牙周膜相关蛋白1在人牙周膜细胞成骨分化过程中的表达

目的 研究牙周膜相关蛋白1（PLAP-1）在人牙周膜细胞（PDLC）成骨分化过程中的表达变化,为明确PLAP-1 在牙周组织中的作用奠定基础.方法 酶消化法培养人PDLC,免疫细胞化学染色鉴定其来源和PLAP-1 的表达,茜素红染色和碱性磷酸酶（ALP）实验鉴定其成骨分化能力,定量反转录聚合酶链反应（RT-PCR）检测其成骨分化过程中,PLAP-1、Periostin、RGD-CAP、RUNX2、OCN 和OPN mRNA 表达变化,Western blot 检测PLAP-1、RUNX2 和OCN 蛋白表达变化并进行统计分析.结果 培养的人PDLC 来源于间充质;人PDLC 矿化诱导21 d 后茜素红染色阳性,矿化诱导后7、14、21 d 时ALP 活性较对照组明显增高（P ＜ 0.05）,具有成骨分化能力;PLAP-1 免疫细胞化学染色阳性;定量RT-PCR 和Western blot 结果显示,人PDLC 在mRNA 和蛋白水平均表达PLAP-1,且mRNA 表达量随人PDLC 成骨分化过程发生变化,诱导14 d上调,诱导21 d 下调,较对照组有明显差异（P ＜ 0.05）,Western blot 检测蛋白表达显示类似的表达趋势.结论 PLAP-1 在基因及蛋白水平均高表达于人PDLC,其表达量随人PDLC 成骨分化成一定模式,提示其参与调控牙周膜的功能与稳定,但其精细的调控功能还有待进一步深入研究.

Expression of periodontal ligament associated protein-1 in human periodontal ligament cellosteogenic differentiation

Objective To examine the expression of periodontal ligament associated protein-1 （PLAP-1） in the human periodontal ligament cells （PDLC） and explore its possible function in PDLC osteogenic differentiation. Methods PDLC were digested and cultivated by a solution containing collagenase type I and dispase, and were preceded to osteogenic induction for 7, 14 and 21 days respectively, and cells groups without induction served as controls （0 d）. The expression of Vimentin and PLAP-1 in PDLC were investigated by immunocytochemistry. Alizarin red staining and alkaline phosphatase （ALP） experiment identificated the osteogenic differentiation capacity of PDLC. The expressions of PLAP-1, Periostin, RGD-CAP, RUNX2, OCN and OPN mRNAs were investigated by quantitative real-time RT-PCR analysis, and statistical analysis was performed to compare the differences of mRNA expression levels among cell samples collected at different time points. The expression of PLAP-1, RUNX2 and OCN proteins was investigated by western-blot. Results The cultured PDLC were of mesenchymal origin. After cultured in the osteogenic medium for 21 days, PDLC formed alizarin red-positive nodules. Compared to the control groups, ALP activity was significantly higher in the 7% 14- and 21-day induction groups （P 〈 0.05）, which means PDLC were capable of osteogenic differentiation capacity. PLAP-1 was expressed in PDLC on both gene and protein levels. The expression of PLAP-1 mRNA increased in the 14-day group in the osteogenic differentiation but decreased in the 21-day group, the differences were significant compared to the control groups （P〈 0.05）. The proteinsshowed analogously temporal expression of mRNAs. Conclusions PLAP-1 was highly expressed in PDLC on both gene and protein levels. The expression of PLAP-1 exhibited a certain mode in the osteogenie differentiation of PDLC, and presented that it was involved in the regulation of function and stability of periodontal ligament. But its fine regulation of the function is still to he in further study.