张、压应力刺激下人牙周膜成纤维细胞早期增殖活性和差异表达基因的变化

目的对比在张应力和压应力两种不同性质力的刺激下人牙周膜成纤维细胞（HPDLF）增殖活性的变化,并对差异表达基因进行筛选,探讨不同应力作用后HPDLF增殖变化的分子机理。方法使用四点弯曲加力装置对细胞进行0.5 Hz、4 000 μstrain的应力加载,加载时间2 h,采用流式细胞术测定张、压应力刺激对HPDLF增殖活性的影响,应用基因芯片技术检测两种应力刺激下细胞的差异表达基因。结果在两种应力刺激作用下,S期细胞百分比和细胞增殖指数均降低;差异表达的基因最主要位于细胞核,集中在转录因子活性相关基因群,参与最多的生物过程为转录因子调控,其中压应力引起的差异表达基因和生物过程的数量较张应力更多。结论1）周期性张、压应力作用下,HPDLF增殖变缓、细胞周期阻滞。2）细胞增殖变缓和细胞周期阻滞可看成是细胞对外界刺激的适应和自我保护,有利于HPDLF有更多的时间决定如何应答外界应力刺激,其应答的结果首先是转录水平上基因表达的改变,最主要的生物学反应是核转录调控。3）HPDLF对周期性压应力更敏感。     

周期性牵张应力作用下KLF5对人牙周膜细胞增殖和成骨分化的影响

目的: 探讨周期性牵张应力(cyclic tensile stress, CTS) 作用下Kruppel样转录因子5 (Kruppel-like factor 5, KLF5) 在人牙周膜细胞 (human periodontal ligament cells, hPDLCs) 中的表达及其对 hPDLCs增殖和成骨分化的影响。方法: 酶解组织块法体外培养hPDLCs,对其施加形变率10%,频率0.5 Hz的周期性牵张应力1、4、8、12、24 h,采用RT-PCR 和Western印迹法检测细胞中KLF5 mRNA和蛋白的表达。转染siRNA（si-KLF5）至hPDLCs沉默KLF5表达,RT-PCR检测KLF5 mRNA的表达。同时,将过表达碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF、FGF2）的pcDNA3.1-FGF2转染至稳定转染si-KLF5的hPDLCs,加力8 h 后,以CCK8法检测各组细胞的增殖活性,碱性磷酸酶(alkaline phosphatase, ALP)试剂盒检测ALP活性,RT-PCR检测成骨分化因子Runx2和Osterix的mRNA表达,Western印迹法检测Runx2、Osterix、FGF2、GSK-3β、P-GSK-3β (ser 9)、β-catenin蛋白的表达。采用SPSS22.0 软件包对数据进行统计学分析。结果: 10% CTS刺激呈时间依赖性地诱导hPDLCs中KLF5 mRNA 和蛋白表达。si-KLF5转染可显著抑制10% CTS诱导的hPDLCs的增殖,降低其ALP活性,减少Runx2和Osterix的mRNA和蛋白表达,并抑制FGF2-GSK-3β/β-catenin信号通路的激活;而过表达FGF2可以部分逆转沉默KLF5对hPDLCs增殖和成骨分化的抑制效应。结论: 周期性牵张应力作用下,KLF5通过FGF2-GSK-3β/ /β-catenin信号通路影响人牙周膜细胞的增殖和成骨分化。     

辛伐他汀对人牙周膜细胞增殖和分化的影响

目的:研究辛伐他汀对人牙周膜细胞增殖和成骨分化的影响.方法:将第4 代人牙周膜细胞在条件矿化培养液中诱导培养,同时加入不同浓度的辛伐他汀(10-9、10-8、10-7、10-6 mol/L),噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖情况,4-硝基苯基磷酸二钠盐(4-nitrophenyl phosphate,hexahydrate,PNPP)偶氮法检测碱性磷酸酶(alkaline phosphatase,ALP)活性.结果:辛伐他汀各浓度组均能促进人牙周膜细胞的增殖和分化,其中10-8、10-7、10-6 mol/L组与对照组比较,差异有统计学意义(P<0.05),10-7 mol/L的辛伐他汀组促进细胞增殖和ALP活性的作用最明显.结论:适宜浓度的辛伐他汀可有效促进人牙周膜细胞的增殖和成骨分化.     

RhoE regulates actin cytoskeleton organization in human periodontal ligament cells under mechanical stress

ObjectivesRhoE and regulator of G-proteins signalling (RGS) 2 were identified as the up-regulated genes in human periodontal ligament (PDL) cells under compression. RhoE belongs to the Rho GTPase family, and RGS2, a novel family of GTPase-activating proteins, turns off the G-protein signalling. Rho family proteins have recently been known to regulate actin cytoskeleton dynamics in various cell types. In this study, we investigated the involvement of RhoE and RGS2 in the regulation of actin filament organization in the PDL cells under mechanical stress.MethodsHuman PDL cells were cultured and subjected to a static compressive force (3.0 g/cm²) for 48 h. To observe changes in the actin cytoskeleton and the expression of RhoE and RGS2 in response to mechanical stress, immunofluorescence analysis was performed. To examine the role of RhoE and RGS2 in actin filament organization, cells were transfected with antisense S-oligonucleotides (ODNs) to RhoE and RGS2.ResultsCompressive force caused a loss and disassembly of actin stress fibres leading to cell spreading. Immunocytochemical study revealed that RhoE and RGS2 expressions were induced by mechanical stress and localized in the perinuclear and in the cell membrane, respectively. The impaired formation of stress fibres caused by compressive forces was recovered by treatment with antisense S-ODN to RhoE to the control levels. However, addition of antisense S-ODN to RGS2 did not affect the stress fibre formation.ConclusionsThese results indicate that the loss and disassembly of stress fibres due to mechanical stress are mediating RhoE signalling, without the exertion of RGS2.     

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。     

真核表达载体pIRES2-EGFP-PELP1的构建及其在人牙周膜干细胞中的表达

目的 克隆人源脯氨酸-谷氨酸-亮氨酸富集蛋白1(proline-,glutanic acid-,leucine-rich proteinl,PELP1)基因全长,构建PELP1基因全长过表达载体,研究PELP1基因表达特点,为PELP1基因在不同组织细胞中的功能性研究奠定基础.方法 采用反转录PCR法从MCF-7细胞总RNA中克隆PELP1基因全长,与含有EcoR I与Xho I双酶切位点的真核过表达载体pIRES2-EGFP质粒载体连接,构建重组质粒pIRES2-EGFP-PELP1.经双酶切及测序鉴定重组质粒中PELP1基因的完整性及正确性.运用脂质体将重组质粒转染至人牙周膜干细胞中,运用实时定量PCR(quantitative real-time PCR,qRT-PCR)及蛋白质印迹法分别从基因和蛋白水平对转染细胞中PELP1的表达进行检测.结果 成功克隆出PELP1基因全长,并将其插入至pIRES2-EGFP过表达载体中,经双酶切鉴定和测序鉴定证实目的质粒片段插入无误.重组质粒pIRES2-EGFP-PELP1转染48 h后PELP1基因水平是转染空质粒人牙周膜干细胞的(148.0±1.3)倍,二者差异有统计学意义(P＜ 0.005).蛋白质印迹法结果显示,与转入pIRES2-EGFP的人牙周膜干细胞中PELP1蛋白相对表达量(0.3300±0.0117)相比,转入pIRES2-EGFP-PELP1重组质粒的人牙周膜干细胞中PELP1蛋白相对表达量(0.6200 ±0.0045)显著增高,二者差异有统计学意义(P＜ 0.005).结论 本项研究成功运用qRT-PCR反应从MCF-7细胞中克隆出PELP1全长基因,并成功构建pIRES2-EGFP-PELP1真核过表达载体.     

Insulin modulates cytokines expression in human periodontal ligament cells

ObjectivePeriodontal disease (PD) has recently been recognized as the ‘sixth’ major complication of diabetes but the mechanisms involved in diabetic PD remain unclear. This study was to elucidate the potential bone-sparing effect of insulin in diabetic PD conditions.DesignThe human periodontal ligament (hPDL) cells were obtained from the healthy hPDL tissue and were treated with high concentrations (25 or 45 mM) of glucose with or without different concentrations (10⁻⁶, 10⁻⁷ or 10⁻⁸ mM) of insulin.ResultsHigh concentrations of glucose increased the production of pro-inflammatory cytokines interleukin-1 beta (IL-1β), tumour necrosis factor alpha (TNF-α), Interleukin-6 (IL-6) at both mRNA and protein levels, and receptor activator of NF-кB ligand (RANKL) at mRNA levels in hPDL cells. Insulin treatment alleviated the stimulatory effects of high glucose on pro-inflammatory cytokines and RANKL expression by hPDL cells. Moreover, insulin up-regulated OPG expression and therefore attenuated the reduction of OPG vs. RANKL ratio.ConclusionInsulin plays significant roles in modulating the periodontal tissue responses to hyperglycemia, and thus exerts its bone-sparing effects on periodontal tissues via altering the inflammatory cytokines expression in hPDL cells.     

Efficient gene transfer to periodontal ligament cells and human gingival fibroblasts by adeno-associated virus vectors

Objectives

We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and human gingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types.

Methods

AAV serotypes 1–5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry.

Results

Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5.

Conclusions

The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.     

氟化钠对人牙周膜细胞增殖及矿化能力的影响

目的:观察氟化钠对体外培养的人牙周膜细胞增殖及矿化能力的影响,为氟添加入牙周组织工程药物中的应用提供依据.方法:原代培养并鉴定人牙周膜细胞,应用CCK8检测不同浓度NaF对hPDLCs增殖的影响,并筛选出4个浓度用于矿化实验.矿化条件下,将0、1×10-5、5×10-4和1×10-3 mol/L的NaF作用hPDLCs后,通过碱性磷酸酶(ALP)染色、茜素红染色和实时荧光定量PCR检测矿化能力及成骨相关基因的表达.采用SPSS20.0软件包对数据进行单因素方差分析.结果:5×10-5、1×10-4、5×10-4 mol/L的NaF均能促进hPDLCs增殖,且以5×10-4 mol/L效果最佳(P＜0.05).而1×104 mol/L的NaF碱性磷酸酶染色阳性面积最大、茜素红染色矿化结节数量最多(P＜0.05).RT-PCR结果根据时间、指标变化程度较大.结论:5×10-5、1×10-4、5×10-4 mol/L的NaF能促进hPDLCs的增殖能力,1×10-5 mol/L的NaF能提高hPDLCs的碱性磷酸酶活性及钙结节形成.     

人牙周膜成纤维细胞增殖与压力关系的初步观察

目的：在体外培养环境下观察压力对人牙周膜成纤维细胞（HPLF）增殖的影响。方法：取第4－6代培养的HPLF,应用可控压力细胞加载装置分别间断性施加30、60、90kPa的压力,分别在培养3、5、7d后用MTT法测定各组的A值。结果：HPLF在30、60、90kPa的压力培养环境下MTT反应的A值显著小于对照组,压力值越大,A值越小。结论：30、60、90kPa间断性压力显著抑制PLF细胞增殖,该抑制作用随压力值增大而增强。     

周期性牵张力调节人牙周膜细胞成骨相关基因表达的研究

目的探讨牵张力诱导体外培养的人牙周膜细胞（human periodontal ligament cells，HPDLC）成骨分化的基因调控机制。方法通过体外细胞加载系统对培养的人牙周膜细胞施加12％形变率、6周／min的周期性牵张力48h，使用点样数为96点的人骨再生基因表达谱芯片检测周期性牵张力加载组细胞成骨相关基因表达的变化。结果HPDLC在周期性牵张力作用下有21种成骨相关基因的表达明显升高，包括10种生长因子和相关分子，10种细胞外基质和相关蛋白，1种细胞黏附分子。有2种基因表达明显下降，包括1种生长因子和相关分子，1种细胞黏附分子。结论周期性牵张力作用于体外培养的HPDLC，可以通过调节部分成骨相关基因的表达，诱导牙周膜细胞的成骨分化。     

RNA干扰抑制人牙周韧带细胞S100A4基因的表达

目的 研究RNA干扰技术(RNAi)下调牙周韧带(PDL)细胞中钙结合蛋白S100A4的表达,获得稳定的细胞单克隆,拟为后续试验提供细胞水平平台.方法 利用RNAi技术,针对人cDNA序列设计了S100A4干扰序列及一个阴性对照,将其连接到RNA干扰载体pGenesil-1上.转染人PDL细胞,通过反转录聚合酶链反应(RT-PCR)和Western blot检测有干扰序列的有效性.结果 干扰片段转染72 h后的mRNA抑制效率为67.83％.结论 RNAi可以有效抑制PDL细胞中S100A4的基因表达,为进一步研究S100A4在PDL细胞中的功能提供新的思路.     

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells

Objective: This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells.

Material and methods: After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24?h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84?key genes encoding cytoskeletal regulators after 6 and 24?h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes.

Results: The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6?h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24?h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated.

Conclusion: The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.     

Inhibition of the proliferation of human periodontal ligament fibroblasts by hyaluronidase

Hyaluronan (HA) exists in various living tissues as one of the major matrix macromolecules, and is well known to play an integral role in cell differentiation and proliferation. The present study was conducted to elucidate whether or not the proliferation of periodontal ligament (PDL) cells are affected specifically by the degradation of HA by hyaluronidasze (HAase). Human PDL fibroblasts were isolated and cultured with and without 15-150U/ml bovine testicular HAase from 1 to 11 days after seeding. The cells were also cultured with anti-CD44 antibody of 2 microg/ml. For the control against the anti-CD44 antibody treatment, 2 microg/ml IgG was used. The HA-dependent pericellular matrix was visualized by particle-exclusion assay. The number of cells was counted by MTT assay during the proliferation. The mRNA levels of HA synthases (HASs), HAases (HYALs) and CD44s were examined by a quantitative real-time PCR analysis. The cell proliferation was inhibited by the treatment with HAase and anti-CD44 antibody in cultured PDL fibroblasts. HASs mRNAs were down-regulated, whereas HYALs mRNAs were up-regulated significantly by the treatment with HAase and anti-CD44 antibody. The CD44s mRNA level exhibited no significant changes. These results suggest that HA may contribute to modulate the proliferation of cultured human PDL cells through a CD44-mediated mechanism.     

银杏叶提取物对脂多糖作用下人牙周膜细胞增殖及分泌细胞因子的影响

目的 观察银杏叶提取物(Ginkgo biloba extract,GBE)对脂多糖(lipopolysaccharide,LPS)作用下人牙周膜细胞(periodental ligament cell,PDLC)增殖及分泌白细胞介素(interleukin,IL)6、IL-1β及肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的影响,为GBE在牙周病防治中的应用提供依据.方法 选取2010年5至7月在遵义医学院附属口腔医院口腔颌面外科门诊因正畸拔除的10例11 ～15岁患者的20颗前磨牙,采用改良组织块培养法体外原代培养人PDLC,实验共分5组:①阴性对照组,含10 ml/L胎牛血清的达尔伯克改良伊格尔培养基(Dulbecco's modified Eagle's medium,DMEM)培养液;②LPS组,含10 ml/L胎牛血清的DMEM培养液+100 mg/L LPS;③LPS+0.1 g/L GBE组,含10 ml/L胎牛血清的DMEM培养液+100 mg/L LPS+0.1g/L GBE;④LPS+0.01 g/L GBE组,含10 ml/L胎牛血清的DMEM培养液+100 mg/L LPS +0.01 g/L GBE;⑤LPS+地塞米松组,含10 ml/L胎牛血清的DMEM培养液+100 mg/L LPS+2 mg/L地塞米松.甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法测定GBE对LPS作用下人PDLC活性的影响,酶联免疫吸附测定法测定各组IL-6、IL-1β及TNF-α的含量,对所得结果分别进行单因素方差分析,采用LSD-t检验进行组间两两比较,检验水准为双侧α=0.05.结果 实验12、24、48及72 h,LPS+0.1 g/L GBE组的吸光度值(分别为0.30±0.03、0.33±0.02、0.34 ±0.02及0.35 ±0.02)均显著高于LPS组(分别为0.20±0.03、0.20±0.02、0.22±0.04及0.24±0.02)(P＜0.05),PDLC IL-6、IL-1 β及TNF-α的分泌量均显著低于LPS组(P＜0.05);实验12、24、48及72 h,LPS+0.01 g/L GBE组吸光度值(分别为0.27±0.05、0.31 ±0.03、0.33±0.03及0.32 ±0.01)均显著高于LPS组(P＜0.05),PDLC IL-6、IL-1β及TNF-α的分泌量均显著低于LPS组(P＜0.05).结论 GBE对LPS作用下的人PDLC有保护作用.     

釉原蛋白对牙周膜干细胞迁移、黏附及增殖的影响

目的：检测釉原蛋白（amelogenin,AML）对牙周膜干细胞（PDLSCs）迁移、黏附和增殖的影响。方法：用0．25、50和100μg／ml 的 AML 培养人 PDLSCs。用划痕实验和 transwell 法检测 AML 对 PDLSCs 迁移的影响。用黏附实验检测 AML 对PDLSCs 黏附的影响。用 MTT 法和计数法检测 AML 对 PDLSCs 增殖的影响。结果：AML 可促进 PDLSCs 的迁移,而且呈剂量效应关系（P ＜0．05）。AML 对 PDLSCs 迁移和黏附有促进作用（P ＜0．05）,且随培养时间延长而增加。AML 可促进 PDLSCs的增殖,且呈剂量效应关系。结论：AML 可促进 PDLSCs 的迁移、黏附和增殖。     

氯霉素对人牙周膜细胞增殖及超微结构的影响

目的：比较0．25％氯霉素和生理盐水对人牙周膜细胞增殖和超微结构的影响,以提高脱位牙再植的疗效。方法：采用组织块法体外培养人牙周膜细胞,取第5代培养细胞,分别加入氯霉素、生理盐水和DMEM培养液作用1h,在作用后0、12、24、48h进行细胞计数．观察对牙周膜细胞增殖的影响。采用透射电镜观察人牙周膜细胞超微结构的变化。数据采用SAS9．1软件包进行方差分析。结果：在0、12、48h时间点．氧霉素组和生理盐水组的细胞数显著低于DMEM培养液对照组（P〈0．0001）;在24h时间点,生理盐水组的细胞数显著低于对照组（P〈0．05）。各时间点氯霉素和生理盐水两组细胞计数无显著差异（P〉0．05）。电镜观察,氯霉素可导致人牙周膜细胞线粒体空泡化．内质网减少。结论：氯霉素对人牙周膜细胞增殖的影响与生理盐水没有区别,但对人牙周膜细胞的超微结构有损伤作用。     

Effects of prostaglandin E2 on clonogenicity,proliferation and expression of pluripotent markers in human periodontal ligament cells

Background and objectiveBased on our earlier work on the response of periodontal ligament (PDL) cells to mechanical stress by induction of cyclooxygenase expression and production of prostaglandin PGE₂ that could regulate mineralization of PDL cells, it was hypothesized that PGE₂ had potential effects on PDL stemness. In this study, we aimed to investigate clonogenicity, proliferation and expression of certain pluripotent markers, considered to be characteristics of PDL stemness, in response to treatment with exogenously-added PGE₂.Material and methodsHuman PDL cells were cultured and treated with various doses of PGE₂, and the aforementioned characteristics of PDL stemness were analyzed.ResultsThe clonogenicity and proliferation were significantly enhanced by PGE₂ at low concentrations (0.01, 0.1 and 1 ng/ml; P < 0.05), but only the proliferation was significantly diminished by PGE₂ at a high concentration (100 ng/ml; P < 0.05). Expression of NANOG and OCT4 mRNA and protein was increased by PGE₂ treatment at 0.1 and 1 ng/ml. Consistently, expression of stage-specific embryonic antigen 4, a putative stem cell marker, was significantly augmented by PGE₂ treatment at 1 ng/ml (P < 0.05).ConclusionOur findings suggest that although a high dose of PGE₂ (100 ng/ml) inhibits proliferation of PDL cells, PGE₂ at low doses appears to play a role in the maintenance of PDL stemness.