【作者】 李冉； 李贤玉； 周密； 韩娜娜； 张旗；

【Author】 Li Ran,Li Xianyu,Zhou Mi,Han Nana,Zhang Qi~*(Hospital of Stomatology Wuhan University)

【机构】 武汉大学口腔医学院；

【摘要】 目的:牙周膜(PDL)纤维结缔组织位于牙槽骨和牙骨质之间,具有成骨分化潜能的牙周膜细胞(PDLCs)却能维持PDL生理宽度而未矿化。因此,推测PDL中存在矿化抑制机制。迄今,一些成骨分化抑制因子已被证实,但PDLCs抑制矿化机制未明。HtrA1(high-temperature requirement protein A1)通过TGFβ/BMP通路抑制成骨分化,本研究旨在探索人PDLCs成骨分化中HtrA1扮演的角色和是否通过BMP-2(TGFβ/BMP通路成员之一)来发挥调节作用。方法:PDLCs取自拔除的健康前磨牙或智齿,在地塞米松、维C和β-磷酸甘油钠的矿化液中诱导28天。分别于0、7、14、21、28天检测ALP活性、钙沉积、矿化结节茜素红染色,于0、3、7、11、14、21、28天提取PDLCs总RNA和总蛋白,实时荧光定量PCR检测ALP、BSP、OCN、Runx2、COLI、HtrA1、BMP-2的mRNA表达,western blotting检测HtrA1、BMP-2蛋白表达。结果:ALP活性和钙含量随诱导逐渐增高,矿化结节在1 4天出现并逐渐增多。ALP、BSP、OCN、Runx2、COLI等成骨相关基因表达显著增高(p<0.05),HtrA1 mRNA于21天达高峰而在28天下降,BMP-2 mRNA表达下降而在28天显著增高(p<0.05)。在蛋白表达水平,HtrA1上调而BMP-2表达下调。结论:二者负性表达关系暗示HtrA1可能通过BMP-2来调节人PDLCs成骨分化,但需要更深入的研究来证实。更多还原

【Abstract】 Objective:Periodontal ligament(PDL),a fibrous connective tissue,is located between two mineralized tissues,i.e.,alveolar bone and cementum.Human periodontal ligament cells(PDLCs) possess osteoblastic differentiation ability.Interestingly,under physiological conditions,PDL remains its unmineralized width.Thus,it has been speculated that PDLCs possess regulatory mechanisms to inhibit mineralization.To date,some regulators have been revealed to inhibit PDLCs osteogenic differentiation.Unfortunately, the precise mechanisms that PDLCs resist mineralization are still not well understood. HtrA1(high-temperature requirement protein A1) has been proved to inhibit osteogenesis by TGFβ/BMP signaling pathways in osteoblasts.However,the potential role of HtrAI in PDLCs cells osteogenic differentiation has not been defined yet.Therefore,this study aims to identify the exact role of HtrA1 during human PDLCs differentiation and whether HtrA1 exerts its function by regulation of BMP-2,a member of TGFβ/BMP signaling pathways. Methods:PDLCs were obtained from extracted healthy premolar or third molar teeth and cultured in mineralizing medium containing dexamethasone(10-8M),ascorbic acid(50μg/ ml) andβ-glycerophosphate(10mM) for 28 days.On days 0,7,14,21 and 28,alkaline phosphatase(ALP) activity was measured with p-nitrophenol phosphate assay,by calcium contents assay extracelluar calcium deposition was tested,and alizarin red staining was applied to detect the mineralized nodules.Total RNA was isolated on days 0,3,7,11, 14,21,28 respectively,and quantitative real-time RT-PCR was performed to evaluate mRNA expression of ALP,BSP,OCN,Runx2,typeⅠcollagen(COLI),HtrA1 and BMP-2. The protein expressions were verified by western blotting for HtrA1 and BMP-2.Results: ALP activity showed a time-dependent increase,as well as the calcium deposition in the extracelluar matrix,mineralized nodules first emerged on day 14 and then gradually increased.The mRNA expression of osteogenic markers was significantly up-regulated, including ALP,OCN,BSP,Runx2 and COLI(p<0.05).Intriguingly,HtrA1 mRNA was increased peaking on day 21 and declined on day 28,whilst mRNA expression of BMP- 2 showed a general decrease and dramatically increased on day 28(p<0.05).Moreover, the protein expressions indicated a general up-regulation in HtrA1 and down-regulation in BMP-2.Conclusion:Our findings suggest that HtrA1 might play a novel role in regulating human PDLCs osteogenic differentiation.The underlying negative relationship between HtrA1 and BMP-2 implies that HtrA1 may exert its regulatory effect via cross-talk with BMP-2.However,to clarify their exact function,further studies are still needed.更多还原