牙龈卟啉单胞菌感染对hPDL细胞中成骨标志基因表达及炎症因子分泌的影响

目的观察牙龈卟啉单胞菌(P.gingivalis)感染对人牙周膜(hPDL)细胞中成骨标志基因表达及炎症因子分泌的影响。方法原代hPDL细胞,分为P.gingivalis感染的P.gingivalis组、常规处理的对照组,成骨诱导后茜素红染色检测矿化结节,PCR法检测成骨标志基因Runt相关转录因子2(RUNX2)、骨钙素(OCN)、骨保护素(OPG)、骨碱性磷酸酶(BALP)的mRNA表达量,ELISA法检测炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6的分泌量,Western blot检测核因子-κB(NF-κB)及NF-κB抑制蛋白(I-κB)的蛋白表达量。结果与对照组比较,P.gingivalis组细胞成骨诱导后茜素红染色的矿化结节明显减少,细胞中RUNX2、OCN、OPG、BALP的mRNA表达量及I-κB的蛋白表达量均明显降低,培养基中TNF-α、IL-1β、IL-6的分泌量及细胞中NF-κB的蛋白表达量均明显增加。结论 P.gingivalis感染hPDL细胞后能够抑制成骨分化、激活炎症反应且该作用与NF-κB通路的激活有关。     

牙龈卟啉单胞菌脂多糖通过TXNIP/Nlrp3炎性通路对小鼠牙周膜成纤维细胞迁移的影响

摘要 目的：研究牙龈卟啉单胞菌脂多糖（Porphyromonas lipopolysaccharide,LPS-PG）对牙周膜成纤维细胞（mouse periodontal Ligament: Normal Fibroblasts,mPDLFs）增殖及迁移的影响,探讨TXNIP/Nlrp3炎性体途径在其中的作用。方法：采用不同浓度的LPS-PG刺激小鼠mPDLFs细胞不同时间,CCK-8法检测细胞增殖抑制率。然后将细胞分为对照组（培养基作用24 h）和LPS-PG组（2 M的LPS-PG作用24 h）,划痕实验检测细胞迁移,ELISA法检测白细胞介素-1β（Interleukin-1β,IL-1β）和高迁移率族蛋白B1（High mobility group protein B1,HMGB1）的水平,Western Blot检测Nod样受体蛋白3（Nod-like receptor pyrin domain3,NLRP3）、凋亡相关斑点样蛋白（Apoptosis-associated speck-like protein,ASC）、活化半胱氨酸蛋白酶（cleaved-caspase-1）和硫氧还蛋白相互作用蛋白（Thioredoxin-interacting protein,TXNIP）的表达,免疫共沉淀检测以上蛋白间的相互作用。结果：与0 M的LPS-PG相比,1 M和2 M的LPS-PG可显著增加mPDLFs的细胞增殖抑制率（P<0.05）。与LPS-PG作用0 h相比,LPS-PG作用12 h、24 h和48 h均可显著增加mPDLFs的细胞增殖抑制率（P<0.05）。LPS-PG组细胞向中间''伤口''迁移的距离及细胞数量远远低于对照组。与对照组相比,LPS-PG组细胞中IL-1β和HMGB1的水平、NLRP3、cleaved-caspase-1和TXNIP蛋白表达均显著增加,且NLRP3和ASC、NLRP3和cleaved-caspase-1、TXNIP和NLRP3的蛋白互作能力显著增强（P<0.05）,而两组ASC蛋白的表达无显著差异（P＞0.05）。结论：LPS-PG可抑制mPDLFs细胞的增殖和迁移,其机制与Nlrp3炎性体的形成与活化有密切的关系。     

牙龈卟啉单胞菌感染通过激活NLRP3小体诱导牙周膜细胞炎症反应及凋亡效应

目的 观察牙龈卟啉单胞菌感染通过激活含NLR家族PYRIN域蛋白3(NLRP3)小体诱导人牙周膜细胞(hPDLCs)炎症反应及凋亡的效应。 方法 取健康前磨牙样本并分离培养hPDLCs,分为牙龈卟啉单胞菌感染的感染组和常规处理的对照组,检测细胞中NLRP3小体[NLRP3、凋亡相关斑点样蛋白(ASC)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-1]、凋亡基因[自杀相关因子(Fas)、Fas配体(FasL)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关x蛋白(Bax)、Caspase-3]的表达量及培养基中炎症细胞因子[白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)]的含量。 结果 感染组hPDLCs中NLRP3、ASC、Caspase-1、Fas、FasL、Bax、Caspase-3的表达量及培养基中IL-1β、IL-18、TNF-α的含量明显高于对照组,细胞中Bcl-2的表达量明显低于对照组。 结论 牙龈卟啉单胞菌感染能够诱导hPDLCs的炎症反应及凋亡且该作用与NLRP3小体的激活有关。     

牙周膜成骨分化研究进展

牙周膜是位于牙根与牙槽骨之间的结缔组织,具有自我更新和多向分化的能力。无论是在正畸治疗还是在牙周组织修复及再生过程中,牙周膜的成骨分化都是必不可少的。近年来,许多国内外学者致力于研究影响牙周膜成骨分化的因素,包括机械力,细胞因子,药物等,这些因素可以单独作用于牙周膜,也可以联合使用加快牙周膜成骨分化,可以为临床上加快牙齿移动和修复牙周组织缺损提供更多新的思路。现就影响牙周膜成骨分化的诸多因素及其主要机制作一综述。     

Wnt 信号通路与骨髓间充质干细胞成骨之间的关系

Wnt信号通路是由Wnts诱发的一系列相互作用的分子组成。Wnt信号对骨髓间充质干细胞的影响在所有研究中均证实有明显作用,其可调节干细胞增殖、分化及凋亡。研究表明,抑制Wnt信号通路转导可使成骨细胞分化进程受阻,从而抑制骨形成;若诱导Wnt家族成员表达则可使成骨细胞特异性基因表达增加,促进骨形成。本文就Wnt信号通路的作用过程及其与骨髓间充质干细胞成骨诱导的关系做一综述。     

miR-26a修饰的人牙周膜干细胞成骨分化能力的研究

目的:研究人牙周膜干细胞(hPDLSCs)在转染miR-26a后成骨分化的促进效果。方法:从因正畸拔除的无龋坏、无牙周疾病离体牙的牙根中部牙周膜组织中分离、胶原酶消化,进行人牙周膜干细胞的体外培养。使用脂质体2000进行miRNA转染,采用激光共聚焦观察转染效果;MTT方法检测转染后的细胞活力;成骨诱导后使用实时定量PCR技术检测miRNA修饰的hPDLSCs成骨分化相关基因的表达;采用BCIP/NBT、天狼星红、茜素红S分别对碱性磷酸酶、胶原以及钙化进行染色观察。结果:采用脂质体2000能够成功转染hPDLSCs,且转染效率较高;转染后的细胞活力有所下降,但仍在80%以上;转染后的细胞在经成骨诱导分化过程中骨钙素(OCN)和骨桥蛋白(OPN)基因表达显著上调,同时碱性磷酸酶活性、胶原分泌以及钙化能力均得到显著提升。结论:miR-26a可以用于修饰hPDLSCs以提高其成骨分化能力。     

TAZ介导的Wnt/β-catenin信号通路与骨髓基质干细胞的成骨分化

骨质疏松症是由于骨重建过程中骨形成和骨吸收失平衡导致骨总量丢失所致,与成骨细胞分化密切相关。Hippo通路影响着哺乳动物体内细胞增殖、分化和凋亡过程。Wnt/β-catenin通路在成骨细胞分化中扮演重要角色。Hippo下游的靶基因转录共激活因子TAZ脱磷酸化后具有促进骨髓基质干细胞(BMSCs)向成骨细胞分化,调节成骨特异基因骨钙素表达,调节骨、肾发育,激活Wnt/β-catenin通路转录反应的功能;而激活的Wnt/β-catenin通路能通过抑制β-catenin降解进而抑制TAZ的降解。因此,TAZ与Wnt/β-catenin通路相互调控。但是,对TAZ与Wnt/β-catenin通路串话是否影响BMSCs成骨能力尚不清楚。因此,深入研究TAZ介导的Wnt/β-catenin通路在骨代谢中的作用,将为深入了解骨质疏松的发病机制具有重要意义。     

小檗碱通过PI3K/AKT信号通路调控人牙周膜干细胞的增殖和成骨分化

该文旨在探讨小檗碱(berberine)对人牙周膜干细胞(hPDLSCs)增殖和成骨分化的影响及潜在的机制。体外培养hPDLSCs,将其分为空白对照(Con)组、PI3K/AKT信号通路抑制剂LY294002(LY)组、小檗碱(Ber)组和小檗碱+PI3K/AKT信号通路抑制剂LY294002(Ber+LY)组,采用细胞计数试剂盒-8(CCK-8)法检测h PDLSCs的增殖活力,流式细胞仪检测细胞周期分布,酶联免疫检测仪检测碱性磷酸酶(ALP)活性,实时荧光定量PCR(q RT-PCR)分析增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)、骨钙蛋白(OCN)、骨膜蛋白(POSTN)和骨桥蛋白(OPN)的表达情况,蛋白免疫印迹法(Western blot)检测PCNA、Cyclin D1、OCN、POSTN、OPN和PI3K/AKT信号通路相关蛋白的表达情况。与Con组相比,Ber组h PDLSCs增殖活力,ALP的活性,S期和G₂/M期细胞比例,PCNA、Cyclin D1、OCN、POSTN、OPN、p-PI3K和p-AKT的表达水平均显著升...     

牙龈卟啉单胞菌影响牙龈上皮细胞miR1555p 通过JAK/STAR信号通路作用牙周炎的机制

目的 通过生物信息学对GEO数据库进行分析筛选miRNA后运用分子生物学手段验证并对机制进行深入探讨,为未来牙周炎治疗的生物标志物筛选及靶向治疗提供理论依据。 方法 通过生物信息学分析GEO数据库发现牙周炎患者中差异表达的miRNA。在DIANA生信预测网站中发现了与JAK/STAT信号传导方式有关的miRNA。随后,TargetScan被用于预测miRNA的靶mRNA,该mRNA不仅在牙周炎中差异表达,而且与JAK/STAT信号传导有关。利用基因集富集分析(GSEA)寻找与JAK/STAT信号转导途径紧密相关的基因集。通过实时定量PCR(qRTPCR)和免疫印迹法(Western blot)检测在牙周炎中差异表达并与JAK/STAT信号有关的miRNA和mRNA的表达。通过免疫组织化学(IHC)观察组织中IL6ST的表达。通过双重荧光素酶测定法证实了miRNA和mRNA之间的关系。此外,采用细胞内氧化活性氧红色荧光检测试剂盒检测活性氧(ROS)的变化。 结果 在牙周炎患者中,与正常组织比较,MiR1555p被下调,mRNA IL6ST被上调且差异均具有统计学意义(t=9.188 7、2.852 1,P=0.000 6、0.015 7)。与对照组比较,miR1555p在P.gingivalis处理组表达情况出现明显下降且IL6ST在处理后表达量出现明显升高,差异均有统计学意义(t=2.125 3、1.852 0,P=0.013 5、0.015 7)。miR1555p具有IL6ST 3′非翻译区的靶结合位点。通过过表达miR1555p能够明显抑制JAK/STAT信号通路pSTAT3、pJAK2、IL6ST蛋白的表达(t=1.924 8、2.530 8、3.107 5,P=0.023 1、0.011 6、0.010 0)。感染牙龈卟啉单胞菌后,细胞中的ROS产生增加(t=3.051 2、9.632 7,均P结论 牙龈卟啉单胞菌可抑制miR1555p表达,激活GECs中的JAK/STAR信号,促进牙周炎的发生和发展。     

The inhibition of periodontal ligament stem cells osteogenic differentiation by IL-17 is mediated via MAPKs

Periodontal disease (PD), a degenerative bacterially induced disease of periodontium, can lead to bone resorption and teeth loss. Development of PD includes a strong inflammatory reaction, which involves multiple immune cells and their secreting factors including interleukin-17 (IL-17), which is not only an important modulator of immune and hematopoietic responses but also affects bone metabolism. In the present study we aimed to determine whether IL-17 affects the regenerative potential of periodontal ligament mesenchymal stem cells (PDLSCs) by investigating its ability to modulate osteogenic differentiation of these cells in vitro along with associated signaling pathways. Our results revealed that IL-17 inhibited both the proliferation and migration of PDLSCs and decreased their osteogenic differentiation by activating ERK1,2 and JNK mitogen-activated protein kinases. Obtained data suggested that IL-17 might contribute to alveolar bone loss in PD.     

A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

One of the most important issues in stem cell research is to understand the regulatory mechanisms responsible for their differentiation. An extensive understanding of mechanism underlying the process of differentiation is crucial in order to prompt stem cells to perform a particular function after differentiation. To elucidate the molecular mechanisms responsible for the hematopoietic differentiation of embryonic stem cells (ESCs), we investigated murine ES cells for the presence of hematopoietic lineage markers as well as Wnt signaling pathway during treatments with different cytokines alone or in combination with another. Here we report that Wnt/beta-catenin signaling is down-regulated in hematopoietic differentiation of murine ES cells. We also found that differentiation induced by the interleukin-3, interleukin-6, and erythropoietin combinations resulted in high expression of CD3e, CD11b, CD45R/B220, Ly-6G, and TER-119 in differentiated ES cells. A high expression of beta-catenin was observed in two undifferentiated ES cell lines. Gene and protein expression analysis revealed that the members downstream of Wnt in this signaling pathway including beta-catenin, GSK-3beta, Axin, and TCF4 were significantly down-regulated as ES cells differentiated into hematopoietic progenitors. Our results show that the Wnt/beta-catenin signaling pathway plays a role in the hematopoietic differentiation of murine ESCs and also may support beta-catenin as a crucial factor in the maintenance of ES cells in their undifferentiated state.     

Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples

Abstract To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia . The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia . However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia . Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.