双氢青蒿素单独和联合甲氨蝶呤在皮肤T细胞淋巴瘤细胞系的抗肿瘤作用  

Anti-tumor Effects of Dihydroartemisinin Alone and in Combination with Methotrexate in Cutaneous T-cell Lymphoma Cells

作　　者：张芊[1] 王艺萌[1] 姜蔚蔚 谷晓广[1] 张春雷[1] Zhang Qian;Wang Yimeng;Jiang Weiwei;Gu Xiaoguang;Zhang Chunlei(Peking Univesity Third Hospital,Beijing 100191,China)

机构地区：[1]北京大学第三医院,北京100191

出　　处：《中国中西医结合皮肤性病学杂志》2018年第2期112-117,共6页Chinese Journal of Dermatovenereology of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金面上项目(81372915);国家自然科学基金青年项目(81402259);北京大学第三医院院临床重点项目(BYSY201209)

摘　　要：目的研究双氢青蒿素(Dihydroartemisinin,DHA)单独和联合甲氨蝶呤(Methotrexate,MTX)对皮肤T细胞淋巴瘤(Cutaneous T cell lymphoma,CTCL)细胞的增殖抑制和凋亡诱导作用,并探讨其分子机制。方法分别采用不同浓度DHA,5.0μmol/L MTX,5.0μmol/L DHA联合5.0μmol/L MTX处理CTCL细胞系HH细胞24、48或72 h后,采用MTS法检测HH细胞的生存率。用流式细胞仪检测细胞周期及细胞凋亡情况。用Western印迹法检测处理HH细胞48 h后凋亡相关基因和DNA损伤相关基因的蛋白表达。统计分析采用SPSS 21.0软件,双侧P<0.05为差异有统计学意义。结果 DHA能明显抑制HH细胞的增殖,并且有时间依赖性和剂量依赖性,5.0μmol/L DHA联合5.0μmol/L MTX对细胞增殖的抑制作用明显强于5.0μmol/L MTX以及5.0μmol/L DHA(P<0.05)。流式细胞仪结果显示,随着DHA浓度增加,作用时间延长,HH细胞凋亡比例不断增加,联合组的凋亡细胞比例均显著高于5.0μmol/L DHA组和5.0μmol/L MTX组(P<0.05)。此外,各浓度DHA组、5.0μmol/L MTX组、联合组的G0/G1期细胞比例明显高于溶剂对照组,而其G2/M期细胞比例均明显降低(P<0.05)。Western印迹结果显示,联合组与DHA组、MTX组、对照组比较,凋亡相关基因p53、Noxa、Bax、caspase 3和DNA损伤相关基因γ-H2AX、ATM蛋白的表达明显增加。结论 DHA通过抑制细胞增殖和促进细胞凋亡来抑制CTCL细胞系生长,联合MTX能明显提高抑制CTCL细胞的生长率。Objective In order to evaluate the anti-tumor effects of dihydroartemisinin(DHA)alone and in combination with methotrexate(MTX)in cutaneous T-cell lymphoma(CTCL)cell lines.Methods HH cells were incubated with different concentrations of DHA and 5.0μmol/L MTX alone or combined with 5.0μmol/L DHA for 24,48 and 72 hours respectively.MTS assay was conducted to estimate cell viability at each time point.Flow cytometry was performed to detect cell apoptosis and analyze cell cycle.Western-blotting analysis was conducted to quantify protein expressions of apoptosis-associated genes as well as DNA damage related genes respectively.Statistical analysis was performed using SPSS 21.0 software,and bilateral P<0.05 was considered statistically significant.Results DHA could significantly inhibit the proliferation of HH cells in a time-dependent and dose-dependent manner(both P<0.05).After cultured for 24,48 and 72 hours the combination of 5.0μmol/L DHA and 5.0μmol/L MTX showed significantly stronger inhibitory effects on cell proliferation,compared with 5.0μmol/L MTX or 5.0μmol/L DHA group(all P<0.05).The results of flow cytometry showed that with the increasing concentration of DHA,the proportion of apoptotic HH cells increased,and that the percentage of apoptotic cells in the combination treatment group was significantly higher than in the 5.0μmol/L DHA group or 5.0μmol/L MTX group(all P<0.05).In addition,compared with the control group,all treatment groups showed significantly increased proportion of cells at G0/G1 phase and decreased G2/M phase(all P<0.05).The protein expression of p53,Noxa,Bax,caspase 3,γ-H2AX,and ATM significantly increased in the combination group compared with those in the 5.0μmol/L DHA group,the 5.0μmol/L MTX group and the control group.Conclusion DHA can inhibit the growth of CTCL cells by inhibiting cell proliferation and inducing cell apoptosis,and in combination with MTX can markedly increase the inhibition of growth rate of CTCL cells.

关 键 词：皮肤T细胞淋巴瘤 双氢青蒿素 甲氨蝶呤 细胞增殖 细胞凋亡 

分 类 号：R739.91[医药卫生—肿瘤]