蛋白的表达。
OBJECTIVE: Long-term application of doxorubicin (ADM) can increase drug tolerance and toxicity. Single-walled carbon nanotubes (SWCNTs) as drug carriers can improve the drug loading rate. In this study, ADM-loaded carbon nanotube complexes were prepared and then applied to ADM-resistant human ovarian cancer cell lines to investigate the effects of carbon nanotube complex on cell killing. Methods The ADM / PEG / SWCNTs complexes were prepared, the surface functional groups of PEG / SWCNTs were characterized by Fourier transform infrared spectroscopy, the drug loading was measured by ultraviolet spectrophotometer, the effect of ADM / PEG / SWCNTs on the proliferation of SKOV3 / ADM and ADM were detected by flow cytometry and Hoechst33258 staining. The effects of ADM / PEG / SWCNTs on apoptosis were detected by PCR and Western blotting. The effect of ADM / PEG / SWCNTs on MDR-1 mRNA and P-glycoprotein Express the impact. Results ADM / PEG / SWCNTs were successfully prepared. The drug-loading rate of ADM in PEG / SWCNTs was (79.5 ± 10.6)% and encapsulation efficiency was (32.0 ± 4.2)%. WST-1 experiments showed that the average IC50 of ADM / PEG / SWCNTs and ADM-treated cells were (18.0 ± 1.2) and (37.5 ± 1.9) μg / mL. The inhibitory rates of ADM / PEG / SWCNTs on SKOV3 / ADM cells were (43.27 ± 3.60)%, (53.40 ± 8.12)% and (68.00 ± 4.26)% at 24, 48 and 72 hours, respectively. Significance, P <0.001. The apoptosis rates of SKOV3 / ADM cells in ADM / PEG / SWCNTs group and ADM group were (14.83 ± 1.86)% and (5.28 ± 0.45)%, F = 137.292, P = 0.001 by flow cytometry. Hoechst33258 staining ADM / PEG / SWCNTs group showed obvious apoptotic morphological changes. ADM / PEG / SWCNTs group could down-regulate the expression of MDR-1mRNA and P-gp protein, the gray scale scanning analysis showed that the difference between the two groups was statistically significant, F = 18.634, P = 0.003. Conclusion ADM / PEG / SWCNTs nanocomposites prepared in vitro have high drug loading rate, inhibit the proliferation of multidrug-resistant human ovarian cancer cells and promote the apoptosis of ADM-resistant human ovarian cancer cells, down-regulate the expression of MDR-1 mRNA and P-gp protein expression.