【摘要】 目的 :研究人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)与经骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)诱导后的人牙髓细胞(human dental pulp cells,hDPCs)共培养对细胞生物学特性的影响。方法:分别取原代培养的hUCMSCs和hDPCs,分别通过流式细胞术、成骨诱导、成脂诱导以及免疫组织化学染色鉴定细胞;使用BMP2诱导hDPCs,14 d后检查其DSPP、ALP、DMP1的mRNA表达情况;按照1∶1、1∶5、5∶1的比例直接共培养hUCMSCs与经BMP2诱导后的hDPCs,实时定量PCR检测其DSPP、ALP、DMP1、OCN、VEGF、HGF、Nanog的mRNA表达情况。根据实时定量PCR结果选取1∶1组与单独培养hUCMSCs组、hDPCs组比较,分别培养21 d后进行茜素红染色,在酶标仪上于562 nm处检测沉淀物形成情况。采用SPSS21.0软件包对数据进行统计学分析。结果:直接共培养14 d后,1∶1细胞组的DSPP、ALP、DMP1、OCN、VEGF、HGF的mRNA表达量比单独培养的hUCMSCs组显著升高(P<0.05),而Nanog mRNA表达量比单独培养的hUCMSCs组降低(P<0.05)。茜素红染色结果显示,1∶1组的OD值显著高于hUCMSCs组(P<0.05)。结论:将hUCMSCs与经BMP2诱导后的hDPCs按照1∶1比例共培养,可以诱导细胞向成牙本质细胞方向分化,并促进血管生成因子的表达。更多还原

【Abstract】 PURPOSE: To investigated the effect of human umbilical cord mesenchymal stem cells(hUCMSCs) and human dental pulp cells(hDPCs) on cell biological behaviors by co-culture system in vitro. METHODS: hUCMSCs and hDPCs were obtained by primary culture. A culture system of hUCMSCs and hDPCs induced by BMP2 was established in vitro. hUCMSCs and hDPCs were co-cultured at the ratio of 1:1, 1:5 and 5:1. The optimum ratio of each group was selected to further experiment. The formation of calcium nodule was stained by alizarin red staining at 21 day. The expression of DSPP,ALP,DMP1,OCN,VEGF,HGF and Nanog gene was detected by real-time quantitative PCR at 7 day and 14 day. 1:1 group and hUCMSCs, hDPCs group were selected for alizarin red staining at 21 day according to PCR results. Statistical analysis was performed using SPSS 21.0 software package. RESULTS: Calcified nodules formation in 1:1 group was significantly higher than in hUCMSCs group(P<0.05), close to that in hDPCs. q PCR showed that the mRNA expression of DSPP, ALP, DMP1, OCN, VEGF and HGF in 1:1 group was significantly higher than that in hUCMSCs(P<0.05); mRNA expression of Nanog in 1:1 group was significantly lower than in hUCMSCs group(P<0.05). The results of alizarin red staining showed that the OD value of 1:1 group was significantly higher than that of hUCMSCs group(P<0.05).CONCLUSIONS: The cells can be induced to differentiate into odontoblastoid-like cells and the mRNA expression of angiogenic factors was stimulated by hUCMSCs co-culure wih hDPCs.更多还原