成骨细胞与矿化液诱导牙髓干细胞向成骨细胞分化的对比研究

Research of osteoblasts and mineralization culture method induced dental pulp stem cells into osteoblasts

目的比较成骨细胞和矿化液对牙髓干细胞（dental pulp stem cells，DPSC）向成骨细胞分化的作用，选择理想的诱导方法。方法利用插入式小室将成骨细胞与DPSC共培养作为共培养组，矿化液培养DPSC作为矿化液组。在光学显微镜和透射电镜下观察DPSC形态学变化；应用茜素红染色法检测矿化结节；采用反转录聚合酶链反应（reverse transcription polymerase chain reaction，RT-RCR）分别检测DPSC骨涎蛋白、Runt相关转录因子2、骨钙蛋白、I型胶原等骨源性基因表达情况。结果茜素红染色显示培养28d后共培养组DPSC矿化结节明显多于矿化液组。培养15d后共培养组骨涎蛋白和I型胶原基因阳性表达水平（分别为9．807±1．135和2．913±0．310）显著高于矿化液组（分别为6．478±0．781和1．703±0．184，P〈0．05）。结论成骨细胞分泌大量细胞因子或可溶性蛋白，对DPSC的诱导作用较矿化液明显。

Objective To find an ideal method inducing dental pulp stem cells （DPSC） osteogenie differentiation. To compare the effect of co-culture method and that of mineralizing culture medium. Methods DPSC were co-cultured with osteoblasts using cell culture inserts system as experiment group, and DPSC were cultured in mineralizing culture medium as control group. The cell morphology and ultrastructure and mineralized nodes were analyzed under phase contrast microscope, transmission electron microscope, and alizarin red S staning. Bone sialoprotein （BSP）, Runx-2, osteocalcin, and collagen-1 （Col-1） osteoblastic genes expressions of DPSC cultivated in special niche of osteoblasts were assayed by reverse transcription polymerase chain reaction （RT-PCR）. Results The mineralization nudoles of experiment group were more than control group. Fifteen days later, BSP and Col-1 genes in the DPSC of cocultures were 9. 807 ± 1. 135 and 2. 913 ± 0. 310, respectively. And those in the DPSC of mineralizing culture medium were 6. 478 ± 0. 781 and 1. 703 ±0. 184, respectively. Co-cultures and mineralizing were significantly different （P 〈 0.05 ）. Conclusions As osteoblasts can secret lots of osteogenic cell cytokines, they have more significant effect than mineralizing culture medium on osteogenesis of DPSC.