BMP-2促进人炎症牙髓干细胞骨向诱导分化的体外研究

Studies of BMP-2 promote osteogenic differentiation of human inflammatory dental pulp stem cells in vitro

目的:研究人骨形态发生蛋白-2(bone morphogenetic protein,BMP-2)对人炎症牙髓组织来源的牙髓干细胞(dental pulp stem cells from inflamed pulps,DPSCs-IPs)体外骨向诱导分化的影响。方法:取第三代DPSCs-IPs,按是否于培养基中加入BMP-2分成:BMP-2+DPSCs-IPs组(实验组)和DPSCs-IPs组(对照组)。无成骨诱导条件培养1周后,对各组分泌的胶原基质行Trichrome染色,观察并比较实验组和对照组之间的骨向诱导分化情况;实时定量RT-PCR检测二者的Ⅰ型胶原蛋白(collagenⅠ,COL-Ⅰ)mRNA表达情况。在成骨诱导条件下,培养3周后对各组分泌的钙化基质行Von Kossa染色,通过实时定量RT-PCR检测转录因子及成骨相关基因的表达。结果:无成骨诱导培养1周后,实验组DPSCs-IPs分泌的胶原基质Trichrome染色较对照组深,COL-ⅠmRNA的表达水平显著上调(P<0.05),说明BMP-2可诱导DPSCs-IPs沉积更多的胶原基质;成骨诱导培养3周后,相对于对照组,实验组DPSCs-IPs沉积了更多的钙化基质,Nanog、八聚体结合转录因子4(octamer-binding transcription factor 4,Oct4)、性别决定区因子(SRY-related high-mobility group box 2,Sox2)等转录因子表达升高,碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)、骨涎蛋白(bone sialoprotein,BSP),牙骨质蛋白1(cementum protein 1,CEMP-1)、COL-Ⅰ等成骨相关基因表达水平显著上调(P<0.05)。结论:BMP-2在成骨诱导条件下可促进DPSCs-IPs进行体外骨向诱导分化。

Objective：To investigate the influence of bone morphogenetic protein-2（BMP-2）on osteogenic differentiation of human inflammatory dental pulp stem cells（DPSCs-IPs）in vitro.Methods：Passage 3 cells dental pulp stem cells from inflamed teeth in medi-um with or without osteogenic matter.According to whether 100 μg /L BMP-2 was added they were divided into four groups：group 1 , DPSCs-IPs culturing with 100 μg /L BMP-2 and osteogenic medium containing;group 2,DPSCs-IPs culturing with 100 μg /L BMP-2 but without osteogenic medium containing;group 3,DPSCs-IPs culturing without 100 μg /L BMP-2 but with osteogenic medium contai-ning;group 4,DPSCs-IPs culturing without 100 μg /L BMP-2 and osteogenic medium containing.After one week of their culture in nonosteogenic condition,we used Trichrome to stain the secretion of collagen matrix,and compared the difference of osteogenic differ-entiation between them.Real-time RT-PCR was used to test the expression of COL-ⅠmRNA.Three weeks after their culture in osteo-genic condition,the cell differentiation was examined by von Kossa staining.Real-time RT-PCR was used to test nuclear transcription factor and osteogenesis related molecules gene expression.Results：One week later,Trichrome staining matrix secretion of DPSCs-IPs with BMP-2 group is more obvious than the control group in the nonosteogenic medium.Real-time RT-PCR tests showed that expression of COL-ⅠmRNA increased （P〈0.05）.The results suggested that BMP-2 could induce DPSCs-IPs deposit more collagen matrix;3 weeks later,more mineralized matrix secretion of DPSCs-IPs can be observed in the osteogenic medium.In addition,real-time RT-PCR tests showed that experimental group cells express more Nanog,Oct4 and SOX2.And the osteogenesis related molecules ALP,OCN, BSP,CEMP-1 and COL-Ⅰ expression increased significantly compared with the control group （P 〈0.05 ）.Conclusions：BMP-2 can promote osteogenic differentiation of human inflammatory dental pulp stem cells in vitro in osteogenic condition.