离心作用下人牙周膜细胞内诱导型一氧化氮合酶和胱硫醚分解酶的表达

目的研究在离心力作用下人牙周膜细胞内的诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）和硫化氢合酶胱硫醚分解酶（cystathionine-γ-lyase,CSE）的表达变化。方法采用组织块一酶消化联合法培养人牙周膜细胞,对细胞施加离心力（167g）,SABC法染色及胞浆透光度检测iNOS和CSE在10、30、60、90、120和240min不同加力时间点的表达变化。结果正常人牙周膜细胞内几乎无iNOS和CSE表达;加力10min,iNOS和CSE有表达（Pd0．01）;之后两种酶表达逐渐加强,加力60min,iNOS和CSE表达达到高峰（P〈0．01）;之后逐渐减弱,加力240min,iNOS和CSE恢复到基础水平。结论离心力可诱导人牙周膜细胞内iNOS和CSE的表达短暂性升高,提示一氧化氮（NO）和硫化氢（Hzs）可能参与了牙周组织改建中的力学信号转导过程。     

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、12 h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4 h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8 h时,Osx mRNA和蛋白表达显著增强（P<0.01）;持续增加至加力12 h。同时,加力4 h后,少量细胞的胞质内开始呈现微弱的绿色荧光;12 h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（Col I）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Col I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。     

Changes in the expression of Cbfa1 in human periodontal ligament stem cells loaded with static tensile stress

目的 检测静态张应力作用对人牙周膜干细胞核心结合因子Cbfa1表达变化的影响.方法 有限稀释法克隆化培养纯化人牙周膜干细胞(hPDLSCs),应用内外双层套筒式硅胶膜细胞加力装置对人牙周膜干细胞加力,加力时间点为0、3、6、12、24小时.采用原位杂交和实时定量PCR检测各加力时间点Cbfa1 mRNA的表达;蛋白免疫印迹法(Western blot)检测人牙周膜干细胞受力后Cbfa1蛋白的表达.结果 人牙周膜干细胞受到静态张应力作用后3小时、6小时、12小时Cbfa1 mRNA表达均升高(P<0.05),且以加力3 小时最为明显,6小时后开始下降,24小时后基本恢复到不加力时的表达水平.加力3小时Cbfa1蛋白表达未见明显升高,加力6小时后Cbfa1蛋白逐渐升高,一直持续到加力24小时(P<0.05).结论 静态张应力作用下,人牙周膜干细胞核心结合因子Cbfa1在RNA水平和蛋白水平的表达均发生变化,说明Cbfa1在机械力促使人牙周膜干细胞向成骨细胞方向分化的过程中起到了重要作用.     

体外周期性单轴压应力下髁突软骨细胞Raf激酶抑制蛋白(RKIP)的动态变化研究

目的:探讨体外周期性单轴压应力对大鼠髁突软骨细胞的Raf激酶抑制蛋白(RKIP)及其mRNA变化的早期影响,以助于阐明力学引起细胞应答反应的分子机制。方法:利用四点弯曲细胞力学加载仪对第3代大鼠髁突软骨细胞进行体外周期性单轴压应力加载,力值为4000μstrain,时间为0、15、30、60、120、240min。采用实时荧光定量PCR技术和Western blot免疫印迹技术研究大鼠髁突软骨细胞在周期性单轴压应力作用下RKIP及其mRNA变化。结果:大鼠髁突软骨细胞RKIP mRNA和蛋白表达呈现几乎相反的趋势。RKIP mRNA的表达于30min上升(P<0.001),60min达到高峰后迅速回落,低于细胞的正常基态水平;而RKIP的蛋白表达30min开始下调(P<0.001),持续至120、240min时回升。结论:RKIP在髁突软骨细胞的早期力学应答机制中可能扮演着相当重要的作用,其蛋白和mRNA水平表达变化不同,转录水平的调控最终要影响蛋白水平的表达。     

机械牵张应变对人牙周膜细胞内游离钙离子浓度的影响

目的:观察机械牵张应变作用下,人牙周膜细胞(HPDLCs)内游离Ca2 浓度的变化。方法:体外培养HPDLCs,利用动态机械应变细胞加载装置,对1%、10%和20%应变组的细胞分别进行30、60和120min的动态牵张加载,通过流式细胞仪和激光共聚焦显微镜检测细胞内游离Ca2 浓度的变化,并应用SPSS13.0软件包对数据进行方差分析。结果:当HPDLCs加载30min时,各组检测到的胞内游离Ca2 浓度与对照组无显著差异(P>0.05);60min时,各个应变组的胞内Ca2 浓度明显升高,与对照组相比均有非常显著的差异(P<0.01);加载时间延长到120min时,各组Ca2 浓度又降至对照组水平(P>0.05)。结论:机械牵张应变可通过钙离子信号系统影响HPDLCs的生物学活性。     

静态张应力作用下人牙周膜干细胞核心结合因子Cbfa1表达的变化

目的检测静态张应力作用对人牙周膜干细胞核心结合因子Cbfa1表达变化的影响。方法有限稀释法克隆化培养纯化人牙周膜干细胞(hPDLSCs),应用内外双层套筒式硅胶膜细胞加力装置对人牙周膜干细胞加力,加力时间点为0、3、6、12、24小时。采用原位杂交和实时定量PCR检测各加力时间点Cbfa1 mRNA的表达;蛋白免疫印迹法(Western blot)检测人牙周膜干细胞受力后Cbfa1蛋白的表达。结果人牙周膜干细胞受到静态张应力作用后3小时、6小时、12小时Cbfa1 mRNA表达均升高(P0.05),且以加力3小时最为明显,6小时后开始下降,24小时后基本恢复到不加力时的表达水平。加力3小时Cbfa1蛋白表达未见明显升高,加力6小时后Cbfa1蛋白逐渐升高,一直持续到加力24小时(P0.05)。结论静态张应力作用下,人牙周膜干细胞核心结合因子Cbfa1在RNA水平和蛋白水平的表达均发生变化,说明Cbfa1在机械力促使人牙周膜干细胞向成骨细胞方向分化的过程中起到了重要作用。     

血小板衍生生长因子-BB与胰岛素样生长因子-Ⅰ联合应用对大鼠正畸牙牙周膜细胞整合素β3表达的影响

目的观察血小板衍生生长因子-BB(PDGF-BB)与胰岛素样生长因子-Ⅰ(IGF-Ⅰ)联合应用对大鼠正畸牙压力侧牙周膜细胞(PDLCs)中整合素β3蛋白表达的影响。方法建立SD大鼠正畸牙移动模型,隔日于正畸牙颊侧牙龈黏膜下单独或联合注射10 ng PDGF-BB及200 ng IGF-Ⅰ,加力10 d后处死大鼠,取材用免疫组织化学方法检测正畸牙压力侧牙周膜细胞中整合素β3的表达。结果 PDGF-BB、IGF-Ⅰ单独或联合应用均能增强压力侧牙周膜细胞中整合素β3表达(P<0.01);与PDGF-BB、IGF-Ⅰ单独应用比较,IGF-Ⅰ加PDGF-BB组牙周膜细胞中整合素β3表达明显增强(P<0.05和P<0.01)。结论外源性PDGF-BB和IGF-Ⅰ在大鼠正畸牙的局部注射能上调压力侧牙周膜细胞中整合素β3表达,二者联合应用具有协同效应。     

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(10 μg/ml)+RES(0、30、60、90tμmol/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;30～90 μmol/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.     

重离子辐照对舌癌Tca8113细胞中转移相关基因RhoC的作用

目的：：探讨重离子辐照对舌癌侵袭转移相关基因RhoC的作用。方法：应用不同剂量重离子束(12 C6+)对体外培养的舌癌Tca8113细胞进行辐照,采用荧光实时定量PCR法及Western Blot法观察细胞中RhoC mRNA及蛋白的表达变化。结果：与空白对照组比较,12C6+辐照后4h,各实验组细胞中RhoC mRNA的表达降低(P<0.05);辐照后12 h,2 Gy及4 Gy组细胞中RhoC mRNA表达增加,随后各组细胞中RhoC mRNA表达再次下降(P<0.05);辐照后12 h,细胞中RhoC蛋白表达量随辐照剂量的增加而增高,在4 Gy时达到高峰;24 h后随剂量的增加逐渐降低;在相同辐照剂量下,细胞中RhoC蛋白的表达随时间推移呈下降趋势,RhoC蛋白表达和RhoC mRNA表达的趋势相同。结论：12 C6+重离子束可以下调舌癌Tca 8113细胞中RhoC基因的表达。     

Expression of tropoelastin in human periodontal ligament fibroblasts after simulation of orthodontic force

The ability of elastic fibers to respond to mechanical stimuli suggests that they play a central role in physiological adaptation to external stimuli including application of orthodontic force. The purpose of this study was to examine the effect of external pressure simulating orthodontic force on tropoelastin gene expression in cultured human periodontal ligament fibroblasts (hPDLF). External pressure simulation was achieved by centrifugation for 10, 30, 60, 90 and 120 min of hPDLF in a horizontal microplate rotor. Semi-quantitative RT-PCR analysis of tropoelastin mRNA was performed and beta-actin was used as an internal invariant control. While centrifugal force on mRNA levels of beta-actin showed almost no change, the mRNA levels of tropoelastin increased significantly to a peak level of more than four-fold after 30 min. Thereafter, at 60 min, the mRNA levels remained at more than three-fold. After 90 min, mRNA levels decreased to control levels. The finding that no changes in mRNA levels of beta-actin occurred during the first 90 min of centrifugation validates its use as an invariant control gene in such an experimental model. This study demonstrated that tropoelastin is expressed in hPDLF and that the pressure caused significant time-dependent upregulation of the tropoelastin gene. The responsiveness of the tropoelastin gene to force shows its possible clinical importance in orthodontic tooth movement. Further studies, however, are essential in order to learn whether the high expression of the gene in vitro will also be followed by corresponding protein synthesis and deposition in vivo in the extracellular matrix (ECM) of the periodontal ligament (PDL).     

应力对纯钛表面成骨细胞核结合因子α1形成和表达的影响

目的观察应力对纯钛表面成骨细胞核结合因子α1（Cbfα1）形成及表达的影响，揭示应力对支抗种植体周骨反应的调控机制。方法用大小为2.205 N的间断离心力刺激接种于纯钛表面的成骨细胞，刺激频率为每培养4 h加力15 min，分别于4、8、12 h后收集细胞，采用免疫荧光法检测Cbfα1的形成，提取总RNA并应用逆转录聚合酶链反应检测Cbfα1 mRNA的表达。结果纯钛表面加力组和不加力组MG-63成骨细胞的细胞核内均有Cbfα1荧光染色，且加力组Cbfα1蛋白荧光强度较不加力组多，差异有统计学意义（P＜0.05）；纯钛表面加力组和不加力组MG-63成骨细胞Cbfα1 mRNA表达均为阳性，且加力组Cbfα1 mRNA表达较不加力组多，差异有统计学意义（P＜0.05）。结论成骨细胞受力后Cbfα1基因形成和表达增强，提示间歇性的牵引应力可以促进纯钛表面成骨细胞的活性。     

The effect of centrifugal force on mRNA levels of collagenase, collagen type-I, tissue inhibitors of metalloproteinases and beta-actin in cultured human periodontal ligament fibroblasts

BACKGROUND: The aim of orthodontic treatment is to relocate teeth abnormally positioned in the jaws. This is achieved by application of continuous force on the tooth, which is immediately being sensed by the periodontal ligament (PDL), bone and the gingiva. Since the bony response is mediated by the PDL, tooth movement is primarily a PDL phenomenon. OBJECTIVES: Thus, the purpose of the present study was to evaluate the direct effect of force (excluding the in vivo tissue response) on the molecular level of matrix metalloproteinase-1 (MMP-1) and collagen type-I (Col-I) in human PDL fibroblasts. METHODS: PDL cell culture flasks were centrifuged for 10, 20, 30, 60, 90 and 120 min by horizontal microplate rotor. The effect of force on mRNA levels of beta-actin, MMP-1, Col-I, tissue inhibitors-1 and -2 (TIMPs) genes was analyzed by RT-PCR. RESULTS: The results showed that force had no effect on the mRNA levels of beta-actin during the first 90 min of application of force, indicating for the first time the use of beta-actin gene as an internal invariant control. It increased the mRNA levels of MMP-1 while almost no effect on Col-I and TIMPs was observed. CONCLUSIONS: The results indicate that PDL remodeling following application of orthodontic force could be partly attributed to the direct effect of the force on MMP-1 gene expression in fibroblasts.     

Neuropeptides modulate RANKL and OPG expression in human periodontal ligament cells

Neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), may be associated with bone remodeling in response to mechanical stress during orthodontic tooth movement. To investigate this hypothesis, we examined the effects of neuropeptides on the expression of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament (PDL) cells under compression in vitro. PDL cells were subjected to compressive force (2.0 g/cm²) continuously in the presence or absence of SP or CGRP for 2–4 days. The expression of the SP receptor, neurokinin 1-receptor (NK₁-R), in PDL cells was confirmed by RT–PCR and immunofluorescent staining. The effects of neuropeptides (SP and CGRP) on the expression of RANKL and OPG mRNA were determined using RT–PCR. PDL cells constitutively expressed NK₁-R on both the mRNA and protein levels. Compressive force decreased OPG mRNA expression and increased RANKL mRNA expression. In the presence of neuropeptides, the OPG level decreased synergistically with compression. Neuropeptides stimulated RANKL expression without compression, whereas they decreased RANKL mRNA expression with compression. These results indicate that PDL cell compression induces the up-regulation of RANKL and down-regulation of OPG, whereas neuropeptides suppress the RANKL expression induced by compression. Therefore, the neuropeptides SP and CGRP may modulate bone remodeling by PDL cells during orthodontic tooth movement.     

Cyclic stretch influenced expression of membrane connexin 43 in human periodontal ligament cell

Objective

Periodontal ligament (PDL) cells play an important role in preserving periodontal homeostasis and periodontal remodelling in response to mechanical stimulations. Gap junction intercellular communication (GJIC) is essential for homeostasis and many other biological processes of multicellular organisms. While the role of GJIC in mechanotransduction of PDL cells remains largely unknown. In the present study, we examined the influence of cyclic stretch on the expression of membrane gap junction protein connexin 43 (Cx43) in cultured human PDL cells.

Design

Cultured human PDL cells were exposed to 1%, 10% and 20% stretch strains for 0.5 h, 1 h and 24 h. Then the membrane Cx43 protein expression was measured by flow cytometry and the Cx43 mRNA level was measured by real-time polymerase chain reaction.

Results

Half hour and 1 h cyclic stretches with strains up to 20% did not change the expression of membrane Cx43 protein, while 24 h cyclic stretches with 10% and 20% strains down-regulated the expression of membrane Cx43 protein in a strain magnitude-dependent manner. Furthermore, cyclic stretch also changed the Cx43 mRNA level and induced realignment in cells.

Conclusion

The present research provide the first evidence that cyclic stretch influenced the membrane Cx43 protein expression in cultured human PDL cells.     

Gene profile in periodontal ligament cells and clones with enamel matrix proteins derivative

AIM: Evaluate enamel matrix proteins derivative effect on gene expression profiles in cultured human periodontal ligament cell population and its clones. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were explanted. Cell cloning was performed and clones classified into fibroblastic (FB) and mineralized tissue forming (MTF) according to their capacity to express alkaline phosphatase and form mineralized tissue. All cell cultures were grown for 7 days, with and without enamel proteins added to the medium. Following RNA extraction, expression profiling was performed by hybridization with a DNA micro-array. Selected genes differed from the control at a significant level smaller than p<0.01. RESULTS: Enamel proteins induced major qualitative changes in mRNA expression in all PDL cell populations, differently affecting the entire PDL cell population and its clones. In the entire PDL cell population, enamel proteins significantly enhanced PDL cell function, with a general effect on enhanced cell functional metabolism. CONCLUSIONS: Enamel proteins enhanced gene expression responsible for protein and mineralized tissue synthesis in the entire PDL population. In the MTF clones, nucleic acid metabolism, protein metabolism and signal transduction related genes were up-regulated, while in the FB clones, up-regulated genes were related to cell adhesion, nucleic acid metabolism and signal transduction.