抗菌肽对肝癌多药耐药细胞Bel-7402/ADM增殖和耐药性影响

郭姗; 赵瑞君; 程璟霞

doi:10.11847/zgggws1117948

抗菌肽对肝癌多药耐药细胞Bel-7402/ADM增殖和耐药性影响

Effect of antimicrobial peptides on proliferation and multidrug resistance in multidrug resistant human hepatocellular carcinoma Bel-7402/ADM cells

摘要

摘要:
目的检测抗菌肽对耐药细胞Bel-7402/ADM增殖和耐药性的影响，为探讨抗菌肽在肝癌治疗方面的前景和应用价值提供参考依据。
方法采用阿霉素浓度梯度递增法体外诱导建立人肝癌多药耐药细胞系Bel-7402/ADM，检测细胞对化疗药物的敏感性；提取家蝇幼虫抗菌肽，采用噻唑蓝比色法（MTT）检测抗菌肽对肝癌细胞的抑制作用，流式细胞仪检测抗菌肽对细胞周期分布和进入细胞内阿霉素浓度的影响，并采用荧光定量聚合酶链反应（PCR）检测抗菌肽对Bel-7402/ADM细胞多药耐药基因MDR1表达的影响。
结果建立了对阿霉素以及多种化疗药物均耐药的肝癌细胞系Bel-7402/ADM，Bel-7402/ADM对5 – 氟尿嘧啶、长春新碱、紫杉醇和阿霉素的耐药指数（RI）分别为15.94、8.45、8.11和10.96。抗菌肽对Bel-7402/ADM细胞的半数抑制浓度（IC₅₀）为（463.67 ± 16.93）μg/mL，低于对Bel-7402细胞IC₅₀的（596.34 ± 19.27）μg/mL（t = 8.959，P < 0.001）。抗菌肽作用后，Bel-7402和Bel-7402/ADM细胞均出现G₀/G₁期比例升高，S期比例下降；Bel-7402/ADM细胞对阿霉素的RI由10.96降至7.10，而Bel-7402细胞对阿霉素的敏感性无明显变化；Bel-7402/ADM细胞内阿霉素的平均荧光强度由（24.16 ± 3.53）增高至（48.27 ± 5.50）（t = 6.390，P = 0.003），而Bel-7402细胞荧光强度增高不明显；Bel-7402/ADM细胞多药耐药基因1（MDR1）的相对表达量由（1.749 ± 0.020）降至（1.071 ± 0.044）（t = 24.297，P < 0.001）。
结论肝癌多药耐药细胞Bel-7402/ADM对抗菌肽不耐药，且抗菌肽可逆转Bel-7402/ADM细胞的耐药性。

Abstract:
Objective To evaluate the effect of antibacterial peptides (AMPs) on proliferation and drug resistance in human hepatocellular carcinoma (HC) cells (Bel-7402/ADM) with multidrug resistance for providing evidences to possible application of AMPs in HC treatment.
Methods We established a MDR HC cell line with the administration of adriamycin in culture medium with gradient increment and then detected the cells′ sensitivity to chemotherapeutic drugs. The AMPs were extracted from Musca domestica larva. The inhibitive effect of the AMPs on HC was determined with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay; the effect of AMPs on cell cycle and concentration of intracellular adriamycin was detected with flow cytometry; and the effect of AMPs on the expression of multidrug resistance gene 1 (MDR1) in Bel-7402/ADM cells was determined with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).
Results Bel-7402/ADM cell line resistant to adriamycin and other chemotherapeutic drugs was established successfully and the resistance index (RI) of the Bel-7402/ADM cells to 5-fluorouracil, vincristine, paclitaxe, and adriamycin were 15.94, 8.45, 8.11, and 10.96, respectively. The 50% inhibitory concentration (IC₅₀) of AMPs against Bel-7402/ADM cells was 463.67 ± 16.93 μg/mL and was lower than that of AMPs against Bel-7402 cells (596.34 ± 19.27 μg/mL). After treatment with AMPs, the proportion of cells in G₀/G₁ phase was significantly increased but the proportion of cells in S phase declined in both Bel-7402 and Bel-7402/ADM cells; the RI of Bel-7402/ADM cells to adriamycin was decreased from 10.69 to 7.10, while the RI of Bel-7402 cells to adriamycin did not changed significantly; the average fluorescence intensity of adriamycin in Bel-7402/ADM cells was increased from 24.16 ± 3.53 to 48.27 ± 5.5, while that of adriamycin in Bel-7402 cells did not increase obviously; the relative expression of MDR1 in Bel-7402/ADM cells was decreased significantly from 1.749 ± 0.020 to 1.071 ± 0.044(t = 24.297, P < 0.001).
Conclusion The multidrug resistant human hepatocellular carcinoma Bel-7402/ADM cells are not resistant to AMPs and AMPs could reverse drug resistance of Bel-7402/ADM cells.

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