骨桥蛋白对人牙髓干细胞成骨分化的旁分泌作用

Paracrine Function of Osteopontin on the Osteogenic Differentiation of Dental Pulp Stem Cells

目的:研究骨桥蛋白(osteopontin,OPN)对牙髓干细胞(dental pulp stem cells,DPSCs)成骨分化的旁分泌作用。方法:利用四甲基偶氮唑盐(MTT)计数测定不同浓度OPN对细胞增殖的影响;测定DPSCs经不同浓度OPN诱导后的碱性磷酸酶(ALP)活性;选取适宜的诱导浓度,在倒置相差显微镜下观察诱导后的DPSCs形态变化;应用茜素红染色法检测矿化结节的形成;采用反转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-RCR)分别检测DPSCs骨涎蛋白(BSP)、Runt相关转录因子2(Runx-2)、骨钙蛋白(OCN)、Ⅰ型胶原(Col-1)等骨源性基因表达情况。结果:MTT计数及ALP活性测定实验表明,随着OPN浓度增加,ALP活性增强,但细胞增殖率降低。茜素红染色显示OPN诱导培养28d后出现矿化结节。OPN诱导培养7d后DPSCs骨源性基因表达均成阳性。其中BSP、Runx-2、Col-1及OCN等基因表达水平均明显高于对照组。结论:OPN对DPSCs的旁分泌作用是促进其成骨向分化。

Objective： To study the paracrine function of Osteopontin （OPN） on the osteogenic differentiation of dental pulp stem cells （DPSCs）. Methods： Methyl thiazolyl tetra - zolium （MTT） assay was used to evaluate the proliferation rate of DPSCs induced by different concentrations of OPN. The activity of Alkaline phosphatase （ALP） after induced by different concentration of OPN was measured. We select appropriate induction concentration, observe the morphological changes of DPSCs by the inverted phase contrast microscope, detect the mineralize nodules stained by Alizarin red, detect the expression levels of osteogenic related genes including bone sialoprotein （BSP） , Runt- related transcription factor 2 （Runx- 2）, osteocalcin （OCN） and collagen-1 （Col-1） by reverse transcription polymerase chain reaction （RT-RCR）. Results： The results of MTT and ALP activity measurement showed that ALP activity increased and cell proliferation rate dropped with the increase of concentration of OPN. Alizarin red staining showed that mineralized nodules have formed after induced by OPN for 28 day. The expression of osteogenic related genes is positive after 7 days. Expression levels of BSP, Runx - 2, Col-land OCN genes were significantly higher than the control group. Conclusion： The paracrine interaction of OPN contributed to osteogenic differentiation of DPSCs.