血清饥饿及融合培养对人牙髓细胞周期同步化和矿化活性的影响

目的:比较血清饥饿及融合培养对人牙髓细胞周期同步化及矿化活性的影响。方法:将人牙髓细胞分别培养至80%及100%融合后,使用含0.5%胎牛血清的培养基继续培养细胞24、48、72 h,使用流式细胞仪检测牙髓细胞的细胞周期。以100%融合培养后饥饿48 h的人牙髓细胞作为实验组,80%融合培养的细胞作为对照组,在基因水平检测碱性磷酸酶、Ⅰ型胶原、骨钙素的表达;在蛋白水平检测碱性磷酸酶活性。采用SPSS13.0软件包对数据进行统计学分析。结果:在人牙髓细胞达到100%融合后,经血清饥饿48 h的G0/G1期细胞比100%融合培养组以及血清饥饿组多（P<0.05）。在基因水平,实验组Ⅰ型胶原、骨钙素的表达与对照组无统计学差异, 但是能促进碱性磷酸酶的表达（P<0.05）,同时在蛋白水平也刺激了人牙髓细胞碱性磷酸酶的分泌（P<0.05）。结论:100%融合培养联合血清饥饿法使更多的人牙髓细胞周期同步于G0/G1期,能更好地促进人牙髓细胞矿化。

Effect of serum starvation and culture to confluence on cell cycle synchronization and mineralization of human dental pulp cells

PURPOSE: To compare the effect of serum starvation and culture to confluence on cell cycle synchronization and mineralization of human dental pulp cells (hDPCs). METHODS: HDPCs were cultured to 80% and 100% confluence respectively, and then cultured for 24, 48 and 72 hours by culture medium containing 0.5% fetal bovine serum(FBS). Cell cycle of hDPCs were identified by flow cytometry. Then hDPCs cultured by serum starvation for 48h after culturing to 100% confluence were used as the experimental group, and hDPCs cultured to 80% confluence were used as the control group. The expression of alkaline phosphatase(ALP), collagen type Ⅰ(COL-Ⅰ) and osteocalcin(OCN) was detected at gene level; activity of ALPase was detected at protein level. SPSS 13.0 software was used for statistical analysis. RESULTS: When hDPCs were cultured by serum starvation for 48h after culturing to 100% confluence, cells at G0/G1 stage were more than culture to 100% confluence and serum starvation group (P<0.05). At the genetic level, the expression of COL-Ⅰand OC in the experimental group was not statistically different from that of the control group, but can promote the expression of ALP(P<0.05), and stimulate the secretion of hDPCs at protein level at the same time (P<0.05). CONCLUSIONS: Culture to confluence combined serum starvation can synchronize more hDPCs at G0/G1 stage and promote mineralization of hDPCs.