Abstract：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 drugs β-nicotinamide mononucleotide (NMN), betaine, rapamycin and vitamin C under 2D culture conditions, and Oct-4 mRNA expression level was detected by realtime 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 realtime 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 10 μmol/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.
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收稿日期: 2022-02-10
基金资助:国家自然科学基金资助项目(81772694)
通讯作者:
龚爱华(通讯作者)，教授，博士生导师，E-mail: ahg5@163.com
作者简介: 房文静(1997—)，女，硕士研究生；
引用本文:
房文静，杨梦婷，张俊尧，等.. 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[J]. 江苏大学学报:医学版, 2022, 32(05): 369-375.
FANG Wenjing, YANG Mengting, ZHANG Junyao, FEI Fei, LI Li, CHEN Qian, LI Zhangzuo, HOU Hanjin, GONG Aihua. Stemness-enhanced mouse embryonic fibroblast-derived extruded vesicles inhibit multiple tumor cell growth. Journal of Jiangsu University(Medicine Edition), 2022, 32(05): 369-375.
［1］Wang JJ, Lei KF, Han F. Tumor microenvironment: recent advances in various cancer treatments［J］.Eur Rev Med Pharmacol Sci, 2018, 22(12): 3855-3864.
［2］Liu J, Huang Z, Yang L, et al. Embryonic stem cells modulate the cancerpermissive microenvironment of human uveal melanoma［J］. Theranostics, 2019, 9(16): 4764-4778.
［3］Hou P, Li Y, Zhang X, et al. Pluripotent stem cells induced from mouse somatic cells by smallmolecule compounds［J］. Science, 2013, 341(6146): 651-654.
［4］Song J, Li J, Yang F, et al. Nicotinamide mononucleotide promotes osteogenesis and reduces adipogenesis by regulating mesenchymal stromal cells via the SIRT1 pathway in aged bone marrow［J］. Cell Death Dis, 2019, 10(5): 336.
［5］Sotthibundhu A, McDonagh K, von Kriegsheim A, et al.Rapamycin regulates autophagy and cell adhesion in induced pluripotent stem cells［J］. Stem Cell Res Ther, 2016, 7(1): 166.
［6］D′Aniello C, Cermola F, Patriarca EJ, et al. Vitamin C in stem cell biology: impact on extracellular matrixhomeostasis and epigenetics［J］. Stem Cells Int, 2017, 8936156.
［7］Ylostalo JH. 3D stem cell culture［J］.Cells, 2020, 9(10): 2178.
［8］Swain N, Thakur M, Pathak J, et al. SOX2, OCT4 and NANOG: The core embryonic stem cell pluripotency regulators in oral carcinogenesis［J］. J Oral Maxillofac Pathol, 2020, 24(2): 368-373.
［9］Grubelnik G, Botjancic E, Pavlic A, et al. NANOG expression in human development and cancerogenesis［J］. Exp Biol Med（Maywood), 2020, 245(5): 456-464.
［10］Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cellderived extracellularvesicles: novel frontiers in regenerative medicine［J］. Stem Cell Res Ther, 2018, 9(1): 63.
［11］Dong L, Pu Y, Zhang L. Human umbilical cord mesenchymal stem cellderived extracellular vesicles promote lung adenocarcinoma growth by transferring miR410［J］. Cell Death Dis, 2018, 9(2): 218.
［12］丁超，董利阳，郑婷婷，等.人脐带间充质干细胞培养上清液对甲状腺乳头状癌细胞诱导人脐静脉内皮细胞血管形成的影响［J］.江苏大学学报(医学版), 2020, 30（3): 229-233.
［13］Wang X, Hu S, Li J, et al. Extruded mesenchymal stem cell nanovesicles are equally potent to natural extracellular vesicles in cardiac repair［J］. ACS Appl Mater Interfaces, 2021, 13(47): 55767-55779.
［14］Ye J, Ge J, Zhang X, Cheng L,et al. Pluripotent stem cells induced from mouse neural stem cells and small intestinal epithelial cells by small molecule compounds［J］.Cell Res, 2016, 26(1): 34-45.
［15］Zheng J, Choi KA, Kang PJ, et al. A combination of small molecules directly reprograms mouse fibroblasts into neural stem cells［J］. Biochem Biophys Res Commun, 2016, 476(1): 42-48.
［16］Yuan Y, Zhang M, Yan G, et al.Nanog promotes stemlike traits of glioblastoma cells［J］.Front Biosci, 2021, 26(3): 552-565.
［17］Mohiuddin IS, Wei SJ, Kang MH.Role of OCT4 in cancer stemlike cells and chemotherapy resistance［J］.Biochim Biophys Acta Mol Basis Dis, 2020,1866(4): 165432.
［18］Paolillo M, Colombo R, Serra M, et al. Stemlike cancer cells in a dynamic 3D culture system: a model to study metastatic cell adhesion and anticancer drugs［J］. Cells, 2019, 8(11): 1434.
［19］Kalimuthu S, Gangadaran P, Rajendran RL, et al. A new approach for loading anticancer drugs into mesenchymal stem cellderived exosome mimetics for cancer therapy［J］. Front Pharmacol, 2018, 9: 1116.
