干性增强的小鼠胚胎成纤维细胞来源的挤压囊泡抑制多种肿瘤细胞生长

Stemness-enhanced mouse embryonic fibroblast-derived extruded vesicles inhibit multiple tumor cell growth

目的:探究小分子化合物联合3D培养对小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)干性的影响,及其挤压囊泡对不同肿瘤细胞生长的影响。方法:首先,分别采用不同浓度的小分子药物β-烟酰胺单核苷酸(nicotinamide mononucleotide,NMN)、甜菜碱、雷帕霉素和维生素C在2D培养条件下处理MEF,实时定量荧光PCR检测八聚体结合转录因子4(octamer-binding transcription factor 4,Oct4)mRNA表达水平,筛选出促进MEF干性的最适浓度;将4种药物按最适作用浓度进行2种、3种、4种组合处理MEF,筛选最适组合。其次,将筛选出的药物组合联合3D培养,通过实时定量荧光PCR和蛋白质免疫印迹法分别检测干性指标Oct-4和Nanog的mRNA和蛋白表达水平,筛选出促进MEF干性的最适组合培养方法。最后,再利用挤压法将MEF制备成囊泡,并处理小鼠肝癌细胞Hepa1-6、肺癌细胞Lewis和胶质瘤细胞GL261,CCK8法分别检测囊泡对3种肿瘤细胞和MEF生长的影响。结果:NMN、甜菜碱和雷帕霉素促进MEF干性的最适浓度为10μmol/L,维生素C的最适浓度为100μmol/L;NMN+甜菜碱组合促进MEF干性的效果最强。与2D培养组相比,NMN+甜菜碱联合3D培养组MEF Oct-4 mRNA表述水平明显增高,提高近600倍(P<0.05)。蛋白质免疫印迹结果显示,甜菜碱+维生素C、NMN+甜菜碱、雷帕霉素+甜菜碱等联合3D培养组Oct-4、Nanog蛋白表达水平明显升高,其中NMN+甜菜碱联合3D培养组表达水平最高(P<0.05)。NMN+甜菜碱联合3D培养组细胞挤压囊泡明显抑制小鼠肝癌细胞Hepa1-6、胶质瘤细胞GL261和肺癌细胞Lewis生长(P<0.05),而对MEF的抑制作用不明显。结论:NMN+甜菜碱联合3D培养能够明显增强MEF干性,其挤压囊泡能够明显抑制Hepa1-6、Lewis和GL261等肿瘤细胞生长。

Objective:To explore the effect of small molecule compound combined with 3D culture on the stemness of mouse embryonic fibroblast(MEF),and the effect of extruded vesicles on the growth of different tumor cells.Methods:First,MEFs were treated with different concentrations of small molecule drugsβ-nicotinamide mononucleotide(NMN),betaine,rapamycin and vitamin C under 2D culture conditions,and Oct-4 mRNA expression level was detected by real-time quantitative PCR,and the optimal concentration for promoting cell stemness was screened out;2,3,4 combinations of 4 drugs were used to treat MEF according to the optimal concentration,and the optimal combination was screened.Secondly,the selected chemical combinations were combined with 3D culture,and the mRNA and protein expression levels of stemness indicators Oct-4 and Nanog were detected by real-time quantitative PCR and Western blotting,respectively,and the optimal combination for promoting cell stemness was screened out.Finally,MEFs were prepared into vesicles by extrusion method,and Hepa1-6,Lewis and GL261 tumor cells were treated with vesicles,and the effect of vesicles on the growth of several tumor cells and MEFs was detected by CCK8 method,respectively.Results:The optimal concentration of NMN,betaine and rapamycin on MEF stemness promotion was 10μmol/L,and the optimal concentration of vitamin C was 100μmol/L;the combination of NMN+betaine had the strongest effect on promoting MEFs stemness.Compared with the 2D culture group,the expression level of Oct-4 mRNA in the NMN+betaine combined with 3D culture group was significantly increased by nearly 600 times(P<0.05).The results of Western blotting showed that the expression levels of Oct-4 and Nanog in the combined 3D culture group of betaine+vitamin C,NMN+betaine,rapamycin+betaine,ect.were significantly increased,and the expression level in NMN+betaine combined 3D culture group was the highest(P<0.05).The extruded vesicles of NMN+betaine combined with 3D culture group greatly inhibited the growth of mouse hepatoma cells Hepa1-6,glioma cells GL261 and lung cancer cells Lewis(P<0.05),but the inhibitory effect on MEFs was not obvious.Conclusion:NMN+betaine combined with 3D culture could significantly enhanced the stemness of MEFs,and its extruded vesicles could markedly inhibit the growth of Hepa1-6,Lewis and GL261 tumor cells.