| BackgroundPeriodontitis is a inflammation and destructive disease of periodontal tissue, with the gums and periodontal ligament damage collagen fibers being dissolved, periodontal attachment being lost, alveolar bone being lost, resulting in loose teeth being fall out. Periodontitis is a serious threat to human health, as cancer and cardiovascular disease, so it is particularly important to clear the role of a variety of host factors in the development and progression of periodontitis and understand the etiology and pathophysiology of periodontitis.The complex pathogenesis of periodontitis, is one of the interested subjects for scholars to explore the in depth study of periodontitis in recent years, bacteria is the initial factor of periodontitis, in addition to systemic risk factors, cytokines can not be ignored. Scholars believe that periodontitis is mainly indirect damage caused by pathogenic factor disorders of host immune and inflammatory responses. A variety of inflammatory cytokine format a network, mostly involved in alveolar bone osteoclasts and osteoblasts, periodontal tissue destruction and repair process. Wherein the differentiation of osteoclast precursor cells into mature osteoclasts during promoting osteoclast differentiation with interleukin1β (Interleukin-1β, IL-1β), tumor necrosis factor-a (Tumor necrosis factor-a, TNF-a), IL-17and other osteoclast differentiation inhibiting and osteogenesis promoting with IL-10, transforming growth factor-P (Transforming growth factor, TGF-P) and the like.Interleukin-1(IL-1) is produced mainly by monocytes-phagocytic cells and vascular endothelial cells, has pro-inflammatory effects, can biodegradable, metabolism, induce synthesis of prostaglandin E2, increase activation of osteoclasts and promote bone resorption. It also can induce mesenchymal cells within the physical production of matrix metalloproteinases, promote collagen matrix degradation and destruction. Studies have shown that IL-1activity is higher in the site of inflammation in GCF than healthy sites, the amount of IL-1and the depth of periodontal pockets showed a positive correlation. Which IL-1β levels are closely correlated with clinical parameters of chronic periodontitis. IL-1β is a pleiotropic cytokine with a wide range of cell immunomodulatory effects, including the promotion in activation, proliferation and differentiation of thymus cells and T cell, stimulating osteoclasts and collagenase, promoting osteoclast formation and bone resorption, leading to bone and periodontal tissue damage, losing the capacity of repairing periodontal tissue.TNF-a is mainly produced by G-bacterial Iipopolysaccharide (Lipopolysaccharide, LPS)-activated macrophages, with a broad variety of biological effects on leukocytes, endothelial cells and connective tissue cells, and activation of differentiation and maturation of osteoclasts, promoting bone resorption, but also enhancing vascular permeability, with a pro-inflammatory effect.IL-8is a potent neutrophil chemotactic factor, can gather and activate neutrophils to release lysozyme, Gamonal have found that IL-8can be expressed in the normal gingival tissue and periodontitis gingival tissue. but the expression in the gingival tissue lesions is significantly higher and significantly decreased after treatment. And studies have found that IL-8can stimulate RANKL mRNA expression and osteoclast formation, promote bone resorption.IL-17is a pro-inflammatory cytokine being discovered in recent years, with capacities of specifically binding to the receptor, promoting inflammation, immune response,and hematopoiesis, and other functions. It also can induce fibroblast cells to release IL-6, IL-8, prostaglandin E2(Prostaglandin E2, PGE2), stimulate macrophages to produce IL-1β, TNF-α, inhibit the production of IL-10to promote bone resorption.Transforming growth factor-β (TGF-β) is a multifunctional regulator of cellular activity, and is closely related to the immune response, inflammation, tissue repair. In the process of development of chronic periodontitis, TGF-β involved in the regeneration of periodontal tissue inflammation and immune responses of hard and soft tissues, affecting the pace of development of the disease and the healing process.Asporin (ASPN), also known as periodontal ligament associated protein1(PLAP1), is a member of small leucine-rich proteoglycans(SLRPs) with other members Biglycan, Decorin, fibromodulin, lumican so on. SLRPs can combine with many different cytokines, surface receptors, growth factors to achieve the regulation of cell function, such as Toll-like receptors, TGF-β, bone morphogenetic protein-4(BMP-4) and so on. Existing research shows that SLRPs plays an important role in the formation, degradation, assembly processes of collagen fibers. Binding collagen and elastic fibers, SLRPs can enhance the stability, protect the fiber from degradation by various collagenase.Asporin as a new member of the SLRPs family found in2001, has leucine repeat area like other members, it is not a strictly proteoglycan, which has a unique amino-terminal aspartic acid residue repeat region. Loughlin J has found that amount of aspartate residues D14allele of Asporin was significantly associated with osteoarthritis susceptibility. Sebastian K studied the interactions with other extracellular matrix and found Asporin and Decorin compete, directly affect osteoblast collagen mineralization. Kei et al reported Asporin inhibiting TGF-β being into cartilage and bone remodeling in the pathogenesis of osteoarthritis, thereby inhibiting bone formation. Yamada et al reported Asporin also play a regulatory role in periodontal collagen fibers, and inhibit osteogenesis through the inhibitory activity of BMP-2. However, Wang L et al reported Asporin promote participation in the mineralization process, binding calcium, and can affect the formation of hydroxyapatite crystals, promote osteoblast collagen mineralization. Eun-hyang Lee found Asporin is highly expressed in dentin and dentin border and in the early stage of Mineralization of human dental pulp stem cells, lowly expressed in post-mineralization by immunohistochemical method, indicating Asporin paly a important role in mineralization process of human dental pulp stem cells.Asporin is highly expressed in cartilage and has a polymorphic Correlation with Osteoarthritis. Can the inflammatory cytokines expressing in cartilage inhibiting bone formation regulate Asporin? Elise Duval found cytokines in osteoarthritis regulate Asporin expression, showed that IL-1β and TNF-a downregulated the expression of Asporin while TGF-β upregulated it.Currently, Asporin has been confirmed, expressing in the normal periodontal tissue, but it has not been reported whether there are changes in periodontal tissue. Is Asporin involved in the pathogenesis of periodontitis? How to exercise its functions? Is there a regulation in periodontal ligament cells by the important cytokines in periodontitis pathogenesis? The subject will detect expression of Asporin in hPDLCs by induced by inflammatory cytokines IL-1β, TNF-a, TGF-p, IL-17and IL-8exploring the role of Asporin in the occurrence and development of periodontitis.This paper is composed of three chapters:Chapter I hPDLCs isolation, culture and identificationHealthy permanent wisdom teeth and premolars extracted for orthodontic reasons were collected from young adults, scraping periodontal ligament tissue from the roots, hPDLCs were isolated and cultured by combination of explants method and enzymatic separation method. To identify the cells we cultured,we detected the growth curve of the cells, then we cleared the cells could differentiate into osteoblast, the expression of college I was positive and the expression of college IV was negative, the mesenchymal cell surface markers were positive by flow cytometry. which confirmed the obtained cells came from mesenchyme, providing a reliable source of cells for subsequent experiments.Chapter II The expression of Asporin in periodontal tissuesClinically healthy periodontal tissues were collected, immunohistochemistry were used to detect the expression of Asporin in periodontal tissues. RT-PCR and Immunocytochemistry were used to detect the expression of Asporin in hPDLCs.ChapterⅢ The effect of inflammatory cytokines on expression of Asporin in hPDLCsqRT-PCR was used to detect the effect of cytokines at different concentrations (0.5,1,2,5,10ng/mL) on expression of Asporin mRNA level in hPDLCs respectively. Based on the results of dose-response experiment, we selected the right concentration of inflammatory cytokines, qRT-PCR was used to detect the effect of cytokines at different times(3,6,24,48h) on expression of Asporin mRNA level in hPDLCs respectively. Immunocytochemistry and western blot was used to detect the protein level, exploring the regulation of Asporin in the occurrence and development of periodontitis.Materials and methodsisolation and culture of hPDLCsHuman PDL cells were obtained from permanent noncarious premolars extracted for orthodontic reasons, the age of person should less than25years old, the teeth should without decay and infection of inflammation, keep saline at4℃, and should be cultured in vitro with enzymatic digestion within4h. The health teeth were rinsed five times in sterile PBS, PDL tissue was removed from the middle third of the root, using a sterile scalpel and rinsed three times in Dulbecco’s Modified Eagle’s Medium (DMEM). Briefly, the PDL tissues were cut into small pieces and digested with0.2%collagenase I for10min, collected by centrifugation at800rpm for3min. After being incubated in a biopsy medium at37℃,5%CO2, and95%humidity for24hours, the cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) with10%fetal bovine serum (FBS),100U/μL penicillin, and100μg/μL streptomycin. After reaching confluence, the cells were passaged with0.25%trypsin/0.1%ethylenediaminetetraacetic acid. When Cells were cultured to80%confluence, we extend culture of the cells at a ratio of1:3, Cells of passages3-5were used in all experiments.MTT assay hDPSCs were cultured to3-5passages, For MTT assays, the cells were seeded in96-well plates at2×103per well. The absorbance of each sample was analyzed at490nm using a microtiter plate reader at the same time in1to7days.then we have a statistical analysis and paint the growth curve.RT-PCRTotal RNA was extracted and reverse transcribed into cDNA,then having PCR amplification, electrophoretic imaging and ultraviolet photographs.Alizarin red stainingThe cells of the third generation were mineralization induced for14days. Then the cells were fixed with4%paraformaldehyde for20minutes, stained for10min with alizarin red. Mineralized nodules were observed under inverted microscope.Flow cytometry analysisAt least1x106cells were prepared in cold PBS for each test. Cell phenotype analysis was performed by flow cytometric detection of CD29/PE, CD90/FITC, CD44/FITC, and CD105/FITC according to the manufacturer’s instructions.ImmunohistochemistryClinically healthy peiodontal tissues were fixed with4%paraformaldehyde, embedded with paraffin, the sections were dewaxed to water, normal goat serum closed, incubated with primary antibodies, secondary antibodies at room temperature, DAB color, hematoxylin, dehydrated in ethanol, xylene, neutral gum seal tablets.qRT-PCRThe total RNA of cells was extracted using the Trizol Reagent, and the first-stand cDNA synthesis was performed according to the relative manufacture’s protocol. SYBR Green kits were used to detect real-time polymerase chain reaction. The relative quantitative method2-ddCt was used to accurate expression of Asporin.ImmunocytochemistryThe cells were fixed with4%Paraformaldehyde, incubated with0.2%Triton X-100at room temperature, normal goat serum closed, incubated with primary antibodies, secondary antibodies at room temperature, DAB color, hematoxylin, dehydrated in ethanol, xylene, neutral gum seal tablets, having microscope photographs. Image-Pro Plus (IPP) software measure its density (IOD/area) values.Western blotCells were rinsed and scrapped in radioimmunoprecipitation assay (RIPA)lysis buffer supplemented with protease inhibitors. The total protein was quantitated using BCA assay. Equivalent protein amounts were separated in10%SDS-polyacrylamide gels and transferred to PVDF membranes. The blots were then hybridized with specific primary Asporin antibodies, GAPDHand secondary antibodies labeled with an IRDye800-conjugated affinity-purified anti-rabbit/mouse immunoglobulin M antibody. The membrane was washed several times and scanned using an Odyssey infrared imaging system at a wavelength of800μm The data were analysed with Odyssey software.Statistical AnalysisAll experiments were repeated at least3times each with different donors, and similar results were obtained. The significance (P<0.05) of differences was assessed using the One-way ANOVA test.Results1. hPDLCs were isolated and cultured Successfully, hPDLCs were isolated by combination of explants method and enzymatic separation method, we verified that the obtained cells can differentiate osteoblast. have a normal growth curve. The expression of collagen I was positive, expression of collagen IV was weakly positive, The mesenchymal cell surface markers were positive (CD29,98.17%; CD44,99.13%; CD90,100%; CD105,78.21%) by Flow cytometry. based on the periodontal ligament tissue, we confirmed the obtained cells could be considered as hPDLCs.2. Asporin is highly expressed in normal periodontal tissues and hPDLCs, and mainly localized in the cytoplasm.3. Dose-response experiments showed that0.5ng/mL TNF-a, IL-1β, IL-8, IL-17for24h is sufficient to repress Asporin mRNA expression, and1ng/mL TNF-a decrease it the most significantly. Similarly, lOng/mL IL-1β,5ng/mL IL-8and IL-17can drop Asporin mRNA expression greatest.0.5ng/mL TGF-β for24h is sufficient to increase Asporin mRNA expression, and5ng/mL TGF-β increase it The most significantly.4.qRT-PCR,immunohistochemistry and Western blot results showed proinflammatory cytokines significantly reduce Asporin expression. This decrease was seen as early as3and6h of treatment with1ng/mL TNF-a or lOng/mL IL-1β,5ng/mL IL-17and IL8, Asporin was significantly reduced after24and48h of incubation with1ng/mL TNF-a,10ng/mL IL-1β,5ng/mL IL-17at both mRNA and protein level. After the induction of5ng/mL IL-8, the expression was significantly decreased at48h.Asporin was significantly increased after24and48h of incubation with5ng/mL TGF-β at botn mRNA and protein level.Conclusions1. The hPDLCs were successfully isolated and cultured using combination of explants method and enzymatic separation method. Based on the periodontal ligament tissue, we confirmed the obtained cells could be considered as hPDLCs.2. Asporin is highly expressed in normal periodontal tissues and hPDLCs, and mainly localized in the cytoplasm.3. proinflammatory cytokines IL-1β, TNF-a, IL-17, IL-8inhibited Asporin expression in hPDLCs, anti-inflammatory factors TGF-β promote Asporin expression in hPDLCs.4. Asporin may induce collagen mineralization and be implicated in progression of periodontitis. Proinflammatory cytokines TNF-a, IL-1β, IL-8, IL-17which are highly existed in the progression of periodontitis and, decrease Asporin expression. Then they may weaken its function and promote bone destruction. Conversely, TGF-β increases Asporin expression, this may stimulate the ability of each to promote collagen mineralization, bone formation and remodeling.this finding suggests that Asporin can be regulated by cytokines related to periodontitis named TNF-a, IL-1β, IL-8, IL-17and TGF-β, which may be one of the key proteins and provide clues as to the underlying pathology of periodontitis. However, the mechanism of interaction of them is not clear and need a further research. |
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