人牙龈成纤维细胞和牙周膜成纤维细胞的培养和生物学特征

目的：探讨采用不同方法建立体外培养人牙龈成纤维细胞和人牙周膜成纤维细胞并对其生物学特性作初步探讨。方法：分别采用消化培养法和组织块培养人的牙龈和牙周膜成纤维细胞。通过倒置显微镜活体观察，以及光镜、透射电镜、免疫组化和生长曲线等方法对其生物学特性作初步观察。结果：消化培养法成功率低于组织块培养法。光镜下和透射电镜下观察两种细胞在形态和结构上相似。免疫组化染色证实此两种细胞均来源于中胚层的纤维母细胞。生长曲线表明牙龈成纤维细胞的倍增时间比牙周膜成纤维细胞稍短，而牙周膜成纤维细胞的增殖活性比牙龈成纤维细胞高。结论：组织块培养法适用于此二种细胞的培养。细胞生物学特征方面有许多相似形，提示这两种细胞来于同一个细胞群。     

温度变化对体外培养人牙周膜成纤维细胞的影响

目的:在体外观察温度升高对人牙周膜成纤维细胞活力的影响,探讨热牙胶根管充填的安全性.方法:将体外培养人牙周膜成纤维细胞悬液于不同温度及时间培养,用噻唑蓝(MTT)比色法、台酚蓝染色测定细胞的存活,流式细胞分析法测定人牙周膜成纤维细胞凋亡及死亡情况.结果:体外培养人牙周膜成纤维细胞的死亡率随着温度的上升逐渐增加.流式细胞分析显示:50℃组较对照组的坏死细胞比例明显上升,差异有统计学意义(P＜0.01).结论:温度升高能诱发人牙周膜成纤维细胞活力降低,死亡率、凋亡率上升.     

人牙周膜成纤维细胞体外培养的研究进展

人牙周膜成纤维细胞是牙周细胞生物学、药理学、毒理学和牙周组织工程学研究的基础,对口腔各个领域意义重大。由于受到牙周组织的解剖结构和取材数量的限制,牙周膜成纤维细胞的体外培养较为困难,因此本文就人牙周膜成纤维细胞的体外培养方法和应用等研究进展作一综述。     

四环素对人牙周膜成纤维细胞的生物学作用

目的探讨四环素对体外培养的人牙周膜成纤维细胞(I1PDLFs)的生物学作用。方法将不同浓度的四环素(1,5,20,100,500,2 500习ml)加人体外培养的HPDLFs中,孵育2d后,在倒置显微镜下观察其对细胞形态的影响,并用M,1法、考马司亮蓝法及“H-TdR掺人法,分别检测四环素对细胞的增殖活性、蛋白合成及DNA合成的影响。结果在1 -. 100 pg/ml的浓度范围内,细胞形态呈正常的梭形或纺锤形。在20- 100留ml的浓度范围内,四环素可促进HPDLFs的增殖及生物合成(P < 0.01)。当四环素的浓度增至2 500 lcg/ml,不仅使细胞的镜下形态发生了明显改变,而且严重抑制了细胞的生物学活性。结论在合适的浓度范围内,四环素能促进HPDLFs的增殖及生物合成,而浓度过高则具有细胞毒性。     

体外培养碱性成纤维细胞因子对人牙周膜成纤维细胞生物学特性的影响

目的：探讨体外培养过程中,碱性成纤维细胞生长因子（bFGF）对人牙周膜成纤维细胞（hPDLFs）生物学特性的影响。方法：体外分离培养人牙周膜成纤维细胞并鉴定,加入不同浓度bFGF（1ng／ml、10ng／ml、50ng／ml、100ng／ml）培养,MTT方法检测细胞增殖情况;并对细胞进行矿化诱导,检测细胞的碱性磷酸酶活性。结果：hPDLFs呈星形或长梭形,免疫组化波丝蛋白阳性,角蛋白阴性,证实该细胞来源可靠。bFGF在一定浓度范围内,与细胞增殖成正比,而在本实验培养条件下（10％FBS）bFGF最大效应浓度为10ng／ml。矿化诱导条件下,碱性磷酸酶活性明显增加,在联合应用bFGF的情况下,100ng／ml组碱性磷酸酶活性明显高于其他组。结论：不同浓度bFGF对人牙周膜成纤维细胞生物学行为的影响不同,在一定浓度条件下,低浓度bFGF促进人牙周膜成纤维细胞的增殖,而高浓度bFGF作用于人牙周膜成纤维细胞可能更易于分化。     

静态拉伸应变对人牙周膜成纤维细胞的影响

目的：研究人牙周膜成纤维细胞(PDLF)在机械拉伸应变作用下，细胞和细胞核投影面积及其细胞骨架的改变情况，探讨PDLF的形态、功能和应变之间的关系。方法：采用自制的细胞体外静态机械加载装置用不同的拉伸应变量加力于细胞上，通过激光扫描共聚焦显微镜观察细胞骨架的形态及其改变情况，并进行统计分析。结果：在8％、12％、16％力值组，细胞和细胞核投影面积随着加力时间和力值的增加而每天递增，肌动蛋白纤维也随之逐渐增粗，排列更有规律；而在20％力值组，其结果则反之。结论：静态拉伸应变可影响牙周膜成纤维细胞的骨架。     

内毒素对人牙髓、牙周膜成纤维细胞DNA含量的影响

具核梭杆菌和牙髓类杆菌内毒素脂多糖(LPS)在50μg/ml浓度下可使人牙髓,牙周膜成纤维细胞核面积值和DNA含量升高,而100μg/ml浓度则可使细胞DNA含量降低,内毒素在不同浓度对细胞DNA含成的影响有显著差异。     

人牙龈成纤维细胞电泳行为的研究

细胞电泳行为的研究是通过对细胞表达面电荷的测定来探讨细胞的结构和功能状态间的关系，其特点是研究对象为活细胞，本实验首次测定了牙龈成纤维细胞的电泳行为，为牙周病学研究提供了一定的实验数据和一种新的研究方法，实验结果，人牙成龈成纤维细胞表面带负电荷，当电压为２０Ｖ，温度为３７℃ｐＨ为７．３时，电泳率值为０．８１３７±０．０３６７μｍ／ｓ／Ｖ／ｃｍ。     

3株产黑色素类杆菌内毒素对人牙髓、牙周膜成纤维细胞细胞毒作用

研究发现:3株感染根管内产黑色素类杆菌、牙髓类杆菌、产黑类杆菌、中间类杆菌的内毒素脂多糖,对体外培养的人牙髓、牙周膜成纤维细胞具有细胞毒作用,其最低抑制浓度为100ug/ml,并表现出时间依赖性和浓度依赖性。     

Human dermal and gingival fibroblasts in a three-dimensional culture: a comparative study on matrix remodeling

Free-floating collagen lattice is considered a useful tool for assessing wound healing in vitro. This work compared extracellular matrix remodeling in collagen lattices populated by gingival or dermal fibroblasts. For 21 days we followed gel contraction and changes in cell number of collagen lattices seeded with l.5 x 10(5) fibroblasts of each tissue. We also used indirect immunodetection to study extracellular matrix components, metalloproteinases (MMPs), and their tissues inhibitors (TIMPs). In addition, the presence of MMPs and TIMPs in the culture media was analyzed by zymography and western blotting. No significant difference was found concerning gel contraction and changes in cell number. We observed the early expression of fibrillin I and collagen type III, apparently codistributed and at the end of the gel contraction their disappearance. Concomitantly we demonstrated the expression of MMPs and TIMPs, initially localized in cellular cytoplasm, then spreading in the extracellular compartment, and even found in the culture medium. This remodeling was more rapid and intense with gingival fibroblasts than dermal fibroblasts. In conclusion, gingival fibroblasts seem more efficient at remodeling the connective tissue than dermal fibroblasts and could lead to the better wound healing observed in vivo.     

人口腔癌相关成纤维细胞的分离培养及鉴定

目的：分离培养及鉴定口腔癌相关成纤维细胞（ CAFs）,为探究CAFs可能在肿瘤微环境促进口腔癌细胞的侵袭转移中发挥重要作用奠定基础。方法：用组织块培养法和酶消化法分离培养、纯化原代口腔癌相关成纤维细胞CAFs和口腔黏膜正常成纤维细胞（ NFs）;细胞免疫荧光技术及免疫蛋白印迹法对口腔CAFs和NFs进行鉴定。结果：口腔CAFs和NFs分离培养成功,口腔CAFs呈长梭形,达到一定密度时呈多层生长,排列紊乱;NFs呈多胞质突的扁平星状,无重叠生长现象,排列有一定方向性。上皮标志物细胞角蛋白18（ CK18）免疫荧光染色在口腔CAFs和NFs均呈阴性,间质标志物波形蛋白（ Vimentin）染色均呈阳性。α?平滑肌动蛋白（α?SMA）、成纤维细胞特异性蛋白?1（FSP?1）,成纤维细胞激活蛋白（FAP）、血小板衍生生长因子受体α（ PDGFR?α）在CAFs染色呈阳性,而在NFs中染色呈阴性。结蛋白（ Desmin）在CAFs和NFs中染色均呈阳性。蛋白表达水平上,相对于NFs,CAFs中α?SMA、PDGFR?α蛋白表达呈升高趋势,FSP?1呈下降趋势,而FAP、Desmin在 NFs和CAFs中表达无明显差异。结论：CAFs与NFs相比,在形态结构、生长方式、蛋白表达等方面均有明显差异。     

人成牙本质细胞样细胞的原代培养

目的：培养人原代牙本质细胞。方法：取引产的8月龄死胎，剥离乳磨牙胚牙乳头，组织块法培养。然后采用滤纸片法挑取了3个与成牙本质细胞形态相似的细胞克隆，扩大培养。对培养细胞从形态学、矿化结节、碱性磷酸酶（alkaline phosphatase,ALP）活性、人牙本质基质蛋白-1（human dentin matrix protein-1,hDMP-1）和人牙本质涎磷蛋白（human dentin sialophosphoprotein,hDSPP）在mRNA水平上的表达等方面进行鉴定。结果：有一个克隆来源的细胞呈梭形、有单侧较长细胞突起，未见核极化，同时它们具矿化功能，表达人成牙本质细胞特异性标志hDMP-1、hDSPPmRNA。结论：该培养细胞为人成牙本质细胞样细胞，为进一步的有关研究奠定了基础。     

Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts

Wada N, Wang B, Lin N‐H, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival and periodontal ligament fibroblasts. J Periodont Res 2011; 46: 438–447. © 2011 John Wiley & Sons A/S Background and Objective: Human induced pluripotent stem (iPS) cells, which have similar properties to human embryonic stem (hES) cells, have been generated from neonatal and adult human dermal fibroblasts by reprogramming. iPS cells have high pluripotency and differentiation potential, and may be a potential autologous stem cell source for future regenerative therapy. Material and Methods: iPS cell lines from human gingival fibroblasts and, for the first time, from periodontal ligament fibroblasts, were generated by reprogramming using a retroviral transduction cocktail of OCT3/4, SOX2, KLF4 and c‐MYC. iPS induction was investigated through expression of the embryonic stem cell markers SSEA4, OCT4, NANOG, GCTM‐2, TG30 and TRA‐1‐60. Following in vitro differentiation, the expression of genes for differentiation markers for ectoderm (SOX1, PAX6), mesoderm [RUNX1, T(Brachyury)] and endoderm (GATA4, AFP) was assessed by real‐time RT‐PCR. The ability to form teratomas following implantation into mouse testes was assessed by histology. Results: Human gingival fibroblast‐ and periodontal ligament fibroblast‐derived iPS cells showed similar characteristics to hES cells. Both sets of iPS cells displayed colony morphology comparable to that of hES cells and expressed the hES cell‐associated cell‐surface antigens, SSEA3, SSEA4, GCTM‐2, TG30 (CD9) and Tra‐1‐60, and the hES cell marker genes, OCT4, NANOG and GDF3. These iPS cells showed differentiation potential to form embryoid bodies in vitro and expressed genes for endoderm, ectoderm and mesoderm. Teratoma formation following implantation into mouse testes was observed. Conclusion: These results demonstrate that iPS cells can be successfully generated from adult human gingival and periodontal ligament fibroblasts.     

两种体外培养人牙周韧带成纤维细胞方法比较

目的探索一种在体外短时间内简便、可靠获取大量人牙周韧带成纤维细胞(human periodontal ligament fibroblast,HPLF)、建立稳定的体外培养体系的方法。方法采用酶消化法和组织贴块法进行HPLF体外原代培养及传代培养的对比研究。通过细胞形态学、超微结构观察及波形蛋白和角蛋白免疫组化染色等对细胞进行定性研究;测定细胞生长曲线了解细胞生长基本规律及其增殖能力。结果采用酶消化法和组织贴块法均可成功的进行HPLF连续传代培养。最高传代数为30代。培养的细胞具有成纤维细胞的典型形态,波形蛋白染色阳性,角蛋白染色阴性,生长稳定期倍增时间为48~72h。组织块培养法需培养时间长,原代培养获取的细胞量较少,较难传代。酶消化法可短时间内获取大量细胞,细胞产量高,但操作手续复杂易污染,细胞易受损伤。结论成功建立了一个稳定的HPLF体外培养体系。除常用的组织块法外,胰蛋白酶及胶原酶联合消化法不失为一种简便、快速、可靠的组织原代分离培养方法。     

Influence of chlorine dioxide on cell death and cell cycle of human gingival fibroblasts

OBJECTIVES: The effects of chlorine dioxide (ClO2), sodium hypochlorite (NaOCl), and hydrogen peroxide (H2O2) on cell death and the cell cycle of human gingival fibroblast (HGF) cells were examined. METHODS: The inhibition of HGF cell growth was evaluated using a Cell Counting Kit-8. The cell cycle was assessed with propidium iodide-stained cells (distribution of cells in G0/G1, S, and G2/M phases) using flow cytometry. The patterns of cell death (necrosis and apoptosis) were analyzed using flow cytometry with annexin V-FITC/PI staining. RESULTS: The lethal doses for 50% of the cells (LD50) of ClO2, NaOCl, and H2O2 were 0.16, 0.79, and 0.11 mM, respectively. All three dental disinfectants induced G0/G1 cell cycle arrest. H2O2 induced apoptosis at concentrations of 0.05 and 0.1 mM, while NaOCl and ClO2 did not induce significant apoptosis at any concentration examined. CONCLUSIONS: These results suggest that ClO2 is sufficient for use as a dental disinfectant compared with H2O2 or NaOCl.     

人牙周膜成纤维细胞在无血清培养液中的生长特性

目的：探讨人牙周膜成纤维细胞在无血清培养液中的生长特性。方法：用倒置显微镜和MTT法观察人牙周膜成纤维细胞在无血清培养液中的生长和增殖变化。结果：人牙周膜成纤维细胞在无血清培养条件下可以生长的增殖，但与含血清培养液相比，其增殖速率变缓，分化明显。结论：无血清培养液可应用于人牙周膜成纤维细胞的体外培养和实验研究中。     

Viability of fibroblasts in cell culture after treatment with different chemical retraction agents

Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination.