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

›› 2013, Vol. 4 ›› Issue (2): 65-69.

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

束丽红,曹灵,闫明,李俊俊,张光东,于金华

南京医科大学口腔医学研究所

收稿日期:2013-04-02 修回日期:2013-05-28 出版日期:2013-06-25 发布日期:2013-06-26
通讯作者: 于金华 E-mail:yuziyi_yjh@sohu.com
基金资助:
江苏省卫生厅医学重点人才项目;江苏高校优势学科建设工程资助项目

Study on the proliferation and osteo/odongenic differentiation of human periodontal ligament stem cells in different developing stages

Received:2013-04-02 Revised:2013-05-28 Online:2013-06-25 Published:2013-06-26

摘要/Abstract

摘要： 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。

Abstract: 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 respectively 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 abilities 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 completion 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.

束丽红曹灵闫明李俊俊张光东于金华. 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[J]. , 2013, 4(2): 65-69.

参考文献

[1]Honda MJ, Tsuchiya S, Shinohara Y, et al.Recent advances in engineering of tooth and tooth structures using postnatal dental cells[J].Jpn Dent Sci Rev,2010,46(1):54-66.
[2]Lei G, Yan M, Wang Z, et al.Dentinogenic capacity: immature root papilla stem cells versus mature root pulp stem cells[J].Biol Cell,2011,103(4):185-196.
[3]Yan M, Yu Y, Zhang G, et al.A journey from dental pulp stem cells to a bio-tooth[J].Stem Cell Rev,2011,7(1):161-171.
[4]Ruparel NB, de Almeida JF, Henry MA, et al.Characterization of a stem cell of apical papilla cell line: effect of passage on cellular phenotype[J].J Endod,2013,39(3):357-363.
[5]Seo BM, Miura M, Gronthos S,et al.Investigation of multipotent postnatal stem cells from human periodontal ligament[J].Lancet,2004,364(9429):149-155.
[6]Iwata T, Yamato M, Tsuchioka H, et al.Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model[J].Biomaterials,2009,30(14):2716-2723.
[7]Yang ZH, Zhang XJ, Dang NN, et al.Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like t2s[J].J Periodontal Res,2009,44(2):199-210.
[8]Wada N, Menicanin D, Shi S, et al.Immunomodulatory properties of human periodontal ligament stem cells[J].J Cell Physiol,2009,219(3):667-676.
[9]Zheng W, Wang S, Ma D, et al.Loss of proliferation and differentiation capacity of aged human periodontal ligament stem cells and rejuvenation by exposure to the young extrinsic environment[J].T9 Eng Part A,2009,15(9):2363-2371.
[10]Yu Y, Mu J, Fan Z, et al.Insulin-like growth factor 1 enhances the proliferation and osteogenic differentiation of human periodontal ligament stem cells via ERK and JNK MAPK pathways[J].Histochem Cell Biol,2012,137(4):513-525.
[11]Inukai T, Katagiri W, Yoshimi R, et al.Novel application of stem cell-derived factors for periodontal regeneration[J].Biochem Biophys Res Commun,2013,430(2):763-768.
[12]Zhou Y, Li Y, Mao L, et al.Periodontal healing by periodontal ligament cell sheets in a teeth replantation model[J].Arch Oral Biol,2012,57(2):169-176.
[13]Foster BL, Popowics TE, Fong HK,et al.Advances in defining regulators of cementum development and periodontal regeneration[J].Curr Top Dev Biol,2007,78:47-126.
[14]Zeichner-David M.Regeneration of periodontal ts: cementogenesis revisited[J].Periodontol 2000,2006,41:196-217.
[15]Ohshima H, Nakasone N, Hashimoto E, et al.The eternal tooth germ is formed at the apical end of continuously growing teeth[J].Arch Oral Biol,2005,50(2):153-157.
[16]Smith AJ.Vitality of the dentin-pulp complex in health and disease: growth factors as key mediators[J].J Dent Educ,2003,67(6):678-689.
[17]Kiyozumi D, Osada A, Sugimoto N, et al.Identification of a novel cell-adhesive protein spatiotemporally expressed in the basement membrane of mouse developing hair follicle[J].Exp Cell Res,2005,306(1):9-23.
[18]Hong D, Chen HX, Yu HQ,et al.Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells[J].Exp Cell Res,2010,316(14):2291-2300.
[19]Kheralla Y, G?tz W, Kawarizadeh A, et al.IGF-I, IGF-IR and IRS1 expression as an early reaction of PDL cells to experimental tooth movement in the rat[J].Arch Oral Biol,2010,55(3):215-222.
[20]Zhang W, Shen X, Wan C, et al.Effects of insulin and insulin-like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK[J].Cell Biochem Funct,2012,30(4):297-302.
[21]Lu Y, Ye L, Yu S,et al.Rescue of odontogenesis in Dmp1-deficient mice by targeted re-expression of DMP1 reveals roles for DMP1 in early odontogenesis and dentin apposition in vivo[J].Dev Biol,2007,303(1):191-201.
[22]Lee SK, Lee KE, Jeon D,et al.A novel mutation in the DSPP gene associated with dentinogenesis imperfecta type II[J].J Dent Res,2009,88(1):51-55.
[23]McKnight DA, Simmer JP, Hart PS, et al.Overlapping DSPP mutations cause dentin dysplasia and dentinogenesis imperfecta[J].J Dent Res,2008,87(12):1108-1111.
[24]Zhang JF, Fu WM, He ML,et al.MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling[J].RNA Biol,2011,8(5):829-838.
[25]Yu J, He H, Tang C, et al.Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging[J].BMC Cell Biol,2010,11:32-.
[26]Nevins ML, Reynolds MA.T2 engineering with recombinant human platelet-derived growth factor BB for implant site development[J].Compend Contin Educ Dent,2011,32(2):18, 20-27.
[27]Bai Y, Bai Y, Matsuzaka K, et al.Formation of bone-like t2 by dental follicle cells co-cultured with dental papilla cells[J].Cell T2 Res,2010,342(2):221-231.