【作者】 秦雁雁；

【导师】 林崇韬；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2012， 硕士

【摘要】 牙周炎是由牙菌斑中微生物所引起的慢性感染性疾病，表现为牙周支持组织炎症、牙周袋形成、进行性附着丧失和牙槽骨吸收，最终导致牙松动和拔除，是我国成人丧失牙齿的主要原因。研究发现，牙周病可能成为全身健康的威胁，而系统性疾病也能加速牙周病的发展。牙周病治疗的根本目的是控制菌斑，消除炎症，促进牙周组织修复再生，但是通过牙周治疗获得的牙周组织再生非常有限。随着组织工程学的发展，将组织工程技术应用于牙周组织再生已受到众多学者的关注。按照组织工程学原理组织再生的三要素是种子细胞、支架材料和生长因子。研究发现牙周膜细胞具有干细胞潜能,它在牙根面的黏附、增殖和分化直接影响牙周组织的再生和修复，是理想的种子细胞之一。碱性成纤维细胞生长因子是具有多种功能的多肽生长因子，在生理和病理过程中参与生长发育和组织损伤的修复。它能够促进体外培养的人牙周膜细胞的增殖、分化和迁移，应用前景广泛，但具体机制尚不明确。组织的损伤愈合，伴随着基质的合成和降解。组织损伤之后，局部释放的大量的多配体蛋白聚糖，调控细胞与细胞外基质、细胞与细胞间的信号交流，介导成纤维细胞的增殖和迁移，促进伤口愈合。多配体蛋白聚糖是成纤维细胞生长因子的辅助受体，它能使成纤维细胞生长因子在局部高浓度聚集，增强信号级联反应。碱性成纤维细胞生长因子通过细胞表面多配体蛋白聚糖-4表达情况调节信号传导，促进细胞的增殖和迁移。降解细胞外基质的蛋白酶在组织损伤愈合过程中也起着关键性作用。ADAMTS1是ADAMTS金属蛋白酶家族的第一个成员，能分泌到细胞外基质中并与之结合，从而参与细胞外基质蛋白的调节。ADAMTS1可通过降解细胞外基质蛋白，如多能聚糖、短蛋白聚糖、聚集蛋白聚糖等，参与多种生理和病理过程。在皮肤损伤愈合的过程中，ADAMTS1在低表达时可以通过降解基质蛋白促进内皮和成纤维细胞迁移，高表达时则结合碱性成纤维细胞生长因子，抑制内皮细胞和成纤维细胞的迁移。ADAMTS家族是与多配体蛋白聚糖-4胞外区脱落有关的酶，ADAMTS1特异性的作用于多配体蛋白聚糖-4，使其胞外区脱落，多配体蛋白聚糖胞外区的脱落使细胞骨架发生改变，丧失粘附功能，获得迁移功能。研究显示在含bFGF的PDLC培养基中脱落的多配体蛋白聚糖-4含量明显增高，牙周膜细胞表面的多配体蛋白聚糖-4明显减少。有关ADAMTS-1是否参与牙周组织修复再生，国内外尚未见报道。基于以上研究背景，本实验选择体外原代培养的人牙周膜细胞为研究对象，探索ADAMTS1在牙周膜细胞中的表达及bFGF对ADAMTS1基因表达的影响，探讨ADAMTS1在牙周组织损伤修复再生中的作用，为探究bFGF促进PDLC增殖和迁移的机制提供新的思路和楔入点。方法：在吉林大学口腔医学院口腔外科门诊收集因正畸治疗需要而拔除的健康前磨牙，年龄10-15岁。利用组织块原代培养法培养人PDLC，首先用无菌生理盐水将牙根面的血迹冲洗干净后，用刀片和龈下刮治器结合刮取牙根中1/3的牙周膜，常规培养。取第3-5代细胞用于实验。采用免疫组化方法检测ADAMTS1在牙周膜细胞中的表达，应用RT-PCR检测不同浓度组0ng/ml、1ng/ml、10ng/ml bFGF刺激牙周膜细胞在24h、48h、72h ADAMTS1mRNA的表达。结果： ADAMTS1在牙周膜细胞胞质中阳性表达。10ng/mlbFGF促ADAMTS1表达最强，且ADAMTS1表达与时间有相关性，48h组ADAMTS1表达强于24h组（P<0.01）、72h组（p<0.01），24h组ADAMTS1表达强于72h组（P<0.01）。结论：ADAMTS1在牙周膜细胞中阳性表达，10ng/ml bFGF能促进牙周膜中ADAMTS1的表达，且具有时间依赖性。更多还原

【Abstract】 Periodontitics is a chronic and infectional disease which is caused bybiofilm in dental plaque. It leads to the supporting tissues destruction such aspockets, alveolar bone defects and attachment loss, even the teeth get loose andlost. Investigators have reexamined periodontal infections can have an adverseeffect on general health and that systemic diseases can accelerate thedevelopment of periodontal diseases.The main goal of periodontal treatment isto remove dental plaque,control inflammation,especially gain tissue repair andregeneration. But the gainning tissue regeneration is limited with presentperiodontal treatment. Recent years,with the development of tissue engineeringwhich is formed by cells,growth factors and scaffold,people put their hope inPeriodontal tissue engineering. Only periodontal ligament cells which is theideal seed cells can be used as stem cells in wound healing of periodontal tissue.The adhesion,proliferation,and differentiation of periodontal ligament cells onthe root surface are the key component of gaining periodontal tissueregeneration.Fibroblast growth factor2(FGF-2) is polypeptide molecule whichmediates a variety of physiological and pathological responses in developmentand wound healing.In vitro studies reveals that FGF-2can promote periodontalligament cells proliferation, migration and adhesion. However the detailedmechanism by that FGF-2regulate periodontal ligament cells remains elusive.Extracellular matrix synthesis and degradation are involved in wound heal.The syndecan can modulate cell proliferation and cell migration and regulatesignaling communication between cell and extracellulur matrix during woundreparation. As co-receptors of FGF, syndecans increase the local concentrationsof growth factors and promote signaling cascades. FGF-2regulate signaling communication, promote cell proliferation and migration by syndecan-4on cellsurface.Protease of ECM plays a key role in wound healing.A disintegrin andmetalloproteinase with thrombospondin motifs-1(ADAMTS-1) is a member ofthe family of multifunctional proteins known as ADAMTS. Several studieshave shown that ADAMTS-1is capable of degrading aggrecan andversican,which are components of extracellular matrix (ECM) barriers,andplays a key role in many physiology and pathology processes. In skin woundheal, low-level ADAMTS1promotes endothelial and fibroblast cells migrationby degrading ECM protein, but high-level ADAMTS1can combine withFGF-2to inhibite endothelial and fibroblast cells migration.ADAMTS familymembers are syndecan-4sheddases.ADAMTS1uniquely cleave thetransmembrane proteoglycan syndecan-4, and the extracellular domainshedding made cell skeletal structure change which help cell to migration. Thecurrent findings show that syndecan-4increases obviously in conditionedmedium of FGF-2-activated HPDLCs and reduces on the HPDLCs surface.It’snot clear that the role of ADAMTS1in periodontal tissue wound heal at homeand abroad.As mentioned above, our study choose human periodontal ligament cellscultured in vitro using tissue explant as subject to investigate the expression ofADAMTS1of PDLCs and the effect of bFGF on ADAMTS1gene expression,and to explore the role of ADAMTS1in periodontal tissue wound heal. Itprovides a new view for the mechanism that how bFGF promote PDLCsproliferation and migration.Methods: We gathered healthy premolars of young people(10-15years old)undergoing tooth extraction for orthodontic treatment in stomatology of JilinUniversity,washed tooth with stroke-physiological saline solution, removeblood and bacteria at surface of the root. Healthy periodontal tissue was removed from the center of the root surface with a surgical scalpel andsubgingival curette. We used tissue explant to cultured human periodontalligament cells in vitro. Experiments were carried out with cells from the third tofifth passages. Immunohisto-chemical method was used to examine theexression of ADAMTS1in PDLCs. Expression of ADAMTS1mRNA ofPDLCs stimulated by bFGF was analyzed by reverse transcriptase-Polylnerasechain reaction on24h,48h,72h.Result: we found ADAMTS1is positive expression in PDLCs.10ng/mlbFGF significantly stimulate ADAMTS1expression in PDLCs compared withthe control. ADAMTS1was expressed at every time point, and the level ofADAMTS1at48h is higher comparing with24h(P<0.01),72h(P<0.01), and24h is higher than72h(P<0.01).Conclusion: ADAMTS1is positive expression in PDLCs.10ng/ml bFGFsignificantly stimulate ADAMTS1expression of PDLCs at the earlier period ofproliferation.更多还原