芝麻素对长春瑞滨诱导人肺腺癌A549细胞凋亡的影响

Effects of sesamin on apoptosis of human lung adenocarcinoma A549 cells induced by vinorelbine

[目的]探讨芝麻素对长春瑞滨诱导人肺腺癌A549细胞株凋亡的影响.[方法]选择人肺腺癌A549细胞株进行体外传代培养,制备成浓度为1.0×105个/mL的细胞悬浮液并接种于96孔板中.实验设空白组(加入DMEM培养液)、对照组(加入DMEM培养液及人肺腺癌A549细胞株)、芝麻素组(芝麻素10、20、30、40、50、60、70、80、90、100μg/mL)、长春瑞滨组(长春瑞滨5、10、15、20、25、30、35μg/mL)及联合用药组(加入IC50浓度的芝麻素和长春瑞滨,IC50为后续联合用药组实验药物浓度).空白组加入160μL的DMEM培养液,对照组及实验组各加入160μL已制备好的人肺腺癌A549细胞株悬浮液,待细胞株贴壁后,空白组、对照组及实验组分别加入20μL的生理盐水和20μL各相关浓度的药物.采用MTT法检测细胞增殖能力,利用倒置显微镜及HE染色法观察细胞形态学变化,采用流式细胞仪检测细胞凋亡情况.[结果]在10~100μg/mL质量浓度范围内,低质量浓度的芝麻素具有抑制A549细胞株增殖的作用,IC50质量浓度为40μg/mL;在5~35μg/mL质量浓度范围内,长春瑞滨具有抑制A549细胞株增殖的作用,且随着药物质量浓度升高细胞抑制率升高,IC50质量浓度为20μg/mL;芝麻素与长春瑞滨联合用药对A549细胞株的抑制率明显高于单药用药(P<0.01).倒置显微镜及HE染色法观察结果显示,与对照组比较,芝麻素(40μg/mL)、长春瑞滨(20μg/mL)及联合用药(芝麻素40μg/mL+长春瑞滨20μg/mL)作用于A549细胞株48 h后贴壁细胞数量均明显减少,细胞间连接疏松,贴壁能力减弱,部分细胞体积变小、变圆或呈不规则形,核染色质凝集,细胞膜起泡形成凋亡小体,失去原有肿瘤细胞多角形或梭形形态,且联合用药组较单药组上述变化更为明显.流式细胞仪检测结果显示,药物作用48 h后,芝麻素组(40μg/mL)、长春瑞滨组(20μg/mL)及联合用药组(芝麻素40μg/mL+长春瑞滨20μg/mL)细胞凋亡率均明显高于对照组(P<0.01),且联合用药组早期凋亡率明显高于单独用药组(P<0.01).[结论]芝麻素可增强长春瑞滨诱导人肺腺癌A549细胞凋亡的作用.

OBJECTIVE To explore the effect of sesamin combined with vinorelbine on apoptosis of human lung adenocarcinoma A549 cell line.METHODS Human lung adenocarcinoma A549 cell line was selected and cultured in vitro,prepared into a cell suspension with a concentration of 1.0×105 cells/mL and inoculated into a 96-well plate.The experiment consisted of blank group(adding DMEM culture medium),control group(adding DMEM culture medium and human lung adenocarcinoma A549 cell line),sesamin group(sesamin 10,20,30,40,50,60,70,80,90,100μg/mL),vinorelbine group(vinorelbine 5,10,15,20,25,30,35μg/mL)and combination group(adding sesamin and vinorelbine with IC50 concentration,IC50 was the experimental drug concentration of the subsequent combination group).The blank group was added with 160μL DMEM culture medium,the control group and the experimental group were added with 160μL prepared human lung adenocarcinoma A549 cell line suspension.After the human lung adenocarcinoma A549 cell line was adherent,20μL normal saline and 20μL drugs with related concentration were added to the blank group,the control group and the experimental group,respectively.MTT assay was used to detect the cell proliferation,inverted microscope and HE staining were used to observe the morphological changes of cells,and flow cytometry was used to detect cell apoptosis.RESULTS In the range of 10-100μg/mL,low concentration of sesamin had the inhibitory effect on the proliferation of A549 cell lines,with an IC50 concentration of 40μg/mL.In the range of 5-35μg/mL,vinorelbine inhibited the proliferation of A549 cell lines,and the cell inhibition rate increased with the increase of the mass concentration of vinorelbine,and the mass concentration of IC50 was 20μg/mL.The inhibition rate of sesamin combined with vinorelbine on A549 cell line was significantly higher than that of single drug(P<0.01).The observation results of inverted microscope and HE staining showed that compared with the control group,after treatment with sesamin(40μg/mL),vinorelbine(20μg/mL)and combined administration(sesamin 40μg/mL+vinorelbine 20μg/mL)for 48 h,the number of adherent cells of A549 cell line significantly decreased,with loose intercellular connections and weakened adherent ability,some cells became smaller,rounder,or irregularly shaped,with nuclear chromatin agglutination and cell membrane foaming to form apoptotic bodies,and lost the original polygonal or spindle shaped morphology of tumor cells,and the above changes were more obvious in the combined drug group than in the single drug group.Flow cytometry results showed that after 48 hours of drug action,the apoptosis rates of the sesame group(40μg/mL),vinorelbine group(20μg/mL)and combination group(sesamin 40μg/mL+vinorelbine 20μg/mL)were significantly higher than that of the control group(P<0.01),and the early apoptosis rate of the combination group was significantly higher than that of single treatment group(P<0.01).CONCLUSIONS Sesame can enhance the apoptosis of human lung adenocarcinoma A549 cells induced by vinorelbine.