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二至丸合桂枝汤对三阴性乳腺癌细胞系顺铂耐药性的调控作用
杨慧芬1      毛娟娟2
1.浙江省杭州市中医院乳腺科,浙江杭州   310007;
2.浙江省宁波市中医院外二科,浙江宁波   315012
Regulating effect of Erzhi Pills and Guizhi Decoction on Cisplatin resistance in triple negative breast cancer cell lines
YANG Huifen1   MAO Juanjuan2
1.Department of Galactophore, Hangzhou Hospital of Traditional Chinese Medicine, Zhejiang Province, Hangzhou   310007, China; 
2.the Second Department of Surgery, Ningbo Hospital of Traditional Chinese Medicine, Zhejiang Province, Ningbo   315012, China
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摘要 目的 研究二至丸合桂枝汤对三阴性乳腺癌(TNBC)细胞系顺铂(CDDP)耐药性的影响。 方法 选取TNBC细胞株MDA-MB-231,给予0.01、0.1、0.5、1、5、10 μmol/L的CDDP干预,制备CDDP耐药细胞株(MDA-MB-231/CDDP),用CCK-8检测不同浓度(0.01、0.1、0.5、1、5、10 μmol/L)CDDP干预下MDA-MB-231与MDA-MB-231/CDDP的细胞活性,同时以未加入CDDP的样孔作为对照组。并用定量反转录PCR(qRT-PCR)、Western blot检测其血管内皮生长因子(VEGF)、缺氧诱导因子-1α(HIF-1α)mRNA和蛋白表达情况,以验证制备的MDA-MB-231/CDDP。MDA-MB-231/CDDP细胞分为空白组(不做任何处理)、二至丸合桂枝汤组(200 μg/ml二至丸合桂枝汤)、CDDP组(10 μmol/L CDDP)、联合组(200 μg/ml二至丸合桂枝汤+10 μmol/L CDDP)。CCK-8和流式细胞术检测各组细胞活力及凋亡。qRT-PCR、Western blot测定各组VEGF、HIF-1α的mRNA及蛋白表达。 结果 MDA-MB-231和MDA-MB-231/CDDP细胞对CDDP的IC50值分别为1.6001 μmol/L和36.4320 μmol/L。CDDP浓度为10 μmol/L的MDA-MB-231、MDA-MB-231/CDDP细胞中VEGF、HIF-1α mRNA表达水平均低于对照组,CDDP浓度为10 μmol/L的MDA-MB-231/CDDP细胞中VEGF mRNA表达水平高于MDA-MB-231细胞,差异有统计学意义(P < 0.05或P < 0.01)。CDDP浓度为10 μmol/L的MDA-MB-231细胞中HIF-1α、VEGF蛋白表达水平低于对照组,MDA-MB-231/CDDP细胞HIF-1α蛋白表达水平低于对照组,差异有统计学意义(P < 0.05或P < 0.01),CDDP浓度为10 μmol/L的MDA-MB-231/CDDP细胞中HIF-1α、VEGF蛋白表达水平高于MDA-MB-231细胞,差异有统计学意义(P < 0.05)。作用24 h后,CDDP组和联合组细胞存活率均低于空白组,联合组细胞存活率低于CDDP组和二至丸合桂枝汤组,差异有统计学意义(P < 0.05或P < 0.01)。作用48 h后,联合组、CDDP组和二至丸合桂枝汤组细胞存活率均低于空白组,联合组细胞存活率低于CDDP组和二至丸合桂枝汤组,差异有高度统计学意义(P < 0.01)。二至丸合桂枝汤组、CDDP组和联合组细胞凋亡率均高于空白组,差异有高度统计学意义(P < 0.01),联合组细胞凋亡率高于二至丸合桂枝汤组及CDDP组,差异有统计学意义(P < 0.05或P < 0.01)。CDDP组和联合组MDA-MB-231/CDDP细胞中HIF-1α、VEGF mRNA及蛋白表达水平均低于空白组,联合组低于二至丸合桂枝汤组,差异有统计学意义(P < 0.05或P < 0.01)。 结论 二至丸合桂枝汤可通过调控HIF-1α信号通路,下调VEGF表达,逆转TNBC细胞系CDDP耐药性。
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杨慧芬1 毛娟娟2
关键词 三阴性乳腺癌二至丸合桂枝汤顺铂耐药性    
Abstract:Objective To study the effect of Erzhi Pills and Guizhi Decoction on Cisplatin (CDDP) resistance of triple negative breast cancer (TNBC) cell lines. Methods TNBC cell line MDA-MB-231 was selected and treated with 0.01, 0.1, 0.5, 1, 5, 10 μmol/L CDDP to prepare CDDP resistant cell line (MDA-MB-231/CDDP). The cell activities of MDA-MB-231 and MDA-MB-231/CDDP were detected by CCK-8 at different concentrations (0.01, 0.1, 0.5, 1, 5, 10 μmol/L) of CDDP. At the same time, the sample wells without CDDP were used as control group. The mRNA and protein expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-lα (HIF-1α) were detected by quantitative reverse transcriptase-mediated PCR(qRT-PCR) and Western blot to verify the prepared MDA-MB-231/CDDP. MDA-MB-231/CDDP cell lines were divided into blank group (without any treatment), Erzhi Pills and Guizhi Decoction group (200 μg/ml Erzhi Pills and Guizhi Decoction), CDDP group (10 μmol/L CDDP), and combined group (200 μg/ml Erzhi Pills and Guizhi Decoction +10 μmol/L CDDP). CCK-8 and flow cytometry were used to detect cell viability and apoptosis. The mRNA and protein expressions of VEGF and HIF-1α were determined by qRT-PCR and Western blot. Results The IC50 values of MDA-MB-231 and MDA-MB-231/CDDP cells to CDDP were 1.6001 μmol/L and 36.4320 μmol/L, respectively. The mRNA expression levels of VEGF and HIF-1α in MDA-MB-231 and MDA-MB-231/CDDP cells with CDDP concentration of 10 μmol/L were lower than those in the control group, the mRNA expression level of VEGF in MDA-MB-231/CDDP cells with CDDP concentration of 10 μmol/L was higher than that in MDA-MB-231 cells, and the differences were statistically significant (P < 0.05 or P < 0.01). The protein expression levels of HIF-1α and VEGF in MDA-MB-231 cells with CDDP concentration of 10 μmol/L were lower than those in the control group, and the protein expression levels of HIF-1α in MDA-MB-231/CDDP cells were lower than those in the control group, with statistical significance (P < 0.05 or P < 0.01). The protein expression levels of HIF-1α and VEGF in MDA-MB-231/CDDP cells with CDDP concentration of 10 μmol/L were higher than those in MDA-MB-231 cells, and the differences were statistically significant (P < 0.05). The survival rates of CDDP group and combined group 24 h after treatment were lower than those of blank group, and the cell survival rate of combined group was lower than that of CDDP group and Erzhi Pills and Guizhi Decoction group, the differences were statistically significant (P < 0.05 or P < 0.01). the cell survival rate of Erzhi Pills and Guizhi Decoction group, CDDP group and combined group 48 h after treatment was lower than that of blank group, and the cell survival rate of combined group was lower than that of CDDP group and Erzhi Pills and Guizhi Decoction group, the differences were highly statistically significant (P < 0.01). The apoptosis rate of Erzhi Pills and Guizhi Decoction group, CDDP group and combined group was higher than that of blank group, and the differences were highly statistically significant (P < 0.01). The apoptosis rate of combined group was higher than that of Erzhi Pills and Guizhi Decoction group and CDDP group, and the differences were statistically significant (P < 0.05 or P < 0.01). The mRNA and protein expression levels of HIF-1α and VEGF in MDA-MB-231/CDDP cells in CDDP group and combined group were lower than those in blank group, and those in combined group were lower than those in Erzhi Pills and Guizhi Decoction group, the differences were statistically significant (P < 0.05 or P < 0.01). Conclusion Erzhi Pills and Guizhi Decoction can reverse CDDP resistance of TNBC cell line by regulating HIF-1α signaling pathway and down-regulating the VEGF expression.
Key wordsTriple negative breast cancer    Erzhi Pills and Guizhi Decoction    Cisplatin    Drug resistance
    
基金资助:浙江省中医药科技计划项目(2020ZB156)。
作者简介: 杨慧芬(1973.5-),女,硕士,副主任医师;研究方向:中西医结合外科。
引用本文:   
杨慧芬1 毛娟娟2. 二至丸合桂枝汤对三阴性乳腺癌细胞系顺铂耐药性的调控作用[J]. 中国医药导报, 2021, 18(25): 19-25.
YANG Huifen1 MAO Juanjuan2. Regulating effect of Erzhi Pills and Guizhi Decoction on Cisplatin resistance in triple negative breast cancer cell lines. 中国医药导报, 2021, 18(25): 19-25.
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