细胞外基质磷酸化糖蛋白mRNA在人牙周韧带细胞成骨分化过程中的表达

Expression of matrix extracellular phosphoglycoprotein mRNA in human periodontal ligament cell osteogenic differentiation

目的检测人牙周韧带细胞（periodontal ligament stem cell, PDLC）诱导矿化过程中碱性磷酸酶（alkaline phosphatase, ALP）、骨钙素（osteocalcin，OCN）和细胞外基质磷酸化糖蛋白（matrix extraeellular phosphoglyeoprotein, MEPE）mRNA的表达，探讨MEPE能否作为人牙周韧带细胞的分化标记及其功能。方法酶消化法培养PDLC并鉴定其来源，取未诱导及矿化诱导7、14和21d的PDLC，茜素红染色检测矿化结节的形成；免疫组织化学染色检测OCN的表达；实时荧光定量反转录聚合酶链反应（RT-PCR）检测ALP、OCN和MEPE mRNA的表达变化，并对结果进行方差分析。结果PDLC矿化诱导后茜素红染色显示钙化结节形成，免疫染色OCN表达增强；PDLC成骨诱导培养前ALP、OCN和MEPEmRNA表达系数分别为72、1．1和534。随着诱导时间延长，ALP、OCN和MEPEmRNA表达上调，诱导7d组的表达系数分别为78、9．56和629．6，诱导14d组的表达系数分别为290、133和638．3，诱导21d组的表达系数分别为1108、925和2261．1。与对照组相比，各诱导组OCNmRNA的表达、诱导14d和21d组舢口mRNA的表达，以及诱导21d组MEPEmRNA的表达，差异均有统计学意义（P〈0．05）。结论在PDLC向成牙骨质或成骨样细胞分化过程中，MEPEmRNA与ALP和OCN呈现相似的表达变化趋势，提示MEPE与PDLC成骨分化有关，有可能作为PDLC向成牙骨质或成骨样细胞分化的标志。

Objective To investigate the mineralization capacity of periodontal ligament stem cells （PDLC） by determining the mRNA expressions of alkaline phosphatase （ALP）, osteocalein （OCN） and matrix extracellular phosphoglycoprotein （MEPE） and to explore the potential of MEPE as a differentiation marker for PDLC, and its possible function in PDLC ostengenic differentiation. Methods PDLC were digested and cultured by a solution containing collagenase type I and dispase. PDLC were preceded to osteogenic induction for 7, 14 and 21 days respectively, and the cells before induction served as controls. Mineralization nodules and the expression of OCN in PDLC were investigated by alizarin red and immunohistochemistry respectively. The expressions of ALP, OCN and MEPE mRNA were investigated by quantitative real-time BT-PCB analysis. Statistical analysis was performed to compare the differences of mRNA expression levels among cell samples collected at different time points. Results The mRNA expressions of ALP, OCN and MEPE in PDLC before induction were 72, 1.1 and 534 respectively, but increased time-dependently in the induction cultures. The mRNA expressions of ALP, OCN and MEPE were 78,9.56 and 629.6 on day 7; 290, 133 and 638.3 on day 14; 1108, 925 and 2261.1 on day 21 respectively. The relative mRNA levels of OCN,ALP on day 14 and 21, MEPE on day 21 were significantly higher than control group （ P 〈 0. 05 ）. Conclusions PDLC showed analogously temperal expression of ALP, OCN and MEPE mRNA while differentiating into cementoblast/osteoblast-like cells in vitro. MEPE may play a regLdatory role in PDLC osteogenic differentiation, and may be a potential osteogenic differentiation marker along with ALP and OCN.