不同发育阶段的人牙周膜干细胞增殖能力和成牙／成骨能力的比较研究

摘要

Objective:To investigate the proliferation and osteo/odongenic differentiation of human periodontal ligament stem cells (PDLSC) in different developing stages,and to provide the candidate cell sources for tooth engineering .Methods:Cells were respec-tively isolated from developing teeth with open apical foramen and developed teeth with closed apical foramen .Methyltetrazolium(MTT) assay was performed to observe the proliferation features of the two kinds of human periodontal ligament stem cells .And their alkaline phosphotase(ALP) activities were evaluated using an ALP assay kit .Alizarin red staining was used to observe the mineralization abili-ties of the two kinds of cells.Furthermore,dentin matrix protein 1(DMP1),dentin sialoprotein(DSP),runt-related transcription factor 2 (RUNX2) and osterix(OSX) were assessed by Western blot to evaluate the osteo /odongenic differentiation capacities of two kinds of PDLSC.Results:PDLSC from developing teeth presented with a faster proliferation rate ,higher ALP activity and mineralization ability , and upregulated proteins expression of DMP1,RUNX2,DSP and OSX in vitro,as compared with PDLSC from developed teeth (P<0.05).Conclusions:The developing stages can affect the proliferation and osteo /odongenic differentiation of PDLSC.After the comple-tion of root development,the proliferation ability and osteo/odongenic differentiation capacity of PDLSC decreased due to the changes in the microenvironment at different developing stages .%　　目的：初步比较人牙周膜干细胞（human periodontal ligament stem cell ，PDLSC）在牙根不同发育阶段时的增殖能力和成牙/成骨能力的差异，为组织工程化牙齿的种子细胞来源提供一定的实验基础。方法：分别分离培养来自人根尖孔未闭合及根尖孔完全闭合牙齿的PDLSC，通过MTT法检测其增殖能力，碱性磷酸酶（alkaline phosphatase，ALP）试剂盒法检测其ALP活性，茜素红染色法检测两者的体外矿化能力，Western blot检测其牙本质基质蛋白1（dentin matrix protein 1，DMP1）、牙本质涎蛋白（dentin sialoprotein，DSP）、核心结合因子（runt－related transcription factor 2，RUNX2）和骨细胞特异性转录因子（osterix， OSX）的蛋白表达情况。结果：来自人根尖孔未闭合牙的PDLSC的增殖活性、ALP活性和矿化能力、DMP1、RUNX2、DSP和OSX蛋白的表达量均高于根尖孔完全闭合牙的PDLSC，差异具有统计学意义（P＜0．05）。结论：PDLSC的增殖和成牙/成骨能力随着牙根发育阶段的不同而变化，随着牙根发育的完全，PDLSC的增殖能力和成牙/成骨能力均下降，这种影响可能是由于牙根不同发育阶段牙根周围微环境的变化而导致的。