成纤维细胞上清提取物对表皮细胞增殖迁移的影响

目的:在人皮肤成纤维细胞培养上清液中寻找有效的生长因子样活性组分.方法:收集超滤成纤维细胞的培养上清液,采用MTT法、划痕试验检测对人皮肤表皮细胞增殖和诱导迁移的影响.结果:上清液中分子量5～10 KD组和5～30 KD组的试验组对表皮细胞有明显促增殖趋势,而5～30 KD组有较强的促进单层细胞伤口愈合的能力,二者与对照组比较有显著性差异(P＜0.01).结论:人成纤维细胞培养上清液的提取物中含有促进表皮细胞增殖及迁移能力的活性物质,可以尝试用其替代部分条件培养基进行对组织工程皮肤种子细胞的实验研究.     

Nd：YAG激光照射对提高人牙周膜成维细胞增殖力的作用

目的观察Nd：YAG激光对人牙周膜成纤维细胞增殖和碱性磷酸酶（ALP）活性的影响。方法采用酶消化组织块法原代培养人牙周膜成纤维细胞，并进行波形蛋白及角蛋白的免疫细胞化学染色，对所培养的细胞进行鉴定。将牙周膜成纤维细胞分组，接受不同能量密度的Nd：YAG激光照射。测定牙周膜成纤维细胞碱性磷酸酶活性并观察其生长情况。结果5个实验组的细胞增殖速度及碱性磷酸酶含量均高于对照组，其中能量密度为60j／cm^2组、80j／cm^2组、100i／cm^2组与对照组相比有统计学的差异。结论低能量密度，短时间的Nd：YAG激光照射能促进牙周膜成纤维细胞的增殖，提高碱性磷酸酶活性。     

牙髓干细胞对皮肤成纤维细胞衰老及增殖能力的影响

目的：研究牙髓干细胞对皮肤成纤维细胞衰老及增殖能力的影响,并初步探讨其机制。方法：从人牙髓中提取,培养牙髓干细胞,与皮肤成纤维细胞分组共培养,分为3组：对照组（单纯皮肤成纤维细胞培养）、条件培养基组（利用牙髓干细胞条件培养基培养成纤维细胞）、直接共培养组（采用Transwell共培养小室）。共培养后,进行 β半乳糖苷酶（SA-β-gal）染色,检测成纤维细胞的衰老情况;利用 CCK-8 法检测各组成纤维细胞的活性;流式细胞仪分析成纤维细胞的细胞周期变化;采用RT-PCR和 Western免疫印迹检测各组成纤维细胞衰老相关蛋白 p21、p53以及pRb 的mRNA和蛋白表达水平。采用SPSS 13.0软件包对数据进行统计学分析。结果：与空白组相比,后2组的皮肤成纤维细胞表达β半乳糖苷酶降低、增殖能力增强、G1期细胞减少而S期和G2期增多, p53、p21 mRNA和蛋白表达水平降低而PRb表达升高。结论：牙髓干细胞及其条件培养基具有抗皮肤成纤维细胞衰老的作用,为牙髓干细胞在抗皮肤衰老方面的临床应用提供了依据。     

转化生长因子-β1和碱性成纤维细胞生长因子对人牙周膜成纤维细胞增殖的影响

目的观察转化生长因子．晶和碱性成纤维细胞生长因子单独或联合应用对人牙周膜成纤维细胞增殖的影响。方法采用组织块培养技术进行人牙周膜成纤维细胞的体外培养，对照组采用含小牛血清的DMEM培养液，实验组分别在培养液中加入不同浓度的转化生长因子-β1或碱性成纤维细胞生长因子或两者联合加入，通过四唑盐比色法观察细胞增殖情况。结果转化生长因子-β1或碱性成纤维细胞生长因子单独作用后，人牙周膜成纤维细胞较对照组有显著的增殖，而两者联合作用后的增殖较各自单独作用时明显（P〈0．05）。结论转化生长因子-β1和碱性成纤维细胞生长因子两者联合具有促进人牙周膜成纤维细胞增殖的作用。     

rhBMP-4与rhIGF-I联合应用对人牙周膜成纤维细胞增殖及分化能力的影响

目的:探讨rhBMP-4与rhIGF/-I联合应用对人牙周膜成纤维细胞增殖及分化能力的影响.方法:将人牙周膜成纤维细胞分别与10 ng/ml rhBMP-4、10 ng/ml rhIGF-I、10 ng/ml rhBMP-4 10 ng/ml rhIGF-I、无血清DMEM共同培养3 d,用MTT法测定细胞增殖情况,用碱性磷酸酶试剂盒检测细胞碱性磷酸酶的活性,用羟脯氨酸试剂盒检测细胞分泌I型胶原的量.结果:第3天,10 ng/ml rhBMP-4 10 ng/ml rhIGF-I组人牙周膜成纤维细胞的牛长增殖能力最强,并与其它3组比较有显著性差异(P＜0.05),但其增强细胞碱性磷酸酶的活性及促进细胞分泌I型胶原的能力并不比两者单独作用强.结论:rhBMP-4与rhIGF-I联合应用,能协同促进人牙周膜成纤维细胞增殖,但不能协同促进细胞分化.     

白细胞介素-1β（IL-1β）和地塞米松对成纤维细胞增殖的影响

目的：检测白细胞介素-1β（IL-1β）和地塞米松（DEX）对体外培养的口腔正常黏膜与口腔扁平苔藓（OLP）黏膜成纤维细胞增殖的影响。方法：应用MTT法检测3种浓度梯度的IL-1β和DEX对体外培养的14例OLP黏膜成纤维细胞和11例正常口腔黏膜成纤维细胞增殖活性的影响。结果：MTT测定结果显示,IL-1β对OLP黏膜成纤维细胞和正常口腔黏膜成纤维细胞的增殖均有促进作用,且随浓度增加促进作用增强;相反,DEX却抑制两种成纤维细胞的增殖,浓度越高抑制作用越强。结论：白细胞介素-1β可促进成纤维细胞的增殖,地塞米松则抑制成纤维细胞的增殖。     

转化生长因子-β_1和碱性成纤维细胞生长因子对人牙周膜成纤维细胞增殖的影响

目的观察转化生长因子-β_1和碱性成纤维细胞生长因子单独或联合应用对人牙周膜成纤维细胞增殖的影响。方法采用组织块培养技术进行人牙周膜成纤维细胞的体外培养,对照组采用含小牛血清的DMEM培养液,实验组分别在培养液中加入不同浓度的转化生长因子-β_1或碱性成纤维细胞生长因子或两者联合加入,通过四唑盐比色法观察细胞增殖情况。结果转化生长因子-β_1或碱性成纤维细胞生长因子单独作用后,人牙周膜成纤维细胞较对照组有显著的增殖,而两者联合作用后的增殖较各自单独作用时明显(P<0.05)。结论转化生长因子-β_1和碱性成纤维细胞生长因子两者联合具有促进人牙周膜成纤维细胞增殖的作用。     

rhBMP-4与rhIGF—Ⅰ联合应用对人牙周膜成纤维细胞增殖及分化能力的影响

目的:探讨rhBMP-4与rhIGF-Ⅰ联合应用对人牙周膜成纤维细胞增殖及分化能力的影响。方法:将人牙周膜成纤维细胞分别与10ng/ml rhBMP-4、10ng/ml rhIGF-Ⅰ、10ng/ml rhBMP-4 10ng/ml rhIGF-Ⅰ、无血清DMEM共同培养3d,用MTT法测定细胞增殖情况,用碱性磷酸酶试剂盒检测细胞碱性磷酸酶的活性,用羟脯氨酸试剂盒检测细胞分泌Ⅰ型胶原的量。结果:第3天,10ng/ml rhBMP-4 10ng/ml rhIGF-Ⅰ组人牙周膜成纤维细胞的生长增殖能力最强,并与其它3组比较有显著性差异(P<0.05),但其增强细胞碱性磷酸酶的活性及促进细胞分泌Ⅰ型胶原的能力并不比两者单独作用强。结论:rhBMP-4与rhIGF-Ⅰ联合应用,能协同促进人牙周膜成纤维细胞增殖,但不能协同促进细胞分化。     

肿瘤间质成纤维细胞对体外培养唾液腺多形性腺瘤细胞生物学行为的影响

目的 探讨肿瘤间质成纤维细胞（TSF）对体外培养唾液腺多形性腺瘤（SPA）细胞增殖、侵袭和迁移的影响。方法 采用组织块培养法培养唾液腺多形性腺瘤细胞（SPAC）、TSF和瘤旁正常成纤维细胞（NF），并通过免疫细胞化学染色法进行细胞鉴定。选取对数期TSF、NF制备条件培养液，培养SPAC并命名为TSF-SPAC组和NF-SPAC组，单纯培养SPAC作为对照组（SPAC组）。采用MTT实验、Transwell实验和划痕实验分别检测3组细胞的增殖、侵袭和迁移能力；酶联免疫吸附测定（ELISA）检测3组培养液中血管内皮生长因子（VEGF）的表达量。结果 免疫细胞化学染色显示：波形蛋白（Vimentin）在TSF和NF中为阳性，而α-平滑肌肌动蛋白（α-SMA）和成纤维细胞活化蛋白（FAP）在NF中为阴性，在TSF中为弱阳性。MTT结果显示：TSF-SPAC组细胞增殖与NF-SPAC组和SPAC组的差异有统计学意义（P<0.05）,NF-SPAC组与SPAC组的细胞增殖的差异无统计学意义（P>0.05）。Transwell侵袭实验和迁移实验显示：3组细胞的侵袭和迁移的差异无统计学意义...     

唑来膦酸对成纤维细胞作用的影响

目的 分析唑来膦酸对成纤维细胞的影响,探讨唑来膦酸对软组织损伤及引起创口软组织愈合困难的可能机制。方法 体外常规培养NIH 3T3成纤维细胞。流式细胞凋亡实验检测加入唑来膦酸后成纤维细胞凋亡的变化。MTT法检测加入唑来膦酸条件下成纤维细胞增殖活性的变化,细胞划痕实验观察唑来膦酸对成纤维细胞迁移能力的影响。采用SPSS 17.0软件包对数据进行统计学分析。结果 在药物浓度为5~50 μmol/L范围时,随着药物浓度升高,唑来膦酸促进成纤维细胞凋亡,抑制增殖、迁移的作用逐渐增强。结论 唑来膦酸能对软组织造成损伤,可引起拔牙创软组织愈合困难。     

Cell-seeding of ligament fibroblasts

This study was undertaken to test the hypothesis that seeded periodontal ligament cells are able to create new attachment. In one beagle dog, a premolar was removed and scrapings of the ligament were cultured. Artificial periodontal defects were made and the cultured ligament cells were seeded on the planed root surfaces and covered with muco-periosteal flaps. The opposite side served as control. After 4 months, the dog was sacrificed and histological and electron microscopical sections were prepared. The seeded root surfaces were almost completely covered with cementoblasts, whereas in controls, epithelial down-growth could be observed. We conclude that seeding of periodontal ligament cells could be a promising technique to create new connective tissue attachment.     

Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts

Migration of fibroblasts from surrounding normal tissue into the wound bed is an important requirement for successful wound healing. This study investigated the motility pattern of buccal, periodontal and skin fibroblasts to determine whether differences in the wound healing efficiency at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer-assisted microscope work-station. For evaluation of cell morphology, cell contours were recognized semiautomatically and used for determination of cell area, cell spreading and number and length of processes. We found that the cellular displacement of the buccal fibroblasts was only approximately 50% of the cellular displacement of periodontal and skin fibroblasts. The decreased cellular displacement of the buccal fibroblasts was found to be due to both lower cellular speed and less persistence in direction. The buccal fibroblasts also displayed smaller areas and longer processes. The differences in cellular morphology and motility pattern amongst the three fibroblast types could not be explained by differences in secretion of extracellular matrix components and are therefore believed to reflect phenotypic differences amongst fibroblast subpopulations.     

Cytoplasmic polarization of periodontal ligament fibroblasts

The fibroblasts of the periodontal ligament and especially those of the transseptal ligament are characteristically elongated with their long axes parallel to the principal fiber bundles. Their cytoplasmic orgamelles are polarized suggesting that secretory products are released from one end of the cell. The nucleus occupies the anterior pole of the cell. Cytoplasmic processes or lobopodia, containing numerous cytoplasmic filaments, extend from the anterior pole of the cell. The Golgi complex is situated in a large area of the cell between the nucleus and the distal end of the cell. Granular endoplasmic reticulum, mitochondria and numerous small vesicles are dispersed throughout most part of the cell. Intracellular collagen, akthough present in many areas of the cell, appears to be most abundant in the distal parts of the cell. Administration of colchicine in vivo, blocked collagen secretion and dereased cell elongation and polartity.
Results of and analysis of the orientation and polarization of the fibroblasts in the periodontal tissues appear to support the concept that these cells undergo active migration in vivo along existing substrata and that migration may be coupled to collagen secretion.     

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.     

IGF-1 signaling enhances cell survival in periodontal ligament fibroblasts vs. gingival fibroblasts

The role of insulin-like growth factors (IGFs) in the regulation of apoptosis has been suggested, yet their impact on specific cells such as periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) remains unknown. The purpose of this study was to test the role of IGF-1 signaling in cell survival in PDLF compared with GF. In periodontal tissue sections, a significantly reduced apoptotic rate was first demonstrated in PDLF compared with GF. In vitro, IGF-1 substantially enhanced cell survival in PDLF compared with GF by the up-regulation of anti-apoptotic molecules and the down-regulation of pro-apoptotic molecules. Furthermore, the differential expression of insulin-like growth factor binding protein 5 (IGFBP-5) was observed in vitro, and its differential distribution was confirmed in vivo. Analysis of the present data suggests an enhanced cell survival in PDLF compared with GF by the up-regulation of IGF-1 signaling pathway.     

Tropoelastin expression by periodontal fibroblasts

Elastic system fibers are load-bearing proteins found in periodontal tissue. There are three types--oxytalan, elaunin, and elastic fibers--which differ in their relative microfibril and elastin contents. Oxytalan fibers are known to be distributed in the periodontal ligaments and gingiva, whereas elaunin and elastic fibers are present only in the gingiva. We examined gene expression and accumulation of tropoelastin in the cell-matrix layers of human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF) in vitro. HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 8 wks. Northern blotting and RT-PCR analyses showed that only HGF expressed mRNA encoding tropoelastin. Western blotting analysis demonstrated 77-kDa protropoelastin and 68-kDa tropoelastin only in the cell-matrix layer of HGF cultured for 8 wks. These results suggest that the different tropoelastin expression patterns reflect the difference between HGF and HPLF phenotypes.     

Transwell滤膜上人牙周膜及牙龈成纤维细胞细胞层完整性的研究

目的 探讨生长在Transwell滤膜上人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPDLF)及牙龈成纤维细胞(human gingival fibroblasts,HGF)细胞层完整性,为研究牙周细胞对药物的跨细胞转运提供依据.方法 将原代培养的HPDLF及HGF分别接种到Transwell聚酯滤膜上,在1到4周内行跨膜电阻测定,连续以光学显微镜观察细胞的生长及排列情况,2周时对滤膜纵切面行光学显微镜及透射电子显微镜观察,并以渗漏标志物荧光素钠检测模型的渗漏.结果 HPDLF及HGF接种1周后细胞基本汇合,到2周时细胞连接紧密且完整,细胞呈复层生长,跨膜电阻分别达(56.14±7.43)及(57.34±7.62)Ω·cm~(-2),并持续到第4周;细胞接种2周后细胞药物转运模型经荧光素钠孵育30 min检测细胞旁渗漏小于1%.结论 HPDLF及HGF在Transwell滤膜上生长2周后细胞排列情况可模拟体内牙周膜和牙龈生理状态,且渗漏标志物的检测及跨膜电阻等结果显示细胞完整,旁渗漏很低,符合药物细胞转运模型的要求,可作为研究牙周细胞跨细胞转运药物的体外模型.     

口腔黏膜上皮细胞和成纤维细胞的复合培养

目的 为癌前病变临床病理研究建立一种简便、迅速和有效的口腔黏膜上皮细胞和成纤维细胞复合培养的方法,实现体外模拟组织工程化口腔黏膜的发生发展。方法 用DispaseⅡ分离上皮和皮下组织,用KGM培养口腔黏膜上皮细胞。用细胞培养法和组织块培养法获取验用的口腔黏膜上皮细胞和成纤维细胞,并采用复合培养法对两种细胞进行共同培养。结果 用DispaseⅡ可成分地分离上皮和皮下组织。KGM可明显促进口腔黏膜上皮细胞的分裂繁殖。复合培养的HE染色切片显示薄层的结缔组织之上有方形的基底细胞、颗粒细胞和角化层。结论 KGM可明显促进口腔黏膜上皮细胞的分裂和成熟。采用组织培养法获取原代成纤维细胞是适合口腔黏膜取材等特点的有效方法。气液相培养的口腔黏膜下结缔组织可促进上皮细胞的分层和分化。