Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate

Retaining or improving periodontal ligament （PDL） function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation （LPLI） on the proliferation and osteogenic differentiation of human PDL （hPDL） cells. Cultured hPDL cel Is were irradiated （660 nm） daily with doses of O, 1, 2 or 4 J .cm-2. Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase （ALP） activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction （RT-PCR）. Our data showed that LPLI at a dose of 2 J.cm-2 significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J.cm-2 showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate （cAMP） is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance the Osteoblastic Differentiation of Periodontal Ligament Stem Cells Under High Glucose Conditions Through the PI3K/AKT Signaling Pathway

Objective High glucose(HG)can influence the osteogenic differentiation ability of periodontal ligament stem cells(PDLSCs).Human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSC-exo)have broad application prospects in tissue healing.The current study aimed to explore whether hUCMSC-exo could promote the osteogenic differentiation of hPDLSCs under HG conditions and the underlying mechanism.Methods We used a 30 mmol/L glucose concentration to simulate HG conditions.CCK-8 assay was performed to evaluate the effect of hUCMSC-exo on the proliferation of hPDLSCs.Alkaline phosphatase(ALP)staining,ALP activity,and qRT-PCR were performed to evaluate the pro-osteogenic effect of hUCMSC-exo on hPDLSCs.Western blot analysis was conducted to evaluate the underlying mechanism.Results The results of the CCK-8 assay,ALP staining,ALP activity,and qRT-PCR assay showed that hUCMSC-exo significantly promoted cell proliferation and osteogenic differentiation in a dosedependent manner.The Western blot results revealed that hUCMSC-exo significantly increased the levels of p-PI3K and p-AKT in cells,and the effect was inhibited by LY294002(PI3K inhibitor)or MK2206(AKT inhibitor),respectively.Moreover,the increases in osteogenic indicators induced by hUCMSC-exo were significantly suppressed by LY294002 and MK2206.Conclusion hUCMSC-exo promote the osteogenic differentiation of hPDLSCs under HG conditions through the PI3K/AKT signaling pathway.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

Effects of Tension Force on Proliferation and Differentiation of Human Periodontal Ligament Cells Induced by Lipopolysaccharides

Human periodontal ligament cells (hPDLCs), with the potential for multi-directional differentiation and reproduction, are the target cells of orthodontic tooth movement. The aim of this study was to examine the effect of mechanical tension force and lipopolysaccharides (LPS) on hPDLCs and whether they induce proliferative and differentiated characters in vitro. Tension force was applied to hPDLCs stimulated with and without LPS for 24 hrs. Real-time polymerase chain reaction (qPCR) was carried out to analyze the mRNA expression of Cyclin 2 (CCND2), WNT1 inducible signaling pathway protein 1 (WISP1), runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP). Analysis of variance (ANOVA) was used for statistical analysis. Significant differences were indicated by P < 0.05. The results showed that tension force promoted the mRNA expression of both the proliferation-related genes (CCND2 and WISP1) and differentiation-related genes (RUNX2 and ALP), and that both were enhanced by the simulation of LPS. In addition, the relative expression ratios CCND2/RUNX2 and CCND2/ALP both increased significantly after the application of tension, and this effect was further enhanced by LPS. All results indicated that with the assessed level of mechanical force loading, tension could promote both the proliferation and differentiation of hPDLCs, which could be enhanced by LPS, and that proliferation is promoted to a greater extent than differentiation. These findings may be valuable for understanding the importance of the application of suitable mechanical force in periodontal remodeling, especially in the process of orthodontic tooth movement with inflammation.

Activation of cannabinoid receptor CB2 regulates LPS-induced pro-inflammatory cytokine production and osteoclastogenic gene expression in human periodontal ligament cells

Background and Objective: It has been found that human periodontal ligament (hPDL) cells express cannabinoid receptor CB2. However, the functional importance of CB2 in hPDL cells exposed to bacterial endotoxins is not known. Here we investigate if the inflammation promoter lipopolysaccharide (LPS) affects CB2 expression and if activation of CB2 regulates LPS-induced pro-inflammatory cytokine production and osteoclastogenic gene expression in hPDL cells. Methods: The hPDL cells were obtained from extracted teeth of periodontally healthy subjects. CB2 expression in hPDL cells exposed to LPS was deter- mined by quantitative real-time PCR analysis. Then, the cells were incubated with or without CB2-specific agonist HU-308 before further stimulation with LPS. In some experiments, the cells were pre-treated with CB2-specific antagonist SR144528. The production of pro-inflammatory cytokines interleukin-1 beta (IL- 1β), interleukin-6 (IL-6) and tumor necrosis factoralpha (TNF-α) was assessed by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of osteoclastogenic genes osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) was examined using quantitative real-time PCR analysis. Results: CB2 expression in hPDL cells was markedly enhanced by LPS. HU-308 significantly suppressed the production of IL-1β, IL-6 and TNF-α exposed to LPS, whereas SR144528 attenuated this effect. The OPG/RANKL ratio decreased when exposed to LPS, furthermore increased significantly with the addition of HU-308 and finally decreased markedly after pretreatment with SR144528. Conclusion: Our study demonstrated that activation of CB2 had anti-inflammatory and anti-resorptive effects on LPS-stimulated hPDL cells. These findings suggest that activation of CB2 might be an effective therapeutic strategy for the treatment of inflammation and alveolar bone resorption in periodontitis.

Application of dental stem cells in three-dimensional tissue regeneration

Dental stem cells can differentiate into different types of cells.Dental pulp stem cells,stem cells from human exfoliated deciduous teeth,periodontal ligament stem cells,stem cells from apical papilla,and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development.The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering.In recent years,three-dimensional(3D)tissue scaffolds have been used to reconstruct and restore different anatomical defects.With rapid advances in 3D tissue engineering,dental stem cells have been used in the regeneration of 3D engineered tissue.This review presents an overview of different types of dental stem cells used in 3D tissue regeneration,which are currently the most common type of stem cells used to treat human tissue conditions.

Effects of Ginsenoside Rg-1 on the Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells

Objective： TO evaluate the effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells （hPDLSCs） and to explore the possible application on the alveolar bone regeneration. Methods： To determine the optimum concentration, the effects of ginsenoside Rg-1 ranging from 10 to 100 μmol/L were evaluated by 3-（4,5）-dimethylthiahiazo（-z-yl）-3,5-di-phenytetrazoliumromide, alkaline phosphatase activity and calcium deposition. Expressions of runt-related transcription factor 2, collagen alpha-2（I） chain, osteopontin, osteocalcin protein were examined using real-time polymerase chain reaction. Results： Compared with the control group, a certain concentration （10 μmol/L） of the Rg-1 solution significantly enhanced the proliferation and osteogenic differentiation of hPDLSCs （P〈0.05）. However, concentrations that exceeds 100 μmol/L led to cytotoxicity whereas concentrations below 10 nmol/L showed no significant effect as compared with the control. Conclusion： Ginsenoside Rg-1 can enhance the proliferation and osteogenic differentiation of hPDLSCs at an optimal concentration of 10 μmol/L.

AMP-activated protein kinase acts as a negative regulator of high glucose-induced RANKL expression in human periodontal ligament cells

Methods We examined the expression of osteoprotegerin in hPDL cells cultured at different concentrations of glucose using real-time polymerase chain reaction （PCR）, and Western blotting analysis. AMPK phosphorylation in hPDL cells was studied using immunoprecipitate kinase assay and Western blotting. The effect of AMPK activation on RANKL expression in hPDL cells was investigated by real-time PCR and Western blotting. Results High glucose levels caused an increase in RANKL mRNA and protein expression in hPDL cells. Moreover, the amount of p-AMPK and AMPK activity was lower in hPDL cells exposed to high glucose levels than in cells exposed to normal glucose levels. Suppression of AMPK by Compound C augmented RANKL expression, and AMPK activation by metformin significantly decreased RANKL expression in hPDL cells. Additionally, metformin down-regulated RANKL expression in hPDL cells exposed to high glucose via AMPK activation. Conclusion High glucose-induced up-regulation of RANKL could be due to decreased AMPK activity, and AMPK activation may be involved in regulating of RANKL expression in hPDL cells.

miR-153-3p靶向调控CHI3L1对LPS所致人牙周膜干细胞炎症反应的影响

目的:探索miR-153-3p是否能通过靶向调控几丁质酶3样蛋白1(CHI3L1)改善脂多糖(LPS)所致人牙周膜干细胞(hPDLSCs)炎症反应。方法:分离培养hPDLSCs,采用LPS处理建立炎症细胞模型,并进行miR-NC、miR-153-3p mimics、si-NC、si-CHI3L1以及miR-153-3p mimics+pc-NC、miR-153-3p mimics+pcDNA-CHI3L1转染。转染48h后qRT-PCR法检测各组细胞中miR-153-3p、CHI3L1 mRNA表达,ELISA法检测细胞上清液中IL-6、IL-1β、TNF-α水平,流式细胞术检测细胞凋亡情况,Western Blot法检测细胞中CHI3L1蛋白表达。双荧光素酶报告基因实验检测miR-153-3p与CHI3L1的靶向关系。结果:LPS处理后hPDLSCs中miR-153-3p表达下调(P<0.05),CHI3L1 mRNA与蛋白表达均显著上调(P<0.05);过表达miR-153-3p或抑制CHI3L1表达可降低LPS诱导的hPDLSCs中IL-6、IL-1β、TNF-α水平,下调CHI3L1 mRNA与蛋白表达,降低细胞凋亡率,差异均具有统计学意义(P<0.05)。双荧光素酶报告基因实验证实miR-153-3p可靶向调控CHI3L1表达(P<0.05)。此外,过表达CHI3L1可显著逆转过表达miR-153-3p对LPS诱导的hPDLSCs凋亡与炎症反应的抑制作用(P<0.05)。结论:miR-153-3p可通过靶向调控CHI3L1表达减轻LPS所致hPDLSCs炎症反应,减少细胞凋亡。

钽涂层对hPDLSCs增殖及成骨分化的影响

目的 探究钽涂层表面对人牙周膜干细胞(human periodontal ligament stem cells, hPDLSCs)增殖及成骨分化的影响。方法 对hPDLSCs进行分离、培养和细胞鉴定。纯钛试件抛光清洗后,经喷砂、酸蚀处理,以等离子喷涂技术制备钽涂层。以抛光钛表面(P组)为对照组,喷砂酸蚀钛(SLA组)、钽涂层钛(Ta组)为实验组。通过扫描电镜、能谱仪分析各组钛表面的微形貌、元素组成;将hPDLSCs接种于各组试件表面,通过CCK-8法测定细胞的增殖情况,经成骨诱导培养后行碱性磷酸酶活性检测和茜素红染色,qPCR检测成骨基因表达。结果 在钛试件上成功制备了钽涂层;成功分离培养并鉴定hPDLSCs;CCK-8实验结果显示,在培养3、5、7 d后,Ta组OD值明显高于SLA组及P组(P<0.05);ALP检测结果显示,在第7天时,三组间的差距无统计学意义;第14天时,Ta组ALP活性明显高于P组和SLA组(P<0.01)。茜素红染色显示,三组材料表面都有红色钙化结节形成,SLA组和Ta组的矿化结节明显多于P组。qPCR结果显示Ta组表面细胞ALP、RUNX2、OCN基因表达水平显著高于P组(P<0.01),RUNX2、OCN基因表达显著高于SLA组(P<0.01)。结论 SLA钛表面钽涂层对hPDLSCs的增殖、成骨分化具有促进作用。

SMURF2/β-catenin轴促进张力条件下人牙周膜细胞成骨分化

目的:探究Smad泛素化调节因子2(SMURF2)在循环牵张力诱导人牙周膜细胞(hPDLCs)成骨分化中的作用和机制。方法:对hPDLCs施加循环牵张力刺激后使用qRT-PCR检测SMURF2的表达和成骨相关基因的表达,使用western blotting检测SMURF2和经典WNT信号相关分子的表达,通过ALP染色检测hPDLCs的ALP活性,并结合成骨相关基因的表达评估hPDLCs的成骨分化。通过使用小干扰RNA沉默hPDLCs中的SMURF2基因探究SMURF2对牵张力诱导的hPDLCs成骨分化和经典WNT信号的影响。结果:循环牵张力刺激hPDLCs后SMURF2、ALP、OPN、OSX、COL-1和β-catenin表达升高,ALP活性也增强。沉默SMURF2基因后,hPDLCs的ALP活性减弱,ALP、OPN和OSX基因表达水平下降,β-catenin蛋白下降,但GSK-3β蛋白无变化。结论:SMURF2可能通过经典WNT信号通路促进循环牵张力诱导的hPDLCs成骨分化。

南蛇藤素对脂多糖诱导的人牙周膜干细胞增殖、凋亡和炎症反应的影响

目的 探讨南蛇藤素(Cel)对微小RNA(miR)-223-3p/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)轴对脂多糖(LPS)诱导的人牙周膜干细胞(hPDLSCs)增殖、凋亡和炎症反应的影响。方法 分别给予不同浓度的Cel(1、2、4μmol/L)培养经1μg/ml LPS诱导的hPDLSCs,以未处理的hPDLSCs作为对照组。采用MTT法检测细胞存活率,选取合适的Cel浓度用于后续实验研究。将对数生长期hPDLSCs分为对照组、LPS组、Cel组(2μmol/L),采用LipofectamineTM2000转染试剂盒在Cel组的基础上转染细胞miR-223-3p inhibitor及阴性对照(inhibitor NC),并命名为Cel+inhibitor NC组、Cel+miR-223-3p inhibitor组。分别检测以上各组细胞凋亡、增殖以及炎症因水平;q RT-PCR检测各组细胞中miR-223-3p表达水平,Western blot检测细胞中NLRP3蛋白及凋亡蛋白-天冬氨酸蛋白水解酶-1(Caspase-1)表达;双荧光素酶报告基因检测实验验证miR-223-3p、NLRP3靶向关系。结果 2μmol/L Cel作用细胞后其活力最高。与对照组相比,LPS组细胞存活率、mi R-223-3p表达显著下降,肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6含量、细胞凋亡率、NLRP3、Caspase-1表达显著升高(P<0.05);与LPS组相比,Cel组细胞存活率、miR-223-3p表达显著升高,TNF-α、IL-1β、IL-6含量、细胞凋亡率、NLRP3、Caspase-1表达显著降低(P<0.05);与Cel+inhibitor NC组相比,Cel+miR-223-3p inhibitor组LPS组细胞存活率、miR-223-3p表达显著下降,TNF-α、IL-1β、IL-6含量、细胞凋亡率、NLRP3、Caspase-1表达显著升高(P<0.05)。结论 Cel可以促进LPS诱导的hPDLSCs增殖,抑制细胞凋亡及炎症反应,可能与上调miR-223-3p、抑制NLRP3表达有关。

机械力作用下人牙周膜细胞Osterix mRNA和蛋白的表达

目的研究在机械力作用下人牙周膜细胞内Osterix(Osx) mRNA和蛋白的表达变化,探讨Osx与正畸牙周组织骨改建的关系。方法组织块法培养人牙周膜细胞,采用离心加力装置对细胞分别加载1、2、4、6、8、12h的机械力。逆转录聚合酶链反应(RT-PCR)、Western blot及细胞免疫荧光化学技术分别检测不同时间点Osx mRNA和蛋白的表达变化及其细胞定位。结果在正常人牙周膜细胞中,Osx mRNA表达微弱,蛋白未见表达;在机械力加载4h后,Osx mRNA表达开始明显增强(P<0.01),蛋白呈现弱表达(P<0.05);加载8h时,OsxmRNA和蛋白表达显著增强(P<0.01);持续增加至加力12h。同时,加力4h后,少量细胞的胞质内开始呈现微弱的绿色荧光;12h后,阳性表达主要集中在胞核内。结论机械力可诱导人牙周膜细胞Osx表达增强及活化。Osx可能参与了细胞内生物力学信号的转导,从而可能在正畸牙周组织的骨改建过程中发挥着重要作用。

Osterix过表达对人牙周膜细胞骨向分化的影响

目的研究Osterix(Osx)过表达对人牙周膜细胞受力后骨向分化的影响,探讨Osx与正畸牙周组织骨改建的关系。方法采用组织块法培养人牙周膜细胞,用重组质粒pcDNA3.1 flag-Osx转染人牙周膜细胞。采用逆转录聚合酶链反应(RT-PCR)和Western blot方法检测未转染组、转染空质粒组、转染Osx组细胞Osx mRNA和蛋白表达水平;并观察核心结合因子α1(Cbfα1)、碱性磷酸酶(ALP)、骨桥素(OPN)、骨钙素(OC)、骨涎蛋白(BSP)和a1(Ⅰ)型胶原蛋白(ColⅠ)的mRNA表达情况。3组细胞加载6 h离心力后,观察上述检测指标的变化。结果转染24 h后,相对未转染组,转染空质粒组Osx mRNA和蛋白水平无明显变化(P>0.05);而转染Osx组Osx mRNA和蛋白表达水平明显升高(P<0.01),同时5个成骨标志基因的mRNA表达亦均明显升高(P<0.05,P<0.01)。加力6 h后,3组Osx和成骨标志基因表达水平均明显升高,但是转染Osx组Osx mRNA和蛋白表达增强更加显著,增加量约为其他两组的2倍,其ALP、OPN、OC、BSP和ColⅠ的mRNA表达亦升高更显著。结论 Osx过表达可以促进人牙周膜细胞在机械力作用下向成骨样细胞分化。Osx可能通过调控多种成骨基因的表达,从而在正畸牙周组织的骨改建过程中发挥着重要的作用。

微重力环境下Smads信号通路对人牙周膜干细胞成骨向分化的影响

目的:了解Smads信号通路在模拟微重力条件下对人牙周膜细胞成骨向分化的影响。方法:自手术拔除的人牙周膜中培养获得牙周膜细胞,利用有限稀释法培养获得人牙周膜细胞(human periodontal ligament cells,hPDLCs)。采用旋转细胞培养系统(rotary cell culture system,RCCS)建立模拟微重力环境,将细胞分为对照组(正常重力组)、模拟微重力组,应用实时定量PCR检测Smads信号分子表达及加入Smads抑制剂后成骨标志物的表达,流式细胞仪检测磷酸化Smad表达。采用SPSS13.0软件包对数据进行统计学处理。结果:与对照组相比,模拟微重力组Smads 2、3、4 mRNA表达量显著增加,呈时间依赖性,在2h达到峰值,随后开始下降。Smads7在2h时开始上升,观测时间内未捕捉到其下降。流式细胞检测发现,p-Smads在30min时开始出现高表达(29.39%),2h时达到峰值,表达量为91.32%。加入Smads抑制剂后,p-Smads表达下降至5.3%,成骨标志物COL1、ALP、OCN表达显著下降(P<0.05)。结论:模拟微重力环境下,Smads信号通路参与了hPDLSCs成骨向分化。

周期性张应力对人牙周膜细胞Periostin表达的影响

目的:研究在机械力作用下人牙周膜细胞(h PDLCs)内Periositn(PN)m RNA和蛋白的表达变化。方法:采用组织块酶消化法培养h PDLCs,经鉴定后将第4代细胞随机分为4个实验组和1个对照组(非加力组),4个实验组分别采用Flexcell-5000 Tension System应力加载系统对h PDLCs加载6、12、24、48 h的周期性张应力;然后采用q RT-PCR、Western blot分别检测各组h PDLCs中PN m RNA和蛋白的表达变化。结果:在正常h PDLCs中表达PN m RNA和蛋白;与对照组相比,加力6 h后,PN m RNA和蛋白的表达水平均明显降低(P<0.05);加力12 h后,PN m RNA和蛋白的表达均逐渐升高,并持续至24 h达最高水平(P<0.05);而在加力48 h时,PN m RNA和蛋白表达均明显下降,并恢复至对照组的表达水平(P>0.05)。结论:机械力可诱导h PDLCs中PN的表达增强及活化,且具有时间依赖性;提示PN可能参与了细胞内生物力学信号的转导。

白藜芦醇减轻LPS诱导的牙周膜细胞损伤并抑制TLR4/NF-κB活化

目的:探究白藜芦醇(RES)对脂多糖(LPS)诱导的人牙周膜细胞(hPDLCs)损伤的保护作用。方法:体外培养并鉴定hPDLCs,将培养的hPDLCs随机分为5组:对照组、LPS(10μg/ml)+RES(0、30、60、90μmol/L)组,MTT法检测各组细胞增殖能力,ELISA检测各组细胞分泌TNF-α/IL-6水平,PCR和Western blot检测各组细胞TLR4/NF-κB mRNA和蛋白表达。结果:分离培养的hPDLCs抗波丝蛋白表达阳性,抗角蛋白表达阴性。与对照组比,LPS处理后细胞增殖能力明显降低,细胞分泌TNF-α/IL-6水平和TLR4/NF-κB mRNA和蛋白表达明显增加;30~90μmol/L白藜芦醇预处理后,细胞增殖能力增加(P<0.05),细胞分泌TNF-α/IL-6水平、TLR4/NF-κB mRNA以及蛋白表达则下调(P<0.05),均呈现一定的浓度依赖性。结论:白藜芦醇可抑制TLR4/NF-κB活化并减轻LPS诱导的牙周膜细胞损伤。

哈巴苷通过抑制miR-1246表达对LPS诱导的人牙周膜细胞损伤的影响

目的:探讨哈巴苷对LPS诱导的人牙周膜细胞(hPDLCs)损伤的影响及分子机制。方法:hPDLCs分为Con组、LPS组、哈巴苷低、中、高浓度组(LPS+HG-L、M、H)、LPS+anti-miR-NC组、LPS+anti-miR-1246组、LPS+HG+miR-NC组、LPS+HG+miR-1246组。ELISA检测TNF-α、IL-1β水平;流式细胞术检测细胞凋亡;Western blot法检测蛋白表达;实时荧光定量PCR(RT-qPCR)检测miR-1246表达水平。结果:LPS诱导的hPDLCs中TNF-α、IL-1β水平升高,细胞凋亡率升高,Bcl-2表达水平降低,Bax表达水平升高,miR-1246表达水平升高(P<0.05)。哈巴苷低、中、高浓度处理后,LPS诱导的hPDLCs中TNF-α、IL-1β水平降低,细胞凋亡率降低,Bcl-2表达水平升高,Bax表达水平降低,miR-1246表达水平降低,且呈浓度依赖性(P<0.05)。抑制miR-1246表达后,LPS诱导的hPDLCs中TNF-α、IL-1β水平降低,细胞凋亡率降低,Bcl-2表达水平升高,Bax表达水平降低(P<0.05)。miR-1246过表达逆转了哈巴苷对LPS诱导的hPDLCs损伤的作用。结论:哈巴苷通过抑制miR-1246表达保护hPDLCs免受LPS诱导的损伤。

康复新治疗牙周炎的机制研究

目的通过研究康复新对牙周组织相关细胞增殖的影响及康复新在细胞炎症反应模型中的抗炎作用,从促进牙周组织再生和抗炎2个方面探讨康复新治疗牙周炎的相关机制。方法(1)采用淋巴细胞增殖检测法(MTS法)检测不同浓度康复新对人牙周膜成纤维细胞(hPDLFs)、人牙龈上皮细胞(hGECs)、人单核巨噬细胞(THP-1)和小鼠胚胎成骨细胞(MC3T3-E1)活性的影响;(2)通过细菌脂多糖(LPS)刺激小鼠巨噬细胞RAW264.7建立细胞炎症模型,采用实时定量聚合酶链反应(qRT-PCR)法检测康复新对细胞白细胞介素-1β(IL-1β)、IL-10、一氧化氮合酶(NOS)和基质金属蛋白酶-13(MMP-13)mRNA表达水平的影响。结果(1)与对照组比较:0.1000、0.0500、0.0250、0.0125 mg/mL的康复新组在24 h和48 h时间点均可刺激hPDLFs、THP-1和MC3T3-E1增殖(P<0.01),且促增殖作用具有浓度依赖性;(2)在炎症细胞模型中,与对照组比较,0.0500 mg/mL和0.0125 mg/mL的康复新组IL-1β和IL-10 mRNA表达水平比较,差异均无统计学意义(P>0.05),NOS和MMP-13 mRNA表达水平降低,差异有统计学意义(P<0.05)。结论康复新在一定浓度范围内能促进hPDLFs、hGECs、THP-1和MC3T3-E1增殖;康复新可抑制LPS诱导的RAW264.7细胞炎症反应,其抗炎机制可能是降低NOS和MMP-13 mRNA表达水平。康复新可能是通过促进牙周组织再生和抗感染治疗牙周炎。

糖基化终末产物对人牙周膜干细胞增殖及相关基因HSG、cyclinD1表达的影响

目的:探讨糖基化终末产物(AGEs)对人牙周膜干细胞(HPDLSC)增殖能力以及增殖相关基因HSG、cyclinD1的影响。方法:体外组织块法和有限稀释法克隆化培养牙周膜干细胞;成骨、成脂诱导牙周膜细胞,对其进行干细胞鉴定;将培养出的牙周膜干细胞与不同浓度的AGEs共培养,MTT检测不同浓度下牙周膜干细胞增殖的改变;实时定量聚合酶链反应(real time PCR)检测AGEs刺激后HSG、cyclin D1表达的改变。结果:牙周膜细胞成骨诱导21d后茜素红染色出现钙化结节;成脂诱导21d后油红O染色出现脂滴;MTT显示不同浓度的AGEs对HPDLSCs增殖能力的影响有所差别,高浓度(100mg/L,200mg/L)明显抑制HPDLSCs的增殖,差异有统计学意义(P<0.05),低浓度(1mg/L,10mg/L)对HPDLSCs的增殖能力影响不大,差异无统计学意义;Real time PCR结果显示:AGEs(100mg/L)刺激3d后HSG mRNA表达水平较对照组升高、cyclinD1mRNA的表达水平较对照组降低,差异有统计学意义(P<0.05)。结论:高浓度的AGEs能抑制人牙周膜干细胞的增殖并能改变HSG、cyclinD1mRNA的表达。