不同来源条件培养基对人牙髓干细胞增殖的影响

Effects of conditioned media of different sources on the proliferation of human dental pulp stem cells

背景:利用条件培养基与人牙髓干细胞共培养,初步探索可以快速促进人牙髓干细胞增殖的方法,为今后细胞治疗、扩增高质量种子细胞提供研究基础。目的:初步探究不同来源条件培养基对人牙髓干细胞增殖的影响。方法:体外分离培养人牙髓干细胞,并用流式细胞术鉴定。然后将胎牛血清超高速低温离心产物、骨折患者尿液超高速低温离心产物、骨髓来源树突状细胞体外培养上清液、人皮肤成纤维细胞培养上清液与低糖DMEM培养液配置成条件培养基后,再分别与人牙髓干细胞共培养,使用细胞无标记培养观察装置(BioStation-T)连续拍摄各组细胞生长72 h的照片、使用实时无标记细胞分析仪(RTCA)动态监测150 h,比较各组细胞的标准化细胞指数值的差异。结果与结论:①人牙髓干细胞为典型的间充质干细胞形态,表达间充质干细胞表面标志物CD90和CD105,不表达CD34和CD45;②细胞无标记培养观察装置动态监测时,发现胎牛血清超高速低温离心组的颗粒物质被吸收时间要显著早于其他实验组和空白对照组;③在实时无标记细胞分析仪检测时,与空白对照组相比,胎牛血清超高速低温离心组、骨髓来源树突状细胞体外培养上清液组、人皮肤成纤维细胞培养上清液组的标准化细胞指数值均显著升高(P<0.001),骨折患者尿液超高速低温离心组的标准化细胞指数值显著降低(P<0.001);④结果表明,胎牛血清超高速低温离心产物、骨髓来源树突状细胞体外培养上清液、人皮肤成纤维细胞培养上清液所配置的条件培养基能促进人牙髓干细胞增殖,其中胎牛血清超高速低温离心产物所配置的条件培养基对细胞还有一定保护作用。

BACKGROUND:Using conditioned medium and human pulp stem cells,we will initially explore methods to rapidly proliferate human dental pulp stem cells to provide a research basis for future cell treatment and expansion of high-quality seed cells.OBJECTIVE:To preliminarily explore the effect of conditioned medium from different sources on the proliferation of human dental pulp stem cells.METHODS:Human dental pulp stem cells were isolated in vitro and identified by flow cytometry.Fetal bovine serum ultra-high-speed cryogenic centrifugal products,urine ultra-high-speed cryogenic centrifugal products of fracture patients,in vitro culture supernatants of bone marrow-derived dendritic cells,human skin fibroblast cultures and low-glucose DMEM medium were configured into conditioned medium.After co-culture with human pulp stem cell cells,images of cell growth in each group were taken continuously for 72 hours using a cell label-free culture observation device(BioStation-T).The cells were dynamically monitored using a real-time label-free cell analyzer for 150 hours.Differences in normalized cell index values were compared between each group of cells.RESULTS AND CONCLUSION:(1)Human dental pulp stem cells are typical of mesenchymal stem cells.Expression of the mesenchymal stem cell surface markers CD90 and CD105 was found,but CD34 and CD45 expression was not observed.(2)Cell label-free culture observation device when dynamically monitoring found that the absorption time in the group was significantly earlier than in other experimental and blank control groups.(3)When detected on a real-time label-free cell analyzer,compared with the blank control group,the normalized cell index values of the fetal bovine serum ultra-high-speed cryogenic centrifugal group,the bone marrow-derived dendritic cell in vitro culture supernatant group,and the human skin fibroblast culture supernatant group were significantly increased(P<0.001).The normalized cell index value of fracture patients of the urine ultra-high-speed cryogenic centrifugal group was significantly decreased(P<0.001).(4)The results show that the conditioned medium configured by ultra-high-speed centrifugation,in vitro culture supernatant of bone marrow-derived dendritic cells,and human skin fibroblasts can promote the proliferation of human dental pulp stem cells.Among them,the conditioned medium configured by the fetal bovine serum ultra-high-speed cryogenic centrifugal products also had some protective effects on the cells.