Copper ability to induce premature senescence in human fibroblasts

Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated β-galactosidase (SA β-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA β-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor β-1 (TGF β1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF β1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-011-9276-7) contains supplementary material, which is available to authorized users.     

富血小板血浆对人真皮细胞增殖及PDGF、TGF-β和VEGF表达的影响

目的:研究富血小板血浆(PRP)对人真皮成纤维细胞(hFbs)增殖、分化及胞质中PDGF、TGF-β和VEGF表达量的影响。方法:2次离心法制备PRP,成骨诱导条件下hFbs培养液中加入不同浓度PRP于第12天、21天钙-钴法染色,第21天茜素红染色;免疫细胞化学法检测加不同浓度PRP后第4天细胞PDGF、TGF-β和VEGF的表达;碱性磷酸酶染色(ALP)检测细胞活性;CCK-8法检测有和无成骨诱导条件下不同浓度PRP对细胞增殖的影响。结果:茜素红染色显示,2.8%PRP组钙结节形成最明显;钙-钴法染色显示,2.8%PRP组矿化作用最强;免疫细胞化学结果表明PDGF表达存在剂量依赖性,TGF-β和VEGF表达虽无剂量依赖性,但有适宜浓度的要求;ALP染色表明3%PRP组细胞增殖明显,活性最强。CCK-8检测表明各浓度PRP对hFbs均有促增殖作用,PRP加成骨诱导组比单纯PRP组表现出更明显的促增殖作用,其中4.8%PRP促增殖作用最强。结论:适宜浓度的PRP可促进hFbs增殖,但相关因子的表达存在适宜浓度的要求,PRP通过促进细胞增殖,增加相关因子表达促进创伤愈合。     

壳聚糖/葡甘聚糖混合膜对人牙周膜细胞作用观察

目的:通过壳聚糖/葡甘聚糖膜单独、以及膜加载碱性成纤维细胞生长因子和第4代人牙周膜细胞共同培养,观察壳聚糖/葡甘聚糖膜对人牙周膜细胞增殖分化的影响。方法:观察不同质量比壳聚糖与葡甘聚糖共混膜对第4代细胞HPDLCs增殖影响及其对碱性磷酸酶活性(ALP)影响。结果:不同质量比的壳聚糖/葡甘聚糖混合膜对HPDLCs的增殖有促进作用,加载bFGF后,HPDLCs增殖得到增强,且壳聚糖/葡甘聚糖膜比3:0和1:2的混合膜还可增强ALP的活性水平。结论:壳聚糖/葡甘聚糖膜是一种潜在的牙周组织再生膜。     

甘草甜素对人颊黏膜成纤维细胞炎症反应的初步研究

目的：观察甘草甜素对经脂多糖刺激后体外培养的人颊黏膜成纤维细胞炎症反应的作用。方法：取正常人颊黏膜,组织块法体外培养颊黏膜成纤维细胞,加入不同浓度的甘草甜素,培养24h、48h、72h后,经MTT法检测每孔的光密度值（OD）。用脂多糖（LPS）对细胞进行干预,用来模拟口腔扁平苔藓（0LP）的炎症状态。以第4代成纤维细胞为对照组、加入脂多糖为炎症组、同时加入脂多糖和甘草甜素为实验组,用ELISA法分别检测三组白细胞介素-6（IL-6）的浓度。结果：MTT法测出甘草甜素在一定范围内浓度越高、时间越长,抑制细胞的增殖作用就越强;ELISA法检测出炎症组较对照组上清液中IL-6浓度明显升高,实验组较炎症组上清液中IL-6浓度明显降低。结论：甘草甜素可明显抑制由脂多糖刺激的人颊黏膜成纤维细胞分泌IL-6。甘草甜素在治疗OLP的炎症反应中有重要作用。     

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目的 研究不同剂型亚甲基蓝(methylene blue,MB)介导的光动力疗法(photodynamic therapy,PDT)对体外培养的人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPDLFs)的毒性作用.方法 将体外培养的HPDLFs分别加入两种不同剂型MB中(水剂型和混合剂型,均设置5组浓度,分别为0.1、1.0、10、25、50 μmol/L),孵育10 min,波长670 nm半导体激光照射5min,输出功率40 mW,功率密度10 mW/cm2.光照结束后继续培养24h,用噻唑蓝(MTT)比色法检测细胞毒性,光镜下观察细胞形态学变化.结果 两种不同剂型MB介导的PDT对体外培养的HPDLFs的毒性作用未见明显差异(P＞0.05).当MB浓度≤25 μmol/L时,细胞毒性为1～2级,光镜下观察细胞未发现明显形态学改变;浓度＞ 25 μmol/L时,细胞毒性为3级,并可观察到细胞皱缩、变圆.结论 PDT对HPDLFs的毒性作用受光敏剂浓度影响,适当浓度的两种剂型MB介导的PDT对体外培养的HPDLFs影响轻微,且两种剂型MB介导的PDT对体外细胞毒性无差异.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

MyD88 or TRAM Knockdown Regulates Interleukin (IL)‐6, IL‐8, and CXCL12 mRNA Expression in Human Gingival and Periodontal Ligament Fibroblasts

Background: In a previous report, it was shown that Toll‐like receptor (TLR) 2 knockdown modulates interleukin (IL)‐6 and IL‐8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). This study investigates whether knocking down two important TLR adaptor molecules, such as myeloid differentiation protein 88 (MyD88) and TRIF‐related adaptor molecule (TRAM), could affect mRNA expression of IL‐6, IL‐8, and CXCL12 in HGF and HPDLF. Methods: After small interfering (si) RNA‐mediated silencing of MyD88 or TRAM, HGF and HPDLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL‐6, IL‐8, and CXCL12 mRNAs were evaluated by quantitative polymerase chain reaction. Results: Knockdown of MyD88 or TRAM partially impaired the IL‐8 mRNA upregulation in both fibroblast subpopulations. Similarly, IL‐6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with Pg LPS, as well as in both fibroblast subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non‐stimulated cells. Conclusions: These results suggest that TLR adaptor molecules knockdown, such as MyD88 or TRAM, can decrease IL‐6 and IL‐8 mRNA and increase CXCL12 mRNA expression in HGF and HPDLF. This can be an important step for better understanding the mechanisms that control the inflammatory cytokine and chemokine expression, which in turn contributes to periodontal pathogenesis.     

慢病毒载体感染人口腔黏膜成纤维细胞稳定表达绿色荧光蛋白的研究

目的：确立慢病毒载体高效感染人口腔黏膜成纤维细胞的感染条件,为进一步应用基因工程技术研究人口腔黏膜成纤维细胞奠定基础。方法：采用组织块法培养原代人口腔黏膜成纤维细胞,免疫组化SP法鉴定细胞类型,将携带绿色荧光蛋白基因的慢病毒载体以不同感染复数（multiplicity of infection,MOI）感染纯化细胞,并设置不同感染条件,感染4 d后于荧光显微镜下观察绿色荧光蛋白表达情况。结果：成功分离纯化得到人口腔黏膜成纤维细胞,当慢病毒感染复数为30,polybrene浓度为8μg/mL,并加入感染增强液时,可达到较高的感染效率,满足后续实验需要。结论：慢病毒载体可高效感染人口腔黏膜成纤维细胞,携带的绿色荧光蛋白基因可在成纤维细胞内稳定表达。