中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (10): 1514-1520.doi: 10.12307/2023.287

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

多发性骨髓瘤细胞条件培养液对人脐带间充质干细胞增殖和分化的影响

杨  姁1,王飞清2,赵嘉宁1,张弛可3,梁惠玲1,漆沙沙3,吴  丹1,刘燕青2,唐东昕2,刘  洋1,2,4,李艳菊3   

  1. 1贵州中医药大学,贵州省贵阳市   550025;2贵州中医药大学第一附属医院,贵州省贵阳市   550001;3贵州医科大学附属医院,贵州省贵阳市   550004;4中国医学科学院成体干细胞转化研究重点实验室,贵州省贵阳市   550004
  • 收稿日期:2022-03-02 接受日期:2022-05-19 出版日期:2023-04-08 发布日期:2022-09-07
  • 通讯作者: 刘洋,博士,副教授,贵州中医药大学,贵州省贵阳市 550025;贵州中医药大学第一附属医院,贵州省贵阳市 550001;中国医学科学院成体干细胞转化研究重点实验室,贵州省贵阳市 550004 李艳菊,博士,主任医师,贵州医科大学附属医院,贵州省贵阳市 550004
  • 作者简介:杨姁,女,1993年生,贵州省遵义市人,汉族,贵州中医药大学在读硕士,主要从事干细胞组织工程研究。
  • 基金资助:
    国家自然科学基金(31660326),项目负责人:刘洋;国家自然科学基金(82160519),项目负责人:李艳菊;国家自然科学基金后补助资金科研创新探索专项(2019YFC171250407),项目负责人:刘洋;国家自然科学基金后补助资金科研创新探索专项(2019YFC171250505),项目负责人:王飞清;中国博士后科学基金(2018M640938),项目负责人:刘洋;中国博士后科学基金(43XB3794XB),项目负责人:李艳菊;贵州省科学技术厅项目(黔科合基础[2016]1019),项目负责人:刘洋;贵州省科学技术厅项目(黔科合LH字[2017]7140号),项目负责人:刘燕青

Effects of multiple myeloma cell conditioned medium on proliferation and differentiation of human umbilical cord mesenchymal stem cells

Yang Xu1, Wang Feiqing2, Zhao Jianing1, Zhang Chike3, Liang Huiling1, Qi Shasha3, Wu Dan1, Liu Yanqing2, Tang Dongxin2, Liu Yang1, 2, 4, Li Yanju3   

  1. 1Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou Province, China; 2First Affiliated Hospital of Guiyang University of Traditional Chinese Medicine, Guiyang 550001, Guizhou Province, China; 3Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 4Key Laboratory of Adult Stem Cell Translational Research, Chinese Academy of Medical Sciences, Guiyang 550004, Guizhou Province, China
  • Received:2022-03-02 Accepted:2022-05-19 Online:2023-04-08 Published:2022-09-07
  • Contact: Liu Yang, MD, Associate professor, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou Province, China; First Affiliated Hospital of Guiyang University of Traditional Chinese Medicine, Guiyang 550001, Guizhou Province, China; Key Laboratory of Adult Stem Cell Translational Research, Chinese Academy of Medical Sciences, Guiyang 550004, Guizhou Province, China Li Yanju, MD, Chief physician, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:Yang Xu, Master candidate, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 31660326 (to LY); the National Natural Science Foundation of China, No. 82160519 (to LYJ); the Research Innovation and Exploration Fund of National Natural Science Foundation of China, No. 2019YFC171250407 (to LY); the Research Innovation and Exploration Fund of National Natural Science Foundation of China, No. 2019YFC171250505 (to WFQ); China Postdoctoral Science Foundation, No. 2018M640938 (to LY);  China Postdoctoral Science Foundation, No. 43XB3794XB (to LYJ); the grant from the Science and Technology Department of Guizhou Province, No. [2016]1019 (to LY); the grant from the Science and Technology Department of Guizhou Province, No. LH[2017]7140 (to LYQ)

摘要:

文题释义:
多发性骨髓瘤细胞条件培养液:人多发性骨髓瘤细胞在生长过程中会分泌各种生长因子、细胞因子等多种活性物质于培养液中,收集多发性骨髓瘤细胞株上清培养基,经离心和过滤处理后,称之为多发性骨髓瘤细胞条件培养液。
人脐带间充质干细胞:具有分化为成骨细胞、成软骨细胞和成脂肪细胞的潜能,且具有取材方便、易扩增、低免疫原性等特点。

背景:近年来,在肿瘤微环境中干细胞与肿瘤的相互作用已成为研究热点,但是多发性骨髓瘤细胞条件培养液对人脐带间充质干细胞影响的实验鲜有报道。
目的:探讨多发性骨髓瘤细胞条件培养液对人脐带间充质干细胞增殖、迁移及分化能力的影响。
方法:以组织块贴壁法提取人脐带间充质干细胞,同时培养并提取人多发性骨髓瘤RPMI8226和U266细胞上清液制备条件培养液。按培养基的不同进行细胞分组:对照组人脐带间充质干细胞仅用L-DMEM完全培养液培养;实验组人脐带间充质干细胞用20% RPMI8226细胞条件培养液或20% U266细胞条件培养液和80% L-DMEM完全培养液培养。培养24 h,通过CCK-8、EDU、结晶紫染色观察细胞增殖情况,划痕法评估细胞迁移情况,成骨成脂诱导实验检测细胞分化能力,Real-time PCR检测细胞中周期蛋白基因p16、p21 mRNA表达。
结果与结论:实验组细胞的增殖数量多于对照组(P < 0.05),迁移率高于对照组(P < 0.05),矿化结节染色面积小于对照组(P < 0.05),脂滴阳性染色面积大于对照组(P < 0.05);实验组的细胞周期基因p16、p21 mRNA表达低于对照组(P < 0.05)。结果表明,人多发性骨髓瘤细胞条件培养液可促进人脐带间充质干细胞的增殖、迁移和成脂分化,抑制成骨分化,可能与其抑制p16、p21基因表达有关。

https://orcid.org/0000-0001-6037-9727 (刘洋) 

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

关键词: 多发性骨髓瘤, 条件培养液, 人脐带间充质干细胞, 增殖, 分化

Abstract: BACKGROUND: In recent years, the interaction between stem cells and tumor in tumor microenvironment has become a hot topic. While few studies have reported the experiments of human umbilical cord mesenchymal stem cells in conditioned culture medium of human multiple myeloma cells.  
OBJECTIVE: To investigate the effects of conditioned medium of human multiple myeloma cells on proliferation, migration and differentiation of human umbilical cord mesenchymal stem cells.
METHODS:  Human umbilical cord mesenchymal stem cells were extracted by tissue block adherent method. At the same time, the supernatant of human multiple myeloma RPMI8226 and U266 cells was cultured and extracted to prepare conditioned culture medium. The cells were grouped according to different media: the control group was only cultured with L-DMEM complete culture medium. In experimental group, human umbilical cord mesenchymal stem cells were cultured with 20% RPMI8226 cell conditioned medium or 20% U266 cell conditioned medium and 80% L-DMEM complete medium. After 24 hours of culture, the proliferation of cells was analyzed by CCK8 assay, EDU, and crystal violet staining. The migration of cells was evaluated by the scrape method. Osteogenic and adipogenic induction experiments were used to detect cell differentiation. Real-time PCR was used to detect the mRNA expressions of cyclin genes p16 and p21 in cells.  
RESULTS AND CONCLUSION: The proliferation of cells in the experimental group was more than that in the control group (P < 0.05); the migration rate was higher than that in the control group (P < 0.05); the staining area of mineralized nodules was smaller than that of the control group (P < 0.05); and the positive staining area of lipid droplets was larger than that of the control group (P < 0.05); the mRNA expression of cell cycle genes p16 and p21 in the experimental group was lower than that in the control group (P < 0.05). These findings exhibit that conditioned culture medium of human multiple myeloma cells can promote the proliferation, migration, and adipogenic differentiation of human umbilical cord mesenchymal stem cells, and inhibit osteogenic differentiation. This may be associated with the inhibition of p16 and p21 gene expression.

Key words: multiple myeloma, conditioned medium, human umbilical cord mesenchymal stem cell, proliferation, differentiation

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