丝氨酸蛋白酶HtrA1在人牙周膜组织中的表达及对人牙周膜细胞增殖的影响

The expression of serine protease HtrA1 in human periodontal ligament tissue and the effect of HtrA1 on the proliferation of human periodontal ligament cells

目的观察丝氨酸蛋白酶HtrA1在人牙周膜组织中的表达，探讨其在人牙周膜细胞（human periodontal ligament cells，hPDLC）增殖中的作用。方法收集武汉大学口腔医学院口腔颌面外科门诊拔除的12至25岁健康前磨牙6颗和第三磨牙3颗，要求牙根完整、无龋坏和牙周组织炎症，反转录PCR和免疫组化染色方法分别检测HtrA1在人牙周膜组织中的mRNA和蛋白的表达，组织块法体外培养原代hPDLC，甲基噻唑基四唑比色法检测细胞增殖。用慢病毒转染的方法获得HtrA1基因过表达和基因沉默稳定细胞株，并以空载体慢病毒为对照，然后分别在培养的第1、3、5、7、9d，细胞计数CCK．8试剂盒（cell countingkit-8，CCK-8）检测HtrA1对hPDLC增殖的影响。结果反转录PCR结果显示HtrA1 mRNA在人牙周膜组织中呈阳性表达，免疫组化染色结果显示HtrA1蛋白在人牙周膜组织中也呈阳性表达，并主要表达于hPDLC胞质和胞外基质中。hPDLC经体外培养后生长曲线符合该细胞生长特征。慢病毒成功将HtrA1基因过表达和基因沉默后，CCK．8检测结果显示体外培养1、3、5、7、9d后，分别与各自的空载体对照组相比，过表达HtrA1使hPDLC的增殖速率显著减弱（A值分别为0．897±0．060、0．890±0．083、1．631±0．038、1．111±0．041、1．110±0．189），而基因沉默HtrA1后hPDLC增殖速率显著加快（A值分别为0．329±0．021、0．529±0．044、0．973±0．056、1．626±0．102、2．344±0．198）（P〈0．05）。结论HtrA1在mRNA和蛋白水平均表达于人牙周膜组织中，并对hPDLC的增殖起重要调节作用。

Objective To observe the expression of serine protease HtrA1 in human periodontal ligament tissue and toexplore the effect of HtrA1 on the proliferation of human periodontal ligament cells （hPDLC）. Methods Six human premolars and three human third molars（patient＇s ages ranging from 12 to 25, with intact root, without caries and/or periodontitis） were obtained in the Department of Maxillofacial Surgery of Wuhan University Hospital of Stomatology. Reverse transcription-PCR（RT-PCR） and immunohistochemistry analysis were applied to investigate the expression of HtrA 1. Primary hPDLC were obtained by tissue-culture method in vitro. The proliferation of hPDLC was determined by methyl thiazolytetrazolium（MTT）. Lentivirus-mediated over-expression and reduction of HtrA1 level was performed. An empty vector was used as negative control. On days 1, 3, 5, 7 and 9, the growth of hPDLC was characterized using cell counting kit-8（CCK-8） assay. Results RT-PCR data indicated that HtrA1 mRNA was expressed in human periodontal ligament tissue. Immunohistochemistry analysis showed HtrA 1 was expressed in human periodontal ligament, mainly in the cytoplasm of hPDLC and the extracellular matrix. The MTT result suggested that the growth curve was consistent with the growth characteristics of hPDLC. The stable over-expression and knockdown cell lines was successfully established by lentivirus with more than 90% transfeetion efficiency. CCK-8 assay showed that HtrA1 over-expression inhibited the proliferation of hPDLC（0.897±0.060, 0.890±0.083, 1.631±0.038, 1.111±0.041, 1.110±0.189）, while cell proliferation increased after down-regulation of HtrAl（0.329±0.021, 0.529±0.044, 0.973±0.056, 1.626±0.102, 2.344± 0. 198）（P〈0.05）. Conclusions HtrA 1 is expressed in human periodontal ligament tissue at both mRNA and protein levels, and may play an important role in regulating the proliferation of hPDLC.