炎症微环境下人牙龈固有层间充质干细胞生物学特性的研究
|
摘要
牙龈是附着在牙颈和牙槽突部分的粘膜组织，呈现粉红色，有光泽，质坚韧，是口腔粘膜的一部分。它担负着牙周组织与口腔环境的粘膜屏障，维持口腔健康。从组织学和功能学上看，由于牙龈具有耐受咀嚼食物所产生的摩擦力的特性，因此它属于咀嚼粘膜，以此和颊侧的被覆粘膜和唇侧粘膜得以区分。牙龈组织由上皮和固有层两部分组成，固有层由颅神经嵴发育而成，与之同源还有颅面其它间充质来源的组织，包括牙乳头和牙周组织。一些文献中报导，大量的颅神经嵴来源的间充质干细胞已经得到证实，它们无论在维持自身内环境稳定上还是在再生治疗中都可以发挥重要的作用。然而，尽管已经确认牙龈来源的细胞是上皮/原始干细胞，但是几乎还没有研究对牙龈来源的细胞鉴定为间充质干细胞。
目前一些对牙龈成纤维细胞的研究，认为还不能从其中区分出已经有证据存在的牙龈间充质干细胞。但是近期有文献报导，已经可以成功从人牙龈组织中分离培养出牙龈间充质干细胞(gingival mesenchymal stem cell，GMSC)。这些GMSC无论是在体内环境还是在体外环境都显示出自我更新，多向分化和免疫调节的能力。而这些特征正好与Fournier等人发表的文章中所阐述的牙龈成纤维细胞显示出原始细胞的特性相似，包括体外低密度细胞形成克隆形成率（colony forming unit-fibroblastic，CFU-F）和体内多向分化。当前，各种人的间充质干细胞已经获得认可，并且应用于临床治疗中。早先的实验认为运用分离和培养技术，间充质干细胞几乎可以从人的器官和组织甚至从它们的胚胎中获得。
我们知道临床上慢性牙龈炎导致的牙龈增生原因复杂，表现也较不均一，而正畸治疗中出现的牙龈炎性增生属于仅与牙菌斑有关的牙龈病，表型稳定，炎症是其发生发展的最主要原因。并且有研究表明，正畸治疗和未经正畸治疗牙龈炎性增生的发生率是没有统计学差异，和矫治力无关，而与口腔卫生相关，。
本课题旨在通过对人正常和牙菌斑导致的炎性增生牙龈的组织学，细胞学观察，探讨它们在原位的组织学特征和存在GMSC干细胞的标记物。然后从这两种牙龈组织中分离培养GMSC：正常牙龈间充质干细胞（gingivalmesenchymal stem cell from normal gingival tissue，N-GMSC）、炎性增生牙龈间充质干细胞（gingival mesenchymal stem cell from inflammation ofgingival hyperplasia tissue，I-GMSC）应用单克隆分离和扩增方法，证明它们都具有克隆形成能力和高度增殖能力，比较N-GMSC和I-GMSC成骨和成脂特性的差异，及其细胞外基质、相关蛋白酶、炎性因子的变化特点。建立炎症微环境这一稳定的系统，研究炎症微环境对牙龈间充质干细胞的影响。初步探讨正畸治疗中牙龈炎性增生的发病机制，为探索新的预防和治疗策略提供理论和实验依据。
第一部分人正常牙龈和炎性增生牙龈组织学观察
实验一、人正常牙龈和炎性增生牙龈组织学观察采用组织学方法观察正常牙龈和炎性增生牙龈组织学特征。结果表明：正常牙龈组织，呈现粉红色，有光泽，质坚韧。炎性增生牙龈组织，牙龈充血，失去正常的解剖外形，并且红肿增生。炎性增生牙龈HE组织切片显示，其上皮钉突明显，固有层大量淋巴细胞浸润。
实验二、人正常牙龈和炎性增生牙龈干细胞特异性标志物的比较采用免疫荧光组织化学方法原位观察两种牙龈组织和细胞爬片。结果发现：正常牙龈组织呈现出的干细胞的筛选标志-1（STROL-1）和早期胚胎糖脂抗-4（stage-specific embryonic antigen4，SSEA-4）含量大于炎性增生牙龈组织，提示N-GMSC和I-GMSC存在各自组织中含量的差异，可能同时也具有骨/脂向分化潜力的差异；炎性增生牙龈组织细胞外基质Ⅰ型胶原（collagen-1，COL-1）含量大于正常牙龈组织，N-GMSC和I-GMSC细胞爬片的结果符合它们所来源的组织免疫荧光组织化学COL-1的染色结果，提示两种组织的细胞外基质含量的差异，可能是造成它们的临床出现的不同表型的原因。
第二部分人正常牙龈和炎性增生牙龈间充质干细胞分离、培养和生物学特性的初步研究
实验一、N-GMSC和I-GMSC的分离、培养及形态学鉴定两种来源牙龈间充质干细胞的分离、培养及形态学鉴定。采用酶消化法分离培养原代牙龈间充质干细胞。结果表明：离体的牙龈组织包含结构完整的间充质组织，表明本实验使用的方法能够分离出GMSC，它可以迅速贴壁，伸展后细胞形态呈长梭形或三角形。
实验二、N-GMSC和I-GMSC体外培养生物学特性检测采用有限稀释法细胞接种于大培养皿中，通过克隆形成率检测，从而明确两种状态来源的牙龈组织中GMSC干细胞克隆形成的差异。流式细胞术（flow cytometric，FCM）分别检测来源于间充质的N-GMSC和I-GMSC表面标志：阳性表达-STRO-1、CD29、CD44、CD90、CD105、CD146；阴性表达-CD34、CD45。检测结果表明：N-GMSC和I-GMSC形成克隆的能力无明显差异，但是在相同时间中N-GMSC克隆个体大于I-GMSC。提示炎症导致的牙龈增生组织中仍然存在间充质干细胞。N-GMSC表达的STRO-1、CD146高于I-GMSC，标志物的表达提示GMSC与来源于其他间充质组织（牙髓）的干细胞具有同源性，并且N-GMSC干细胞表达大于I-GMSC。
实验三、N-GMSC和I-GMSC多向分化能力检测采用碱性磷酸酶（alkaline phosphatase，ALP）活性检测、矿化结节染色、脂滴染色及逆转录聚合酶链式反应（reverse transcription polymerase chain reaction，RT-PCR）检测成骨/成脂相关基因表达的方法，检测了N-GMSC和I-GMSC在体外的分化能力。结果表明：N-GMSC在体外显示了较I-GMSC更高的矿化和成脂能力。提示N-GMSC干细胞能力强于I-GMSC，I-GMSC干细胞能力降低，是因为在炎症微环境中会促使GMSC提前进入瞬时扩增细胞（transit-amplyfing cells，TA）的状态。
第三部分炎症微环境对人正常牙龈组织来源的间充质干细胞影响
实验一、N-GMSC和I-GMSC增殖差异和以此特性建立体外稳定的炎症微环境。甲基噻唑基四唑检测(methyl thiazolyl tetrazolium，MTT)以及细胞周期(cell cycle，CC)检测明确N-GMSC和I-GMSC增殖能力差异，根据它们的增殖特点，确定用外源性炎性因子：白介素-1（nterleukin-1beta，IL-1β）、肿瘤坏死因子-α （tumor necrosis factor-alpha，TNF-α）建立体外稳定的炎症微环境的浓度，观察N-GMSC在炎症微环境中凋亡能力的变化。结果表明：I-GMSC增殖能力强于N-GMSC，符合炎性增生牙龈的组织学特点。炎症环境对干细胞的影响是其中的炎性因子对它的影响，外源性炎性因子IL-1β(5ng/ml)和TNF-α (10ng/ml)建立稳定的炎症微环境：MTT检测N-GMSC在IL-1β(5ng/ml)和TNF-α(10ng/ml)炎性因子作用下增殖最高，符合MTT和细胞周期检测结果显示I-GMSC具有强增殖能力。我们还发现N-GMSC在炎性因子作用下凋亡增加，在成骨诱导分化阶段，凋亡数量都会随着作用时间的延长呈现增多的趋势。
实验二、N-GMSC和I-GMSC细胞因子基因表达差异的比较RT-PCR检测N-GMSC和I-GMSC基因表达的差异。结果显示：I-GMSC的基质金属蛋白酶1/2（matrix metalloproteinase1/2，MMP1/2）表达量减少；基质金属蛋白酶抑制剂1/2（metallopeptidase inhibitor1/2，TIMP1/2）、IL-1α/6、COL-1、TNF-α表达量增多。提示是否这些表达变化是GMSC在炎症状态中特有的变化趋势？
实验三、N-GMSC和对照组正常牙髓干细胞（normal dental pulp stemcell，N-DPSC）在炎症微环境中细胞因子基因表达的比较RT-PCR检测N-GMSC和对照组N-DPSC在炎性微环境中基因表达。结果显示：N-GMSC在体外炎症微环境中，MMP1/2的表达量变化与TIMP1/2的表达量变化成负相关，即炎性因子作用8h时TIMP1/2表达量最高，而同一时间测定的MMP1/2表达量最低。对照组的N-DPSC： MMP1/2随着炎性因子作用时间，基因表达量逐渐增加，而TIMP1/2基因表达量逐渐减少。提示为GMSC在炎症微环境中MMPs与TIMPs表达变化规律具有其特异性。随着炎性因子的作用时间IL-1α/6、COL-1、TNF-α表达量增加。GMSC表达的IL-1α相对DPSC的要强，而DPSC表达的IL-6相对GMSC的强。提示两种组织来源的干细胞（GMSC、DPSC）在炎症微环境中的表达IL-1α，IL-6表达量的差异，是不是因此造成它们所表达MMPs、TIMPs变化趋势不同的原因呢?所以最终导致牙龈和牙髓在炎症中表现出的不同表型，一个为组织的增生，一个为组织的液化。
实验四、N-GMSC、N-GMSC (外源性炎性因子)、N-DPSC、N-DPSC(外源性炎性因子)细胞膜片裸鼠皮下移植的实验研究将N-GMSC、N-GMSC(外源性炎性因子)、N-DPSC、N-DPSC(外源性炎性因子)制成细胞膜片移植入裸鼠皮下，2周后取材。对其进行组织免疫荧光组织化学染色，以探讨组织细胞膜片在体内MMP1、TIMP1表达含量符合体外基因表达的特点。牙龈与牙髓组织在炎症中的表型不相同，它们的干细胞属于组织特异的干细胞，在炎性微环境中它们的MMPs与TIMPs表达量的变化趋势也是不同的。可以认为GMSC在炎症微环境中MMPs与TIMPs表达变化规律具有其特异性，符合临床观察到的牙龈炎性增生，失去正常的解剖外形，牙龈充血，红肿增生。牙髓往往会出现牙髓的液化和坏死的炎性反应。
Human gingival tissues are prone to develop hyperplasia under multipleexternal stimili. We have recently shown that normal and hyperplastic gingivaltissue contains mesenchymal stem cells (GMSCs), but it is not known how thesecells are influenced by commonly seen inflammatory conditions. We isolatedGMSCs from human normal (N-GMSC) and inflammatory gingival tissues(I-GMSC). Meanwhile, an in vitro culture with TNF-α and IL-1β wasestablished to simulate the in vivo inflammatory situation. In these two settingswe observed consistent results that GMSC affected by inflammatory factorsproliferated more actively than those under normal condition, while lostpotential of osteogenic and adipogenic differentiation. The expression of matrixmetalloproteinases (MMP)-1/2, IL-1α, IL-6, TNF-α and COL-1wassignificantly higher in I-GMSC than that in N-GMSC. The comparison to dentalpulp stem cells disclosed their intrinsic differences with regard to in vitro geneexpression and in vivo extracellular matrix formation and regulation. Thesefindings suggest that inflammatory factors turn GMSC to a more differentiated and pro-fibrotic phenotype, which could underlie the clinical hyperplasticappearance of inflammatory gingiva.
Section1, Histological observation of human normal andinflammation gingival tissues
Experiment1, inflammation gingival tissues exhibited macroscopic tissueover-growth (FIG.1A,B) and thickened layers and elongated rete ridges withabundant cellularity and extracellular matrix within lamina propria by H&Estaining (FIG.1C, D).
Experiment2, immunofluorescence assay showed that inflammation gingivaltissues and cells expressed more abundant collagen I (COL-I, FIG.1K-M; N-O).We analyzed the expression of STRO-1, aputative mesenchymal stem cellmarker and SSEA-4, which was deemed as an early embryonic glycolipidantigen specific for pluripotent embryonic stem cells and had recently been usedto identify and purify stem cells from adult bone marrow in human normal andhyperplastic gingival tissues. Both normal and inflammation gingival propriadisplayed positive STRO-1(FIG.1E-G) and SSEA-4(FIG.1H-J) staining,with the latter marker being more sporadically distributed. These resultsindicated that despite the existence of differences between human normal andhyperplastic gingival tissues, they both showed evidence for harboring cells withfeatures of mesenchymal stem cells.
Section2, Study of separation, culture and biological characteristicsof GMSC
Experiment1, clonogenic MSC can be isolated from gingival tissue. Tocharacterize whether gingival propria-derived MSC is clonogenic, we generatedand cultured single cell suspensions from human normal and inflammationgingival tissues after separating and discarding gingival epithelium byincubating with3mg/ml dispase for30min. With2×103initially seeded cells,both cultures were observed with cells attaching onto the plastic in2days andthen they proliferate rapidly in next5days to form colonies. These colonies were stained with toluidine blue and exhibited typical fibroblastic morphology(FIG.3A). With above results, we termed these clonogenic populations gingivalmesenchymal stem cells (GMSC), with N-GMSC and I-GMSC referring toGMSC derived from normal and inflammation gingival propria respectively.The ability of gingival tissue derived cells to form adherent clonogenic cellclusters had no significant difference (FIG.3B).
Experiment2, by using limiting dilution technique, single cell-derivedcolony cultures were obtained. For N-GMSC,5batches of culture produced42single cell-derived colonies. For I-GMSC,3batches of culture produced34single cell-derived colonies. These colonies were randomly selected forsubsequent analysis. We used flow cytometric analysis to characterize N-GMSCand H-GMSC by surface molecules. Both GMSC within10passages showedthe characteristic pattern of mesenchymal surface markers including STRO-1,CD29, CD44, CD90, CD105, CD146and negatively expressed hematopoieticmarkers CD34and CD45(Fig.3C).
Experiment3, GMSC possesses self renewal and multilineage differentiationability in vitro. The multi-differentiation potential of both GMSC wasdetermined. Under osteogenic induction conditions for3weeks, both GMSCcould specifically differentiate and formed distinct nodules as stained byAlizarin Red S. After feeding GMSC with osteogenic inducing media, dark redmineralised bone matrix (bone nodules) was shown in alizarin red stainedsections. The cells were positively stained with ALP(FIG.4A①). PCR andRT-PCR analysis showed that the expression of the osteogenic markers bonesialoprotein runt-related transcription factor2(RUNX2), osteocalcin (OCN),alkaline phosphatase (ALP) were increased after osteogenic induction(FIG.4B③④). After3weeks of culture in adipogenic induction medium, GMSCproduced lipid droplets, the hallmark of functional adipogenesis. Alkalinephosphatase activity staining (2weeks) showed I-GMSC lighter thanN-GMSC.(FIG.4A④).
Adipogenic differentiation of GMSC was confirmed by Oil Red-O staining.After feeding GMSCs with adipogenic inducing media for21days, oil dropletswere presented in cytoplasm(FIG.4B①). Adipogenic differentiation was furtherconfirmed by the increased expression of specific adipogenic markers includingperoxisome proliferator-activated receptor γ (PPARγ). All are determined byRT-PCR (FIG.4A⑤B④). Through quantitative comparison of areas fromrandomly selected staining images, or grayscale analysis of RT-PCR bands, wehave found significant differences between N-and I-GMSC groups (datashown). To further compare the enzymatic activity related to ECM remodeling,we performed immunostaining and found that Collagen type1(COL-1)expression within N-GMSC was significantly lower than that in I-GMSC (FIG.1NO), suggesting role of COL-1expression within I-GMSC in mediating thepathological processes.
Section3, N-GMSC and I-GMSC show some special characteristicboth in vitro and in vivo
Experiment1, MTT assay and Cell Cycle showed that the growth tendencyof I-GMSC waw higher than that on the healthy group (FIG.5A,B). Accordingto MTT assay, concentration sitmulates the most N-GMSC proliferation was asthe final extrinsic source inflammatory factor IL-1β (5ng/ml) and TNF-α(10ng/ml)(FIG.5C). With extrinsic source inflammatory factor to N-GMSCshowed more apoptosis than that without inflammatory factor, and especiallywith extrinsic source inflammatory factor and Osteogenesis to N-GMSC showedthe most apoptosis (FIG.5D). N-GMSC possessed self renewal and multilineagedifferentiation ability in inflammatory microenvironment being reduced byRT-PCR analysis and alkaline phosphatase activity staining (FIG.6A,B,C).
Experiment2, GMSC expresses some cytokine in vivo. The present studyexamined HGFs from healthy and inflammatory gingival tissues using DNAmicroarray analysis. The expression levels of TIMP1, TIMP2, IL-1α, IL-6,TNF-α, and COL-1in the HGFs of the Inflammatory group were higher than those in the healthy group on DNA microarray analysis, MMP1MMP2lowerthan those in the N-GMSC (figure3.2.1).
Experiment3, these results showed that the DPSCs in inflammatory produceelevated levels of these MMP1/2, IL-1α, IL-6, TNF-α and COL-1proteins(figure3.3.1). Cytokines are soluble, biologically active glycoproteins, whichare secreted by host immuno-inflammatory cells. They have many functions andexert their effects in a paracrine or autocrine fashion to modulate inflammatoryand immune responses. It is likely that elevated levels of pro-inflammatorycytokines play a role in stimulating osteoclast activity. These cytokines are thekey of mediators in immunological and inflammatory responses, and have beenfound in measurable quantities in areas of active gingical inflammation.Therefore, the gingival tissues may produce various inflammatory cytokines.We demonstrated that the I-GMSC of inflammatory gingival tissues expressedsignificantly higher levels of IL-1α, IL-6, and TNF-α than the N-GMSC ofgingival tissues from healthy individuals did. Our data in the present studysuggest that cytokines are involved in chronic inflammatory conditions such asmechanical irritation-induced human gingival hyperplasia or gingivalinflammation.
Experiment4, to better confirm that the N-GMSC and I-GMSC showedsome special characteristic in vitro, we proposed cell sheet engineering in vivo.A cell sheet was constructed firstly; then the cell sheet shrank and condensedinto an intact cell pellet; finally in vivo differentiation assay ofN-GMSC/N-GMSC (exogenous inflammatory factor) and N-DPSC/N-DPSC(exogenous inflammatory factor) pellets was performed using immunodeficientmice. All the cell pellets had good biology properties. It also found that MMPand TIMP had different changing tendency with N-GMSC (exogenousinflammatory factor) and N-DPSC (exogenous inflammatory factor). Theseresults showed that the DPSCs in inflammatory produced elevated levels ofthese MMP1, MMP2oppositing to that of GMSC (FIG.7C,D).
This study showed that the expression of IL-1α, IL-6, col-1and TNF-α ofboth GMSC and DPSC increased in a time-dependant manner in vitro under aninflammatory microenvironment. However, as both GMSC and DPSC were twotissue-specific stem cells, so the expression of IL-1α in GMSC was relativelystronger than that in DPSC and the expression of IL-6in DPSC was relativelystronger than that in GMSC. Stem cells turned into TA in advance. After that,TA involved in the inflammatory reaction and its autocrine inflammatory factorwent into a positive feedback loop again. Do the expression differences of IL-1and IL-6between the stem cells derived from2kinds of tissue origins (GMSCand DPSC) under inflammatory microenvironment cause the difference ofchange trend of MMPs and MIMPs expression?
In the study, it was found that the I-GMSC was actually the TA which wasturned from N-GMSC in advance by the action of inflammatory factor. It stillcould be cloned and the osteogenic and adipogenic ability decreased in themulti-directional differentiation potential stage. It showed strong proliferativecapacity, and increased the extracellular matrix, being consistent with theclinical phenotype. The inflammatory factors could increase the apoptosis ofN-GMSC and more at the differentiation potential stage. The phenotype wasregulated by MMPs, TIMPs, and inflammatory factors. Currently, the influenceof inflammation on GMSC is clear. While it is still unknown that how dospecific expression of the MMPs, TIMPs and inflammatory factors have aneffect on their clinical proliferative phenotypes, and it can be screened in thefuture.
目前一些对牙龈成纤维细胞的研究，认为还不能从其中区分出已经有证据存在的牙龈间充质干细胞。但是近期有文献报导，已经可以成功从人牙龈组织中分离培养出牙龈间充质干细胞(gingival mesenchymal stem cell，GMSC)。这些GMSC无论是在体内环境还是在体外环境都显示出自我更新，多向分化和免疫调节的能力。而这些特征正好与Fournier等人发表的文章中所阐述的牙龈成纤维细胞显示出原始细胞的特性相似，包括体外低密度细胞形成克隆形成率（colony forming unit-fibroblastic，CFU-F）和体内多向分化。当前，各种人的间充质干细胞已经获得认可，并且应用于临床治疗中。早先的实验认为运用分离和培养技术，间充质干细胞几乎可以从人的器官和组织甚至从它们的胚胎中获得。
我们知道临床上慢性牙龈炎导致的牙龈增生原因复杂，表现也较不均一，而正畸治疗中出现的牙龈炎性增生属于仅与牙菌斑有关的牙龈病，表型稳定，炎症是其发生发展的最主要原因。并且有研究表明，正畸治疗和未经正畸治疗牙龈炎性增生的发生率是没有统计学差异，和矫治力无关，而与口腔卫生相关，。
本课题旨在通过对人正常和牙菌斑导致的炎性增生牙龈的组织学，细胞学观察，探讨它们在原位的组织学特征和存在GMSC干细胞的标记物。然后从这两种牙龈组织中分离培养GMSC：正常牙龈间充质干细胞（gingivalmesenchymal stem cell from normal gingival tissue，N-GMSC）、炎性增生牙龈间充质干细胞（gingival mesenchymal stem cell from inflammation ofgingival hyperplasia tissue，I-GMSC）应用单克隆分离和扩增方法，证明它们都具有克隆形成能力和高度增殖能力，比较N-GMSC和I-GMSC成骨和成脂特性的差异，及其细胞外基质、相关蛋白酶、炎性因子的变化特点。建立炎症微环境这一稳定的系统，研究炎症微环境对牙龈间充质干细胞的影响。初步探讨正畸治疗中牙龈炎性增生的发病机制，为探索新的预防和治疗策略提供理论和实验依据。
第一部分人正常牙龈和炎性增生牙龈组织学观察
实验一、人正常牙龈和炎性增生牙龈组织学观察采用组织学方法观察正常牙龈和炎性增生牙龈组织学特征。结果表明：正常牙龈组织，呈现粉红色，有光泽，质坚韧。炎性增生牙龈组织，牙龈充血，失去正常的解剖外形，并且红肿增生。炎性增生牙龈HE组织切片显示，其上皮钉突明显，固有层大量淋巴细胞浸润。
实验二、人正常牙龈和炎性增生牙龈干细胞特异性标志物的比较采用免疫荧光组织化学方法原位观察两种牙龈组织和细胞爬片。结果发现：正常牙龈组织呈现出的干细胞的筛选标志-1（STROL-1）和早期胚胎糖脂抗-4（stage-specific embryonic antigen4，SSEA-4）含量大于炎性增生牙龈组织，提示N-GMSC和I-GMSC存在各自组织中含量的差异，可能同时也具有骨/脂向分化潜力的差异；炎性增生牙龈组织细胞外基质Ⅰ型胶原（collagen-1，COL-1）含量大于正常牙龈组织，N-GMSC和I-GMSC细胞爬片的结果符合它们所来源的组织免疫荧光组织化学COL-1的染色结果，提示两种组织的细胞外基质含量的差异，可能是造成它们的临床出现的不同表型的原因。
第二部分人正常牙龈和炎性增生牙龈间充质干细胞分离、培养和生物学特性的初步研究
实验一、N-GMSC和I-GMSC的分离、培养及形态学鉴定两种来源牙龈间充质干细胞的分离、培养及形态学鉴定。采用酶消化法分离培养原代牙龈间充质干细胞。结果表明：离体的牙龈组织包含结构完整的间充质组织，表明本实验使用的方法能够分离出GMSC，它可以迅速贴壁，伸展后细胞形态呈长梭形或三角形。
实验二、N-GMSC和I-GMSC体外培养生物学特性检测采用有限稀释法细胞接种于大培养皿中，通过克隆形成率检测，从而明确两种状态来源的牙龈组织中GMSC干细胞克隆形成的差异。流式细胞术（flow cytometric，FCM）分别检测来源于间充质的N-GMSC和I-GMSC表面标志：阳性表达-STRO-1、CD29、CD44、CD90、CD105、CD146；阴性表达-CD34、CD45。检测结果表明：N-GMSC和I-GMSC形成克隆的能力无明显差异，但是在相同时间中N-GMSC克隆个体大于I-GMSC。提示炎症导致的牙龈增生组织中仍然存在间充质干细胞。N-GMSC表达的STRO-1、CD146高于I-GMSC，标志物的表达提示GMSC与来源于其他间充质组织（牙髓）的干细胞具有同源性，并且N-GMSC干细胞表达大于I-GMSC。
实验三、N-GMSC和I-GMSC多向分化能力检测采用碱性磷酸酶（alkaline phosphatase，ALP）活性检测、矿化结节染色、脂滴染色及逆转录聚合酶链式反应（reverse transcription polymerase chain reaction，RT-PCR）检测成骨/成脂相关基因表达的方法，检测了N-GMSC和I-GMSC在体外的分化能力。结果表明：N-GMSC在体外显示了较I-GMSC更高的矿化和成脂能力。提示N-GMSC干细胞能力强于I-GMSC，I-GMSC干细胞能力降低，是因为在炎症微环境中会促使GMSC提前进入瞬时扩增细胞（transit-amplyfing cells，TA）的状态。
第三部分炎症微环境对人正常牙龈组织来源的间充质干细胞影响
实验一、N-GMSC和I-GMSC增殖差异和以此特性建立体外稳定的炎症微环境。甲基噻唑基四唑检测(methyl thiazolyl tetrazolium，MTT)以及细胞周期(cell cycle，CC)检测明确N-GMSC和I-GMSC增殖能力差异，根据它们的增殖特点，确定用外源性炎性因子：白介素-1（nterleukin-1beta，IL-1β）、肿瘤坏死因子-α （tumor necrosis factor-alpha，TNF-α）建立体外稳定的炎症微环境的浓度，观察N-GMSC在炎症微环境中凋亡能力的变化。结果表明：I-GMSC增殖能力强于N-GMSC，符合炎性增生牙龈的组织学特点。炎症环境对干细胞的影响是其中的炎性因子对它的影响，外源性炎性因子IL-1β(5ng/ml)和TNF-α (10ng/ml)建立稳定的炎症微环境：MTT检测N-GMSC在IL-1β(5ng/ml)和TNF-α(10ng/ml)炎性因子作用下增殖最高，符合MTT和细胞周期检测结果显示I-GMSC具有强增殖能力。我们还发现N-GMSC在炎性因子作用下凋亡增加，在成骨诱导分化阶段，凋亡数量都会随着作用时间的延长呈现增多的趋势。
实验二、N-GMSC和I-GMSC细胞因子基因表达差异的比较RT-PCR检测N-GMSC和I-GMSC基因表达的差异。结果显示：I-GMSC的基质金属蛋白酶1/2（matrix metalloproteinase1/2，MMP1/2）表达量减少；基质金属蛋白酶抑制剂1/2（metallopeptidase inhibitor1/2，TIMP1/2）、IL-1α/6、COL-1、TNF-α表达量增多。提示是否这些表达变化是GMSC在炎症状态中特有的变化趋势？
实验三、N-GMSC和对照组正常牙髓干细胞（normal dental pulp stemcell，N-DPSC）在炎症微环境中细胞因子基因表达的比较RT-PCR检测N-GMSC和对照组N-DPSC在炎性微环境中基因表达。结果显示：N-GMSC在体外炎症微环境中，MMP1/2的表达量变化与TIMP1/2的表达量变化成负相关，即炎性因子作用8h时TIMP1/2表达量最高，而同一时间测定的MMP1/2表达量最低。对照组的N-DPSC： MMP1/2随着炎性因子作用时间，基因表达量逐渐增加，而TIMP1/2基因表达量逐渐减少。提示为GMSC在炎症微环境中MMPs与TIMPs表达变化规律具有其特异性。随着炎性因子的作用时间IL-1α/6、COL-1、TNF-α表达量增加。GMSC表达的IL-1α相对DPSC的要强，而DPSC表达的IL-6相对GMSC的强。提示两种组织来源的干细胞（GMSC、DPSC）在炎症微环境中的表达IL-1α，IL-6表达量的差异，是不是因此造成它们所表达MMPs、TIMPs变化趋势不同的原因呢?所以最终导致牙龈和牙髓在炎症中表现出的不同表型，一个为组织的增生，一个为组织的液化。
实验四、N-GMSC、N-GMSC (外源性炎性因子)、N-DPSC、N-DPSC(外源性炎性因子)细胞膜片裸鼠皮下移植的实验研究将N-GMSC、N-GMSC(外源性炎性因子)、N-DPSC、N-DPSC(外源性炎性因子)制成细胞膜片移植入裸鼠皮下，2周后取材。对其进行组织免疫荧光组织化学染色，以探讨组织细胞膜片在体内MMP1、TIMP1表达含量符合体外基因表达的特点。牙龈与牙髓组织在炎症中的表型不相同，它们的干细胞属于组织特异的干细胞，在炎性微环境中它们的MMPs与TIMPs表达量的变化趋势也是不同的。可以认为GMSC在炎症微环境中MMPs与TIMPs表达变化规律具有其特异性，符合临床观察到的牙龈炎性增生，失去正常的解剖外形，牙龈充血，红肿增生。牙髓往往会出现牙髓的液化和坏死的炎性反应。
Human gingival tissues are prone to develop hyperplasia under multipleexternal stimili. We have recently shown that normal and hyperplastic gingivaltissue contains mesenchymal stem cells (GMSCs), but it is not known how thesecells are influenced by commonly seen inflammatory conditions. We isolatedGMSCs from human normal (N-GMSC) and inflammatory gingival tissues(I-GMSC). Meanwhile, an in vitro culture with TNF-α and IL-1β wasestablished to simulate the in vivo inflammatory situation. In these two settingswe observed consistent results that GMSC affected by inflammatory factorsproliferated more actively than those under normal condition, while lostpotential of osteogenic and adipogenic differentiation. The expression of matrixmetalloproteinases (MMP)-1/2, IL-1α, IL-6, TNF-α and COL-1wassignificantly higher in I-GMSC than that in N-GMSC. The comparison to dentalpulp stem cells disclosed their intrinsic differences with regard to in vitro geneexpression and in vivo extracellular matrix formation and regulation. Thesefindings suggest that inflammatory factors turn GMSC to a more differentiated and pro-fibrotic phenotype, which could underlie the clinical hyperplasticappearance of inflammatory gingiva.
Section1, Histological observation of human normal andinflammation gingival tissues
Experiment1, inflammation gingival tissues exhibited macroscopic tissueover-growth (FIG.1A,B) and thickened layers and elongated rete ridges withabundant cellularity and extracellular matrix within lamina propria by H&Estaining (FIG.1C, D).
Experiment2, immunofluorescence assay showed that inflammation gingivaltissues and cells expressed more abundant collagen I (COL-I, FIG.1K-M; N-O).We analyzed the expression of STRO-1, aputative mesenchymal stem cellmarker and SSEA-4, which was deemed as an early embryonic glycolipidantigen specific for pluripotent embryonic stem cells and had recently been usedto identify and purify stem cells from adult bone marrow in human normal andhyperplastic gingival tissues. Both normal and inflammation gingival propriadisplayed positive STRO-1(FIG.1E-G) and SSEA-4(FIG.1H-J) staining,with the latter marker being more sporadically distributed. These resultsindicated that despite the existence of differences between human normal andhyperplastic gingival tissues, they both showed evidence for harboring cells withfeatures of mesenchymal stem cells.
Section2, Study of separation, culture and biological characteristicsof GMSC
Experiment1, clonogenic MSC can be isolated from gingival tissue. Tocharacterize whether gingival propria-derived MSC is clonogenic, we generatedand cultured single cell suspensions from human normal and inflammationgingival tissues after separating and discarding gingival epithelium byincubating with3mg/ml dispase for30min. With2×103initially seeded cells,both cultures were observed with cells attaching onto the plastic in2days andthen they proliferate rapidly in next5days to form colonies. These colonies were stained with toluidine blue and exhibited typical fibroblastic morphology(FIG.3A). With above results, we termed these clonogenic populations gingivalmesenchymal stem cells (GMSC), with N-GMSC and I-GMSC referring toGMSC derived from normal and inflammation gingival propria respectively.The ability of gingival tissue derived cells to form adherent clonogenic cellclusters had no significant difference (FIG.3B).
Experiment2, by using limiting dilution technique, single cell-derivedcolony cultures were obtained. For N-GMSC,5batches of culture produced42single cell-derived colonies. For I-GMSC,3batches of culture produced34single cell-derived colonies. These colonies were randomly selected forsubsequent analysis. We used flow cytometric analysis to characterize N-GMSCand H-GMSC by surface molecules. Both GMSC within10passages showedthe characteristic pattern of mesenchymal surface markers including STRO-1,CD29, CD44, CD90, CD105, CD146and negatively expressed hematopoieticmarkers CD34and CD45(Fig.3C).
Experiment3, GMSC possesses self renewal and multilineage differentiationability in vitro. The multi-differentiation potential of both GMSC wasdetermined. Under osteogenic induction conditions for3weeks, both GMSCcould specifically differentiate and formed distinct nodules as stained byAlizarin Red S. After feeding GMSC with osteogenic inducing media, dark redmineralised bone matrix (bone nodules) was shown in alizarin red stainedsections. The cells were positively stained with ALP(FIG.4A①). PCR andRT-PCR analysis showed that the expression of the osteogenic markers bonesialoprotein runt-related transcription factor2(RUNX2), osteocalcin (OCN),alkaline phosphatase (ALP) were increased after osteogenic induction(FIG.4B③④). After3weeks of culture in adipogenic induction medium, GMSCproduced lipid droplets, the hallmark of functional adipogenesis. Alkalinephosphatase activity staining (2weeks) showed I-GMSC lighter thanN-GMSC.(FIG.4A④).
Adipogenic differentiation of GMSC was confirmed by Oil Red-O staining.After feeding GMSCs with adipogenic inducing media for21days, oil dropletswere presented in cytoplasm(FIG.4B①). Adipogenic differentiation was furtherconfirmed by the increased expression of specific adipogenic markers includingperoxisome proliferator-activated receptor γ (PPARγ). All are determined byRT-PCR (FIG.4A⑤B④). Through quantitative comparison of areas fromrandomly selected staining images, or grayscale analysis of RT-PCR bands, wehave found significant differences between N-and I-GMSC groups (datashown). To further compare the enzymatic activity related to ECM remodeling,we performed immunostaining and found that Collagen type1(COL-1)expression within N-GMSC was significantly lower than that in I-GMSC (FIG.1NO), suggesting role of COL-1expression within I-GMSC in mediating thepathological processes.
Section3, N-GMSC and I-GMSC show some special characteristicboth in vitro and in vivo
Experiment1, MTT assay and Cell Cycle showed that the growth tendencyof I-GMSC waw higher than that on the healthy group (FIG.5A,B). Accordingto MTT assay, concentration sitmulates the most N-GMSC proliferation was asthe final extrinsic source inflammatory factor IL-1β (5ng/ml) and TNF-α(10ng/ml)(FIG.5C). With extrinsic source inflammatory factor to N-GMSCshowed more apoptosis than that without inflammatory factor, and especiallywith extrinsic source inflammatory factor and Osteogenesis to N-GMSC showedthe most apoptosis (FIG.5D). N-GMSC possessed self renewal and multilineagedifferentiation ability in inflammatory microenvironment being reduced byRT-PCR analysis and alkaline phosphatase activity staining (FIG.6A,B,C).
Experiment2, GMSC expresses some cytokine in vivo. The present studyexamined HGFs from healthy and inflammatory gingival tissues using DNAmicroarray analysis. The expression levels of TIMP1, TIMP2, IL-1α, IL-6,TNF-α, and COL-1in the HGFs of the Inflammatory group were higher than those in the healthy group on DNA microarray analysis, MMP1MMP2lowerthan those in the N-GMSC (figure3.2.1).
Experiment3, these results showed that the DPSCs in inflammatory produceelevated levels of these MMP1/2, IL-1α, IL-6, TNF-α and COL-1proteins(figure3.3.1). Cytokines are soluble, biologically active glycoproteins, whichare secreted by host immuno-inflammatory cells. They have many functions andexert their effects in a paracrine or autocrine fashion to modulate inflammatoryand immune responses. It is likely that elevated levels of pro-inflammatorycytokines play a role in stimulating osteoclast activity. These cytokines are thekey of mediators in immunological and inflammatory responses, and have beenfound in measurable quantities in areas of active gingical inflammation.Therefore, the gingival tissues may produce various inflammatory cytokines.We demonstrated that the I-GMSC of inflammatory gingival tissues expressedsignificantly higher levels of IL-1α, IL-6, and TNF-α than the N-GMSC ofgingival tissues from healthy individuals did. Our data in the present studysuggest that cytokines are involved in chronic inflammatory conditions such asmechanical irritation-induced human gingival hyperplasia or gingivalinflammation.
Experiment4, to better confirm that the N-GMSC and I-GMSC showedsome special characteristic in vitro, we proposed cell sheet engineering in vivo.A cell sheet was constructed firstly; then the cell sheet shrank and condensedinto an intact cell pellet; finally in vivo differentiation assay ofN-GMSC/N-GMSC (exogenous inflammatory factor) and N-DPSC/N-DPSC(exogenous inflammatory factor) pellets was performed using immunodeficientmice. All the cell pellets had good biology properties. It also found that MMPand TIMP had different changing tendency with N-GMSC (exogenousinflammatory factor) and N-DPSC (exogenous inflammatory factor). Theseresults showed that the DPSCs in inflammatory produced elevated levels ofthese MMP1, MMP2oppositing to that of GMSC (FIG.7C,D).
This study showed that the expression of IL-1α, IL-6, col-1and TNF-α ofboth GMSC and DPSC increased in a time-dependant manner in vitro under aninflammatory microenvironment. However, as both GMSC and DPSC were twotissue-specific stem cells, so the expression of IL-1α in GMSC was relativelystronger than that in DPSC and the expression of IL-6in DPSC was relativelystronger than that in GMSC. Stem cells turned into TA in advance. After that,TA involved in the inflammatory reaction and its autocrine inflammatory factorwent into a positive feedback loop again. Do the expression differences of IL-1and IL-6between the stem cells derived from2kinds of tissue origins (GMSCand DPSC) under inflammatory microenvironment cause the difference ofchange trend of MMPs and MIMPs expression?
In the study, it was found that the I-GMSC was actually the TA which wasturned from N-GMSC in advance by the action of inflammatory factor. It stillcould be cloned and the osteogenic and adipogenic ability decreased in themulti-directional differentiation potential stage. It showed strong proliferativecapacity, and increased the extracellular matrix, being consistent with theclinical phenotype. The inflammatory factors could increase the apoptosis ofN-GMSC and more at the differentiation potential stage. The phenotype wasregulated by MMPs, TIMPs, and inflammatory factors. Currently, the influenceof inflammation on GMSC is clear. While it is still unknown that how dospecific expression of the MMPs, TIMPs and inflammatory factors have aneffect on their clinical proliferative phenotypes, and it can be screened in thefuture.
引文
1. Nanci A, Somerman M, Squier CA, et al. The Periodontium: Development,Structure and Function. Ten Cate’s Oral Histology.2003;276-279.
2. Talic NF, Evans CA, Daniel JC, Zaki AE. Proliferation of epithelial rests ofMalassez during experimental tooth movement. Am J Orthod DentofacialOrthop.2003;123:527-533.
3.程国芬.固定矫治患者口腔保健方法的探讨.浙江临床医学.2008;30(4):393-394.
4.陈文英,黄吉娜，徐学军等.口腔正畸固定矫治器应用中菌斑的临床调查.浙江临床医学.2004;6（4）:270.
5. Trosscllo VK, Gianclly AA. Orthodontic treatment and periodontal status JPeriodontal.1979.50:665.
6.李松,孙卫斌,吴亚菲.牙龈上皮细胞凋亡的研究凋亡调节蛋白的表达与药物性牙龈增生.国外医学口腔医学分册.2004;31(6):447-451.
7. Kataoka M, Kido J, Shinohara Y, et a1. Drug-induced gingival overgrowth-areview. Biol Pharm Bull.2005;28(10):1817-1821.
8. Cetinkaya BO, Acikgoz G, Ayas B, et al. Increased expression of vascularendothelial growth factor in cyclosporin A-induced gingival overgrowth inrats. J Periodontol.2006;77(1):54-60.
9. Meng HX. Periodontology.3rd ed. Beijing：People，S Medical PublishingHouse.2008;12-13,18,145.
10. Liu PH, MA Su. Pathogenesis of Ciclosporin-induced gingival epithelialhyperplasia. Int: J Stomatol.2009;36(1):81-83.
11. Barclay S, Thomason JM, Idle JR, et al. The incidence and severity ofnifedipine induced gingival overgrowth. J Clin Periodontal.1992;19(5):311-314.
12. Kantarei A, Augustin P, Firatli E, et a1. Apoptosis in gingival overgrowthtissues. J Dent Res.2007;86(9):888-892.
13. Williamson MS, Miller EK, Plemons J, et al. Cyclosporine A upregulatesInterleukin-6gene expression in human gingival: possible mechanism forgingival overgrowth. J Periodontol.1994;65(10):895-903.
14. Ates S, Ng MC, Dill RE, et al.Cyclosporine A upregulateds platelet derivedgrowth factor B chain in hyperplastic human gingiva. J Periodontol.1996;67(3):271-278.
15. Das SJ, Newman HN, Olsen I, Keratinocyte growth factor receptor isup-regulated in cyclosporine A–induced gingival hyperplasia. J Dent Res.2002;81(10):683-687.
16. Bartram U, Speer CP. The role of transforming growth factor beta in lungdevelopment and disease. Chest.2004:125(2):754-765.
17. Chen S, Jim B, Ziyadeh FN. Diabetic nephropathy and transforming growthfactor-beta： transforming our view of glomerulosclerosis and fibrosisbuild-up. Semin Nephrol.2003;23(6):532-543.
18. Calabrese F, Valente M, Giacometti C, et al. Parenchymal transforminggrowth factor beta-1: its type II receptor and Smad signaling pathwaycorrelate with inflammation and fibrosis-in chronic liver disease of viraletiology. J Gastroenterol I-Iepatol.2003;18(11): l302-1308.
19. Kuru L, Yilmz S, Kuru B, et a1. Expression of growth factors in thegingival crevice fluid of patients with phenyt0in-induced gingivalenlargement. Arch Oral Biol.2004;49(11):945-950.
20. Xiao R, Liu FY, Luo JY, et a1. Effect of small interfering RNA on theexpression of connective tissue growth factor and type I and III collagen inskin fibroblasts of patients with systemic sclerosis. J Dermatol.2006;155(6):1145-1153.
21. Uzel MI, Kantarci A, Hong HH, et a1. Connective tissue growth factor indrug-induced gingival overgrowth. J Periodontol.2001;72(7):921-93l.
22. Iacopino AM， Doxey D，Cutler CW，et a1． Phenytoin and cyclosporineA specifically regulate macrophage and expression of platelet-derivedgrowth factor and interleukin--1in vitro and in vivo：possible mechanism ofdrug-induced overgrowth. J Periodontol.1997;68(1):73-83.
23.李松,孙卫斌,吴亚菲．凋亡调节蛋白的表达与药物性牙龈增生.国外医学.口腔医学分册.2004;31(6):447-448..
24. Buduneli E, Genel F, Atilla G, et a1. Evaluation of p53, bcl-2, andinterleukin-15levels in gingival crevicular fluid of cyclosporin A-treatedpatients. J Periodontal.2003;74(4):506-511.
25. Shimizu Y, Kataoka M, Seto H, et a1. Nifedipine induces gingi-valepithelial hyperplasia in rats through inhibition of apoptosis. J Periodontol.2002;73(8):861-867.
26.钱虹，肖殷.纤维调节素在大鼠和人类牙龈组织中的差异性表达.口腔医学研究.2006;22(4):357-358.
27. Hoang AM, Chen D, Oates TW, et a1. Development and characterization ofa transformed human periodontal ligament cell line. Periodontol.1997;68(11):1054-1062.
28. Bartold PM, Walsh LJ, Narayanan AS, et a1. Molecular and cell biology ofthe gingival. Periodontology.2000;24(1):28-55.
29. FujitaT, twataT, Shiba H, et al. ldentification of marker genes distinguishinghuman periodontal ligament cells from human mesenehymal stem cells andhuman gingival fibroblasts. J Periodontal Res.2007;42(3):283-286.
30. Kirschner M, Montazem A, Hilaire HS, Radu A. Long-term culture ofhuman gingival keratinocyte progenitor cells by down-regulation of14-3-3sigma. Stem Cells Dev.2006;15:556-565.
31. Gagari E, Rand MK, Tayari L, Vastardis H, Sharma P, Hauschka PV,Damoulis PD. Expression of stem cell factor and its receptor, c-kit, inhuman oral mesenchymal cells. Eur J Oral Sci.2006;114:409-415.
32. Dooner GJ, Colvin GA, Dooner MS, Johnson KW, Quesenberry PJ. Geneexpression fluctuations in murine hematopoietic stem cells with cell cycleprogression. J Cell Physiol.2008;214:786-795.
33. Naveau A, Reinald N, Fournier B, Durand E, Lafont A, Coulomb B, GoglyB. Gingival fibroblasts inhibit MMP-1and MMP-3activities in an ex-vivoartery model. Connect Tissue Res.2007;48:300-308.
34.许彦枝,王坤.骨碎补对人牙龈成纤维细胞超微结构和碱性磷酸酶活性的影响.中国实用口腔科杂志.2008;1(3):157-160.
35.任娟,李霞,孙克勤,等.人牙周膜成纤维细胞的原代培养和鉴定.中国药物与临床.2007;9:672-673.
36. Ivanovski S, Li H, Haase H, et a1. Expression of bone associated maemmolecules by gingival and periodontal ligament fibroblasts. J PeriodontalRes.2001;36(3):131-141.
37. Carnes DL, Maeder CL, Graves DT. Cells with osteoblastic phenotypes canbe explanted from human gingival and periodontal ligament. J Periodontol.1997;68(7):701-706.
38. J Hillmann G, Steinkamp-Zucht A, Geurtsen W, et a1. Culture of primaryhuman gingival fibroblasts on biodegradable membranes. Bio-materials.2002;23(6):1461-1469.
39. Murphy MG, Mailhot J, Burke J, et a1. The efects of rhBMP-2on humanosteosarcoma cells and human gingival fibroblasts in vitro. J OralImplantol.2001;27(1):16-24.
40.成党校,黄桂君,钱桂生.新型基因转染阳离子脂质体研究进展.国外医学药学分册.2000;27(5):257-260.
41. Katagiri T, Yamaguehi A, Komaki M, et a1. Bone morphogenetic protein-2conveys the diferetiation pathway of C2C12myoblasts into osteoblastlineage. J Cell Biol.1994;127(6): l755-l766.
42. Nakajima K, Abe T, Tanaka M, Hara Y. Periodontal tissue engineering bytrans-plantation of multilayered sheets of phenotypically modified gingivalfibroblasts. J Periodontal Res.2008;43:681-688.
43. Kook SH, Son YO, Choe Y, Kim JH, Jeon YM, Heo JS, Kim JG, Lee JC.Mechanical force augments the anti-osteoclastogenic potential of humangingival fibroblasts in vitro. J Periodontal Res.2009;44:402-410.
44. Zhang Q, Shi S, Liu Y, Uyanne J, Shi Y, Shi S, Le AD. Mesenchymal stemcells derived from human gingiva are capable of immunomodulatoryfunctions and ameliorate inflammation-related tissue destruction inexperimental colitis. J Immunol.2009;183:7787-7798.
45. Fournier BP, Ferré F, Couty L, et al. Multipotent progenitor cells in gingivalconnective tissue. Tissue Eng Part A.2010:[Epub ahead of print] doi:
10.1089/ten.TEA.2009.0796.
46. Liang Tang, Nan Li, Han Xie, Yan Jin. Characterization of MesenchymalStem Cells from Human Normal and Hyperplastic Gingiva. J Cell Physiol.2010;226(3):832-842.
47. Geetanjali BT, Rupesh K. Srivastava, et a1. Human gingiva-derivedmesenchymal stem cells are superior to bone marrow-derived mesenchymalstem cells for cell therapy in regenerative medicine. BBRC.2010;393(3):377-383.
48. da Silva CL, Gon alves R, Porada CD, Ascens o JL, Zanjani ED, CabralJM. Almeida-Porada G. Differences amid bone marrow and cord bloodhematopoietic stem/progenitor cell division kinetics. J Cell Physiol.2009;220:102-111.
49. Valtieri M, Sorrentino A. The mesenchymal stromal cell contribution tohomeostasis. J Cell Physiol.2008;217:296-300.
50. Arthur A, Zannettino A, Gronthos S. The therapeutic applications ofmultipotential mesenchymal/stromal stem cells in skeletal tissue repair. JCell Physiol.2009;218:237-245.
51. Andreasen JO, L vschall H. Response of Oral Tissues to Trauma. In:Textbook and Color Atlas of Traumatic Injuries to the Teeth,4th Edition.Copenhagen, Denmark: Wiley-Blackwell.2007; p.62-113.
52. Rossmann JA, Ingles E, Brown RS. Multimodal treatment of drug-inducedgingival hyperplasia in a kidney transplant patient. Compendium.1994;15(10):1266,1268-1270,1272-1274; quiz1276.
53. Botler RT. Drug-induced gingival hyperplasia: phenytoin, cyclosporine, andnifedipine. JADA.1987; I14(1):56-60.
54. Guo J, Wang W, Yao L, Yan F. Local inflammation exacerbates cyclosporinea-induced gingival overgrowth in rats. Inflammation.2008;31(6):399-407.
55. Sodek J. A comparison of collagen and non-collagenous protein metabolismin rat molar and incisor periodontal ligaments. Arch Oral Biol.1978;23(11):977-982.
56. Sodek J, Ferrier JM. Collagen remodeling in rat periodontal comensationfor precursor reutilization confirms rapid tumover of collagen. Coll RdlatRes.1988;8(1):11-21.
57. Arora PD, Silvestri L, Ganss B, et al. Mechanism of cyclosporin-inducedinhibition of intracellular collagen degradation. J Biol Chem.2001;276(17):14100-14109.
58. Nares S, Ng MC, Dill RE, et al. Cyclosponne A upregulates platelet-derivedgrowth factor B chain in hyperplastic human gingival. J Periodontol.1996;67(3):271-278.
59. Trackman PC, Kantarci A. Connective tissue metabolism and gingivalovergrowth. Crit Rev Oral Med.2004;15(3):165-175.
60. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatalstem cells from human periodontal ligament. Lancet.2004;364:149-155.
61. Liu HW, Yacobi R, Savion N, Narayanan AS, Pitaru S. A collagenouscementum-derived attachment protein is a marker for progenitors of themineralized tissue-forming cell lineage of the periodontal ligament. J BoneMiner Res.1997;12(10):1691-1699.
62. Dean JW, Blankenship JA. Migration of glngival fibroblasts on fibronectinand laminin. J Periodontol.1997;68(8):750-757.
63.刘斌,吴军正,司徒镇强,等.人牙龈成纤维细胞系的建立及其生物学特性．牙体牙髓牙周病学杂志.1999;9(1):34-36．
64.司徒振强,吴军正.细胞培养[M].西安：世界图书出版社:1996;65,71.
65. Yoshida CA, Furuichi T, Fujita T, et al. Core-binding factor beta interactswith Runx2and is required for skeletal development. Nat Genet.2002;32:633-638.
66. Lu H, Kraut D, Gerstenfeld LC, Graves DT. Diabetes interferes with thebone formation by affecting the expression of transcription factors thatregulate osteoblast differentiation. Endocrinology.2003;144:346-352.
67. Xiao G, Jiang D, Ge C, et al. Cooperative interactions between activatingtranscription factor4and Runx2/Cbfa1stimulate osteoblast-specificosteocalcin gene expression. J Biol Chem.2005;280,30689-30696.
68. Kumar S, Mahendra G, Ponnazhagan S. Determination of osteoprogenitor-specific promoter activity in mouse mesenchymal stem cells byrecombinant adeno-associated virus transduction. Biochim Biophys Acta.2005;1731:95-103.
69. Beklen A, Ainola M, Hukkanen M, et al. MMPs, IL-1, and TNF areregulated by IL-17in periodontitis. J Dent Res.2007;86(4):347-351.
70. Carlos Rossa Jr, Min Liub, Chetan Patilc, Keith L. MKK3/6-p38MAPKnegatively regulates murine MMP-13gene expression induced by IL-1βandTNF-αin immortalized periodontal ligament fibroblasts. Matrix Biology.2005;24(7):478-488.
71. Kasasa SC, Soory M. The response of human gingival fibroblasts tointerleukin-1in their metabolic conversion of two androgenic substrates.Arch Oral Biol.1995;40(10):979-981.
72.于艳玲,许丽华,王俊霞,等.内毒素对牙龈成纤维细胞增殖活性及分泌基质金属蛋白酶的影响. J Modem Stomatol.2010;24(1):60-63.
73. Kantarci A, Augustin P, Firatli E, et al. Apoptosis in Gingival OvergrowthTissues. J Dent Res.2007;86(9):888-892.
74. Jung JY, Jeong YJ, Jeong TS, et al. Inhibition of apoptotic signals inovergrowth of human gingival fibroblasts by cyclosporin A treatment. ArchOral Biol.2008;53(11):1042-1049.
75. Waga L, Nakamura M, Honda Z, et al. Two distinct signal transductionpathway for the activation of guinea-pig macrophges and neutrophils byendotoxin. Biochem Biophysic Res Commun.1993;197(2):465-472.
76. Introna M, Hamiton TA, Kaufman RE. Treatment of murine peritonealmacrophages with bacterial lipopolysaccharide alters expression of c-Fosand c-myc oneogenes. J Immunol.1986;137(8):2711-2715.
77. Nakane A, YoshideT, NakataK, et a1. Efects of lipepelysaecharide onhuman dental pulp cells. J Endodon.1995;2l(3):128-135.
78. Harada Y. Efect of lipopolysaccharide on thymidine salvage as related tomacrephage activation. Immunology.1995;84:247-253.
79. Kim JY, ParkSH, Cho KS, et al. Mechanism of azithromycin treatment ongingival overgrowth. J Dent Res.2008;87(11):1075-1079.
80. Guo XK, ZhaWQ, Kondo C, et al. Tissue inhibitors of metalloproteinases-1(TIMP-1) and-2(TIMP-2) are major serum factors that stimulate theTIMP-1gene in human gingival fibroblasts. Biochimica Biophysica Acta.2006;1763(3):296-304.
81. Al Haj Zen A, Lafonta A, Duranda E, et al. Effect of adenovirus-mediatedoverexpression of decorin on metalloproteinases, tissue inhibitors ofmetalloproteinases and cytokines secretionby human gingival fibroblasts.Matrix Biol.2003;22(3):251-258.
82. Tewari M, Tuncay OC, Milchman A, er al. Association of interleukin-1-induced, NFκB DNA-binding activity with collagenasegene expressionin human gingival fibroblasts. Archs Oral Biol.1996;41(5):461-468.
83. Wang PL, Azuma Y, Shinohara M, Ohura K.Toll-like Receptor4-MediatedSignal Pathway Induced by Porphyromonas gingivalis Lipopolysaccharidein Human Gingival Fibroblasts. Biochimica Biophysica Acta.2000;273(3):1161-1167.
84. Dahan M, Nawrocki B, Elkaim R, et a1. Expression of matrixmetalloproteinases in healthy and diseased human gingival. J ClinPeriodontol.2001;28(2):128-136.
85. Cury PR, de Arau′jo VC, Canavez F, et al. The effect of epidermal growthfactor on matrix metalloproteinases and tissue inhibitors ofmetalloproteinase gene expression in cultured human gingival fibroblasts.Archs Oral Biol.2007;52(6):585-590.
86.陆怀秀,康雅斌,牛忠英,等. IL1-ra对IL-1β诱导人牙龈成纤维细胞分泌IL-6的拮抗作用. J Pract Stomatol.2002;18(1):17-19.
87. Dom′nguez, A, Go′mez C, Garc′a-Kass AI, Garc′a-Nun ez JA. IL-1beta,TNF-alpha, total antioxidative status and microbiological findings inchronic periodontitis treated with fluorescence-controlled Er:YAG laserradiation. Lasers Surg Med.2010;42(1):24-31.
88. Kent LW, Dyken R A, RahemtullaA F, Allison AC, Michalek SM. Effect ofin vitro passage of healthy human gingival fibroblasts on cellulamorphology and cytokine expression. Arch Oral Biol.1996;41(3):263-270.
89. Tu¨lay Yucel-Lindberg, Brunius G. Epidermal growth factor synergisticallyenhances interleukin-8production in human gingival fibroblasts stimulatedwith interleukin-1β. Archs of Oral Biol.2006;51,892-898.
90. Takigawa M, Takashiha S, Takahashi K, et a1. Prostaglandin E2inhibitsinterleukin-6release but not its transcription in human gingival fibroblastsstimulated with intedeukin-1beta or tuinor necrosis factor-alpha. JPeriodontol.1994;65:1122-1127.
91. Noguchi K, Shitashige M, Endo H, et a1. Binary regulation ofinterleukin(IL)-6production by EP1and EP2／EP4subtypes of PGEzreceptors in IL-1beta-stimulated human gingival fibmblasts. J PeriodontalRes.2002;37:29-36.
92. Czuszak CA, Sutherland DE, Billman MA, et al. Prostaglandin E2potentiates interleukin-1beta mduced interleukin-6production by humangingival fibroblasts. J CIin Period onto1.1996;23:635-640.
93.和璐, Nagasawa T, Ishikawa I. IL-1α刺激人牙龈成纤维细胞产生IL-11和IL-6的比较研究. J Modem Stomatol.2006;20(2):157-160.
94. Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulpstem cells. J Dent Res.2002;81(8):531-535.
95. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stemcells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A.2000;97(25):13625-13630.
96. Huang G. T.–J, Grantors S, Shi. S. Mesenchymal Stem Cells Derived fromDental Tissues vs. Those from Other Sources: Their Biology and Role inRegenerative Medicine. J Dent Res.2009;88(9):792-806.
97. Akahane M, Nakamura A, Ohgushi H, et al. Osteogenic matrix sheet-celltransplantation using osteoblastic cell sheet resulted in bone formationwithout scaffold at an ectopic site. J Tissue Eng Regen Med.2008;2:196-201.
98. Ohgushi H, Dohi Y, Katuda T, et al. Invitro bone formation by rat marrowcell culture. J Biomed Mater Res.1996;32:333-340.
2. Talic NF, Evans CA, Daniel JC, Zaki AE. Proliferation of epithelial rests ofMalassez during experimental tooth movement. Am J Orthod DentofacialOrthop.2003;123:527-533.
3.程国芬.固定矫治患者口腔保健方法的探讨.浙江临床医学.2008;30(4):393-394.
4.陈文英,黄吉娜，徐学军等.口腔正畸固定矫治器应用中菌斑的临床调查.浙江临床医学.2004;6（4）:270.
5. Trosscllo VK, Gianclly AA. Orthodontic treatment and periodontal status JPeriodontal.1979.50:665.
6.李松,孙卫斌,吴亚菲.牙龈上皮细胞凋亡的研究凋亡调节蛋白的表达与药物性牙龈增生.国外医学口腔医学分册.2004;31(6):447-451.
7. Kataoka M, Kido J, Shinohara Y, et a1. Drug-induced gingival overgrowth-areview. Biol Pharm Bull.2005;28(10):1817-1821.
8. Cetinkaya BO, Acikgoz G, Ayas B, et al. Increased expression of vascularendothelial growth factor in cyclosporin A-induced gingival overgrowth inrats. J Periodontol.2006;77(1):54-60.
9. Meng HX. Periodontology.3rd ed. Beijing：People，S Medical PublishingHouse.2008;12-13,18,145.
10. Liu PH, MA Su. Pathogenesis of Ciclosporin-induced gingival epithelialhyperplasia. Int: J Stomatol.2009;36(1):81-83.
11. Barclay S, Thomason JM, Idle JR, et al. The incidence and severity ofnifedipine induced gingival overgrowth. J Clin Periodontal.1992;19(5):311-314.
12. Kantarei A, Augustin P, Firatli E, et a1. Apoptosis in gingival overgrowthtissues. J Dent Res.2007;86(9):888-892.
13. Williamson MS, Miller EK, Plemons J, et al. Cyclosporine A upregulatesInterleukin-6gene expression in human gingival: possible mechanism forgingival overgrowth. J Periodontol.1994;65(10):895-903.
14. Ates S, Ng MC, Dill RE, et al.Cyclosporine A upregulateds platelet derivedgrowth factor B chain in hyperplastic human gingiva. J Periodontol.1996;67(3):271-278.
15. Das SJ, Newman HN, Olsen I, Keratinocyte growth factor receptor isup-regulated in cyclosporine A–induced gingival hyperplasia. J Dent Res.2002;81(10):683-687.
16. Bartram U, Speer CP. The role of transforming growth factor beta in lungdevelopment and disease. Chest.2004:125(2):754-765.
17. Chen S, Jim B, Ziyadeh FN. Diabetic nephropathy and transforming growthfactor-beta： transforming our view of glomerulosclerosis and fibrosisbuild-up. Semin Nephrol.2003;23(6):532-543.
18. Calabrese F, Valente M, Giacometti C, et al. Parenchymal transforminggrowth factor beta-1: its type II receptor and Smad signaling pathwaycorrelate with inflammation and fibrosis-in chronic liver disease of viraletiology. J Gastroenterol I-Iepatol.2003;18(11): l302-1308.
19. Kuru L, Yilmz S, Kuru B, et a1. Expression of growth factors in thegingival crevice fluid of patients with phenyt0in-induced gingivalenlargement. Arch Oral Biol.2004;49(11):945-950.
20. Xiao R, Liu FY, Luo JY, et a1. Effect of small interfering RNA on theexpression of connective tissue growth factor and type I and III collagen inskin fibroblasts of patients with systemic sclerosis. J Dermatol.2006;155(6):1145-1153.
21. Uzel MI, Kantarci A, Hong HH, et a1. Connective tissue growth factor indrug-induced gingival overgrowth. J Periodontol.2001;72(7):921-93l.
22. Iacopino AM， Doxey D，Cutler CW，et a1． Phenytoin and cyclosporineA specifically regulate macrophage and expression of platelet-derivedgrowth factor and interleukin--1in vitro and in vivo：possible mechanism ofdrug-induced overgrowth. J Periodontol.1997;68(1):73-83.
23.李松,孙卫斌,吴亚菲．凋亡调节蛋白的表达与药物性牙龈增生.国外医学.口腔医学分册.2004;31(6):447-448..
24. Buduneli E, Genel F, Atilla G, et a1. Evaluation of p53, bcl-2, andinterleukin-15levels in gingival crevicular fluid of cyclosporin A-treatedpatients. J Periodontal.2003;74(4):506-511.
25. Shimizu Y, Kataoka M, Seto H, et a1. Nifedipine induces gingi-valepithelial hyperplasia in rats through inhibition of apoptosis. J Periodontol.2002;73(8):861-867.
26.钱虹，肖殷.纤维调节素在大鼠和人类牙龈组织中的差异性表达.口腔医学研究.2006;22(4):357-358.
27. Hoang AM, Chen D, Oates TW, et a1. Development and characterization ofa transformed human periodontal ligament cell line. Periodontol.1997;68(11):1054-1062.
28. Bartold PM, Walsh LJ, Narayanan AS, et a1. Molecular and cell biology ofthe gingival. Periodontology.2000;24(1):28-55.
29. FujitaT, twataT, Shiba H, et al. ldentification of marker genes distinguishinghuman periodontal ligament cells from human mesenehymal stem cells andhuman gingival fibroblasts. J Periodontal Res.2007;42(3):283-286.
30. Kirschner M, Montazem A, Hilaire HS, Radu A. Long-term culture ofhuman gingival keratinocyte progenitor cells by down-regulation of14-3-3sigma. Stem Cells Dev.2006;15:556-565.
31. Gagari E, Rand MK, Tayari L, Vastardis H, Sharma P, Hauschka PV,Damoulis PD. Expression of stem cell factor and its receptor, c-kit, inhuman oral mesenchymal cells. Eur J Oral Sci.2006;114:409-415.
32. Dooner GJ, Colvin GA, Dooner MS, Johnson KW, Quesenberry PJ. Geneexpression fluctuations in murine hematopoietic stem cells with cell cycleprogression. J Cell Physiol.2008;214:786-795.
33. Naveau A, Reinald N, Fournier B, Durand E, Lafont A, Coulomb B, GoglyB. Gingival fibroblasts inhibit MMP-1and MMP-3activities in an ex-vivoartery model. Connect Tissue Res.2007;48:300-308.
34.许彦枝,王坤.骨碎补对人牙龈成纤维细胞超微结构和碱性磷酸酶活性的影响.中国实用口腔科杂志.2008;1(3):157-160.
35.任娟,李霞,孙克勤,等.人牙周膜成纤维细胞的原代培养和鉴定.中国药物与临床.2007;9:672-673.
36. Ivanovski S, Li H, Haase H, et a1. Expression of bone associated maemmolecules by gingival and periodontal ligament fibroblasts. J PeriodontalRes.2001;36(3):131-141.
37. Carnes DL, Maeder CL, Graves DT. Cells with osteoblastic phenotypes canbe explanted from human gingival and periodontal ligament. J Periodontol.1997;68(7):701-706.
38. J Hillmann G, Steinkamp-Zucht A, Geurtsen W, et a1. Culture of primaryhuman gingival fibroblasts on biodegradable membranes. Bio-materials.2002;23(6):1461-1469.
39. Murphy MG, Mailhot J, Burke J, et a1. The efects of rhBMP-2on humanosteosarcoma cells and human gingival fibroblasts in vitro. J OralImplantol.2001;27(1):16-24.
40.成党校,黄桂君,钱桂生.新型基因转染阳离子脂质体研究进展.国外医学药学分册.2000;27(5):257-260.
41. Katagiri T, Yamaguehi A, Komaki M, et a1. Bone morphogenetic protein-2conveys the diferetiation pathway of C2C12myoblasts into osteoblastlineage. J Cell Biol.1994;127(6): l755-l766.
42. Nakajima K, Abe T, Tanaka M, Hara Y. Periodontal tissue engineering bytrans-plantation of multilayered sheets of phenotypically modified gingivalfibroblasts. J Periodontal Res.2008;43:681-688.
43. Kook SH, Son YO, Choe Y, Kim JH, Jeon YM, Heo JS, Kim JG, Lee JC.Mechanical force augments the anti-osteoclastogenic potential of humangingival fibroblasts in vitro. J Periodontal Res.2009;44:402-410.
44. Zhang Q, Shi S, Liu Y, Uyanne J, Shi Y, Shi S, Le AD. Mesenchymal stemcells derived from human gingiva are capable of immunomodulatoryfunctions and ameliorate inflammation-related tissue destruction inexperimental colitis. J Immunol.2009;183:7787-7798.
45. Fournier BP, Ferré F, Couty L, et al. Multipotent progenitor cells in gingivalconnective tissue. Tissue Eng Part A.2010:[Epub ahead of print] doi:
10.1089/ten.TEA.2009.0796.
46. Liang Tang, Nan Li, Han Xie, Yan Jin. Characterization of MesenchymalStem Cells from Human Normal and Hyperplastic Gingiva. J Cell Physiol.2010;226(3):832-842.
47. Geetanjali BT, Rupesh K. Srivastava, et a1. Human gingiva-derivedmesenchymal stem cells are superior to bone marrow-derived mesenchymalstem cells for cell therapy in regenerative medicine. BBRC.2010;393(3):377-383.
48. da Silva CL, Gon alves R, Porada CD, Ascens o JL, Zanjani ED, CabralJM. Almeida-Porada G. Differences amid bone marrow and cord bloodhematopoietic stem/progenitor cell division kinetics. J Cell Physiol.2009;220:102-111.
49. Valtieri M, Sorrentino A. The mesenchymal stromal cell contribution tohomeostasis. J Cell Physiol.2008;217:296-300.
50. Arthur A, Zannettino A, Gronthos S. The therapeutic applications ofmultipotential mesenchymal/stromal stem cells in skeletal tissue repair. JCell Physiol.2009;218:237-245.
51. Andreasen JO, L vschall H. Response of Oral Tissues to Trauma. In:Textbook and Color Atlas of Traumatic Injuries to the Teeth,4th Edition.Copenhagen, Denmark: Wiley-Blackwell.2007; p.62-113.
52. Rossmann JA, Ingles E, Brown RS. Multimodal treatment of drug-inducedgingival hyperplasia in a kidney transplant patient. Compendium.1994;15(10):1266,1268-1270,1272-1274; quiz1276.
53. Botler RT. Drug-induced gingival hyperplasia: phenytoin, cyclosporine, andnifedipine. JADA.1987; I14(1):56-60.
54. Guo J, Wang W, Yao L, Yan F. Local inflammation exacerbates cyclosporinea-induced gingival overgrowth in rats. Inflammation.2008;31(6):399-407.
55. Sodek J. A comparison of collagen and non-collagenous protein metabolismin rat molar and incisor periodontal ligaments. Arch Oral Biol.1978;23(11):977-982.
56. Sodek J, Ferrier JM. Collagen remodeling in rat periodontal comensationfor precursor reutilization confirms rapid tumover of collagen. Coll RdlatRes.1988;8(1):11-21.
57. Arora PD, Silvestri L, Ganss B, et al. Mechanism of cyclosporin-inducedinhibition of intracellular collagen degradation. J Biol Chem.2001;276(17):14100-14109.
58. Nares S, Ng MC, Dill RE, et al. Cyclosponne A upregulates platelet-derivedgrowth factor B chain in hyperplastic human gingival. J Periodontol.1996;67(3):271-278.
59. Trackman PC, Kantarci A. Connective tissue metabolism and gingivalovergrowth. Crit Rev Oral Med.2004;15(3):165-175.
60. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatalstem cells from human periodontal ligament. Lancet.2004;364:149-155.
61. Liu HW, Yacobi R, Savion N, Narayanan AS, Pitaru S. A collagenouscementum-derived attachment protein is a marker for progenitors of themineralized tissue-forming cell lineage of the periodontal ligament. J BoneMiner Res.1997;12(10):1691-1699.
62. Dean JW, Blankenship JA. Migration of glngival fibroblasts on fibronectinand laminin. J Periodontol.1997;68(8):750-757.
63.刘斌,吴军正,司徒镇强,等.人牙龈成纤维细胞系的建立及其生物学特性．牙体牙髓牙周病学杂志.1999;9(1):34-36．
64.司徒振强,吴军正.细胞培养[M].西安：世界图书出版社:1996;65,71.
65. Yoshida CA, Furuichi T, Fujita T, et al. Core-binding factor beta interactswith Runx2and is required for skeletal development. Nat Genet.2002;32:633-638.
66. Lu H, Kraut D, Gerstenfeld LC, Graves DT. Diabetes interferes with thebone formation by affecting the expression of transcription factors thatregulate osteoblast differentiation. Endocrinology.2003;144:346-352.
67. Xiao G, Jiang D, Ge C, et al. Cooperative interactions between activatingtranscription factor4and Runx2/Cbfa1stimulate osteoblast-specificosteocalcin gene expression. J Biol Chem.2005;280,30689-30696.
68. Kumar S, Mahendra G, Ponnazhagan S. Determination of osteoprogenitor-specific promoter activity in mouse mesenchymal stem cells byrecombinant adeno-associated virus transduction. Biochim Biophys Acta.2005;1731:95-103.
69. Beklen A, Ainola M, Hukkanen M, et al. MMPs, IL-1, and TNF areregulated by IL-17in periodontitis. J Dent Res.2007;86(4):347-351.
70. Carlos Rossa Jr, Min Liub, Chetan Patilc, Keith L. MKK3/6-p38MAPKnegatively regulates murine MMP-13gene expression induced by IL-1βandTNF-αin immortalized periodontal ligament fibroblasts. Matrix Biology.2005;24(7):478-488.
71. Kasasa SC, Soory M. The response of human gingival fibroblasts tointerleukin-1in their metabolic conversion of two androgenic substrates.Arch Oral Biol.1995;40(10):979-981.
72.于艳玲,许丽华,王俊霞,等.内毒素对牙龈成纤维细胞增殖活性及分泌基质金属蛋白酶的影响. J Modem Stomatol.2010;24(1):60-63.
73. Kantarci A, Augustin P, Firatli E, et al. Apoptosis in Gingival OvergrowthTissues. J Dent Res.2007;86(9):888-892.
74. Jung JY, Jeong YJ, Jeong TS, et al. Inhibition of apoptotic signals inovergrowth of human gingival fibroblasts by cyclosporin A treatment. ArchOral Biol.2008;53(11):1042-1049.
75. Waga L, Nakamura M, Honda Z, et al. Two distinct signal transductionpathway for the activation of guinea-pig macrophges and neutrophils byendotoxin. Biochem Biophysic Res Commun.1993;197(2):465-472.
76. Introna M, Hamiton TA, Kaufman RE. Treatment of murine peritonealmacrophages with bacterial lipopolysaccharide alters expression of c-Fosand c-myc oneogenes. J Immunol.1986;137(8):2711-2715.
77. Nakane A, YoshideT, NakataK, et a1. Efects of lipepelysaecharide onhuman dental pulp cells. J Endodon.1995;2l(3):128-135.
78. Harada Y. Efect of lipopolysaccharide on thymidine salvage as related tomacrephage activation. Immunology.1995;84:247-253.
79. Kim JY, ParkSH, Cho KS, et al. Mechanism of azithromycin treatment ongingival overgrowth. J Dent Res.2008;87(11):1075-1079.
80. Guo XK, ZhaWQ, Kondo C, et al. Tissue inhibitors of metalloproteinases-1(TIMP-1) and-2(TIMP-2) are major serum factors that stimulate theTIMP-1gene in human gingival fibroblasts. Biochimica Biophysica Acta.2006;1763(3):296-304.
81. Al Haj Zen A, Lafonta A, Duranda E, et al. Effect of adenovirus-mediatedoverexpression of decorin on metalloproteinases, tissue inhibitors ofmetalloproteinases and cytokines secretionby human gingival fibroblasts.Matrix Biol.2003;22(3):251-258.
82. Tewari M, Tuncay OC, Milchman A, er al. Association of interleukin-1-induced, NFκB DNA-binding activity with collagenasegene expressionin human gingival fibroblasts. Archs Oral Biol.1996;41(5):461-468.
83. Wang PL, Azuma Y, Shinohara M, Ohura K.Toll-like Receptor4-MediatedSignal Pathway Induced by Porphyromonas gingivalis Lipopolysaccharidein Human Gingival Fibroblasts. Biochimica Biophysica Acta.2000;273(3):1161-1167.
84. Dahan M, Nawrocki B, Elkaim R, et a1. Expression of matrixmetalloproteinases in healthy and diseased human gingival. J ClinPeriodontol.2001;28(2):128-136.
85. Cury PR, de Arau′jo VC, Canavez F, et al. The effect of epidermal growthfactor on matrix metalloproteinases and tissue inhibitors ofmetalloproteinase gene expression in cultured human gingival fibroblasts.Archs Oral Biol.2007;52(6):585-590.
86.陆怀秀,康雅斌,牛忠英,等. IL1-ra对IL-1β诱导人牙龈成纤维细胞分泌IL-6的拮抗作用. J Pract Stomatol.2002;18(1):17-19.
87. Dom′nguez, A, Go′mez C, Garc′a-Kass AI, Garc′a-Nun ez JA. IL-1beta,TNF-alpha, total antioxidative status and microbiological findings inchronic periodontitis treated with fluorescence-controlled Er:YAG laserradiation. Lasers Surg Med.2010;42(1):24-31.
88. Kent LW, Dyken R A, RahemtullaA F, Allison AC, Michalek SM. Effect ofin vitro passage of healthy human gingival fibroblasts on cellulamorphology and cytokine expression. Arch Oral Biol.1996;41(3):263-270.
89. Tu¨lay Yucel-Lindberg, Brunius G. Epidermal growth factor synergisticallyenhances interleukin-8production in human gingival fibroblasts stimulatedwith interleukin-1β. Archs of Oral Biol.2006;51,892-898.
90. Takigawa M, Takashiha S, Takahashi K, et a1. Prostaglandin E2inhibitsinterleukin-6release but not its transcription in human gingival fibroblastsstimulated with intedeukin-1beta or tuinor necrosis factor-alpha. JPeriodontol.1994;65:1122-1127.
91. Noguchi K, Shitashige M, Endo H, et a1. Binary regulation ofinterleukin(IL)-6production by EP1and EP2／EP4subtypes of PGEzreceptors in IL-1beta-stimulated human gingival fibmblasts. J PeriodontalRes.2002;37:29-36.
92. Czuszak CA, Sutherland DE, Billman MA, et al. Prostaglandin E2potentiates interleukin-1beta mduced interleukin-6production by humangingival fibroblasts. J CIin Period onto1.1996;23:635-640.
93.和璐, Nagasawa T, Ishikawa I. IL-1α刺激人牙龈成纤维细胞产生IL-11和IL-6的比较研究. J Modem Stomatol.2006;20(2):157-160.
94. Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulpstem cells. J Dent Res.2002;81(8):531-535.
95. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stemcells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A.2000;97(25):13625-13630.
96. Huang G. T.–J, Grantors S, Shi. S. Mesenchymal Stem Cells Derived fromDental Tissues vs. Those from Other Sources: Their Biology and Role inRegenerative Medicine. J Dent Res.2009;88(9):792-806.
97. Akahane M, Nakamura A, Ohgushi H, et al. Osteogenic matrix sheet-celltransplantation using osteoblastic cell sheet resulted in bone formationwithout scaffold at an ectopic site. J Tissue Eng Regen Med.2008;2:196-201.
98. Ohgushi H, Dohi Y, Katuda T, et al. Invitro bone formation by rat marrowcell culture. J Biomed Mater Res.1996;32:333-340.