人牙周膜成纤维细胞与牙龈成纤维细胞差异表达一条新基因的克隆

目的：克隆人牙周膜成纤维细胞（ｐｅｒｉｏｄｏｎｔａｌ ｌｉｇａｍｅｎｔ ｆｉｂｒｏｂｌａｓｔ,ＰＤＬＦ）与牙龈成纤维细胞（ｇｉｎｇｉｖａｌ ｆｉｂｒｏｂｌａｓｔ,ＧＦ）差异表达的新基因。方法：采用基于ＰＣＲ和消减杂交的基因克隆技术构建体外培养的人ＰＤＬＦ与ＧＦ差异表达基因的扣除文库,采用酶切和反向杂交的方法筛选目的的克隆,并进行测序和计算机分析。结果：从构建的人ＰＤＬＦ与ＧＦ细胞差异表达基因的扣除     

人牙周膜成纤维细胞与牙龈成纤维细胞差异表达基因的批量克隆及其特征分析

目的：克隆人牙周膜成纤维细胞（ｐｅｒｉｏｄｏｎｔａｌ ｌｉｇａｍｅｎｔ ｆｉｂｒｏｂｌａｓｔ，ＰＤＬＦ）与牙龈成纤维细胞（ｇｉｎｇｉｖａｌ ｆｉｂｒｏｂｌａｓｔ，ＧＦ）差异表达基因并初步分析其中已知基因的功能特征。方法：采用基于ＰＣＲ和消减杂交的基因克隆技术构建人ＰＤＬＦ与ＧＦ差异表达基因的扣除文库，克隆人ＰＤＬＦ与ＧＦ差异表达基因，对已知基因的功能特征进行分析。结果：成功克隆到１４个人ＰＤＬＦ与     

人牙髓细胞与牙龈成纤维细胞cDNA消减文库的构建

目的 建立人牙髓细胞(human dental pulp cell,HDPC)和人牙龈成纤维细胞(human gingival fibroblast,HGF)cDNA消减文库.方法 体外培养HDPC和HGF,分别提取mRNA并反转录为cDNA,应用基于PCR的改良消减杂交技术建立HDPC和HGF cDNA消减文库.结果 成功构建了HDPC和HGF cDNA消减文库,文库容量为4×102.结论 基于PCR的改良消减杂交技术是建立消减文库、寻找差异表达基因的一种高效方法,该消减文库的建立为进一步在分子水平研究牙髓细胞的生长、分化机理奠定了基础.     

人成骨样细胞机械牵拉力敏感基因cDNA扣除文库的构建

目的 :构建人成骨样细胞机械牵拉力敏感基因cDNA扣除文库。方法 :对培养的人成骨样细胞Saos -2进行二维方向上的加载 ,细胞平均变形幅度为 12 % ,牵拉频率为 12次 /min ,加载 12h后提取应力作用后细胞及正常对照组细胞的mRNA ,制备各自的cDNA ,用基于PCR的消减杂交技术构建人成骨样细胞机械牵拉力作用下差异表达基因文库 ,即机械牵拉力敏感基因cDNA扣除文库 ,并进行初步的序列测定。结果 :成功地构建出人成骨样细胞机械牵拉力敏感基因cDNA文库 ,库容量约为 2 0 0 ;初步的测序结果表明文库内的基因多与应力作用下细胞发生的生物学变化有关 ,并成功地得到一个新的基因片段。结论 :用基于PCR的消减杂交技术可以有效构建人成骨细胞样机械牵拉力敏感基因cDNA扣除文库     

牙周韧带细胞与牙龈成纤维细胞生长特性及其胶原表达的比较研究

利用体外细胞培养技术及四唑盐比色法观察了牙周韧带细胞(PDLC)和牙龈成纤维细胞(GF)在体外生长、增殖的情况,用免疫荧光法观察了其细胞外基质表达的差异。结果显示GF较PDLC更容易成活,其增殖速度更快,且GF中Ⅰ、Ⅲ型胶原的表达均较PDLC强,提示在相同生长环境下,GF较PDLC可能有更强的生长能力。     

人牙髓细胞与牙龈成纤维细胞差异表达基因的克隆及特征分析

目的批量克隆人牙髓细胞（HDPC）与人牙龈成纤维细胞（HGF）的差异表达基因并对其特征进行分析,研究HDPC的生物学特性。方法体外培养HDPC和HGF,应用基于PCR的改良消减杂交技术构建HDPC和HGF的cDNA消减文库,批量克隆HDPC和HGF的差异表达基因并测序,使用GenBank的BLAST对测序结果进行同源序列比较。结果经过序列测定,获得12个差异表达基因的序列,经BLAST分析有2个为未知基因。在已知基因中,含有4个与细胞信号转导机制相关的基因;2个与细胞转运机制相关的基因（包括细胞膜及细胞核膜转运）;2个与细胞RNA剪接机制相关的基因。结论HDPC的生物学特性是由某些特定基因的差异表达所决定的,其生长、分化机制可能与相对旺盛的蛋白合成及分泌活性相关。     

牙周组织中成纤维细胞异质性的研究进展

牙周组织中的成纤维细胞包括牙龈成纤维细胞(gingival fibroblast,GF)和牙周韧带细胞(periodontal ligament cell,PDLC)。目前的研究表明:不但GF与PDLC之间存在异质性(heterogeneity),而且同一个GF或PDLC细胞群也是由不同的细胞亚型(subsets,subtypes,subpopulations)组成。本文综述了支持牙周组织成纤维细胞异质性的有关证据、表现及其生物学意义。     

碱性成纤维细胞生长因子对牙周细胞生物学活性的影响

目的：观察基因重组入碱性成纤维细胞生长因子（rh-bFGF)牙龈成纤维细胞（GF）、人牙周韧带成纤维细胞（PDLF）及人牙槽骨细胞（ABC）的增殖、碱性磷酸酶活性、总蛋白含量及对3种细胞矿化结节形成能力的影响。方法：采用细胞培养、MTT比色测定、碱性磷酸酶测定法、考马宙蓝法及茜素红染色法。结果bFGF能促进3种细胞的增殖,但对PDLF和ABC的ALP活性,蛋白含量及矿化结节的形成有抑制作用。结论bFGF可促进细胞的增殖,抑制细胞的分化成熟,从而促进牙周再生。     

用消减杂交技术克隆人成骨样细胞机械牵张力敏感基因的研究

目的　对人成骨样细胞机械牵张力敏感基因进行克隆研究。方法　对人成骨样细胞Saos-2进行二维方向上的加载,变形幅度为12%,牵拉频率为6周期/分钟,加载12 h后分别提取牵张力作用后Saos-2细胞及未作处理的正常Saos-2对照细胞的mRNA,反转录制备cDNA,将cDNA进行消减杂交,构建加载细胞与未加载细胞的差异表达基因的扣除cDNA文库,克隆后进行序列测定。结果　克隆到的基因片段中,功能已知基因片段15个,功能未知基因片段2个,其中牵张力敏感基因1(SSG1)位于9号染色体上,功能未知;牵张力敏感基因2(SSG2)位于18号染色体上,与转录因子2,4高度同源。结论　用消减杂交技术可以对人成骨细胞样机械牵张力敏感基因进行有效的克隆研究。     

用消减杂交技术克隆人成骨样细胞机械牵张力敏感基因的研究

目的 :对人成骨样细胞机械牵张力敏感基因进行克隆研究。方法 :对人成骨样细胞Saos 2进行二维方向上的加载 ,变形幅度为 12 % ,牵拉频率为 6周 /分钟 ,加载 12小时后分别提取牵张力作用后Saos 2细胞及未作处理的正常Saos 2对照细胞的mR NA ,反转录制备cDNA ,将cDNA进行消减杂交 ,构建加载细胞与未加载细胞的差异表达基因的扣除cDNA文库 ,克隆后进行序列测定。结果 :克隆到的基因片段中 ,功能已知基因片段 15个 ,功能未知基因片段 2个 ,其中牵张力敏感基因 (SSG1)位于 9号染色体上 ,功能未知 ;牵张力敏感基因 (SSG2 )位于 18号染色体上 ,与转录因子 2 ,4高度同源。结论 :用消减杂交技术可以对人成骨细胞样机械牵张力敏感基因进行有效的克隆研究。     

牙根发育不全疾病致病差异表达基因文库的建立

目的：建立先天性牙根发育不全疾病患儿与其正常兄弟间致病差异表达基因的cDNA文库。方法：采用改良消减杂交技术，以先天性牙根发育不全疾病患儿与其正常兄弟外周静脉血的总mRNA为对比材料，筛选出候选相关致病基因差异表达的cDNA，将其与pGEM-T载体进行T／A连接构建文库，然后转化高效感受态大肠杆菌进行文库扩增，随机挑取100个白色克隆进行酶切鉴定。结果：扩增消减cDNA文库获得400余个白色阳性克隆，随机挑取的100个白色克隆经酶切后90％有插入片段。结论：用改良消减杂交技术成功构建了先天性牙根发育不全疾病致病差异表达基因的消减cDNA文库，该文库的建立为进一步筛选、克隆该疾病的候选致病相关基因奠定了基础。     

Identification of genes differentially expressed in cultured human periodontal ligament fibroblasts vs. human gingival fibroblasts by DNA microarray analysis

Despite their similar spindle-shaped appearance, periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) appear to display distinct functional activities in the maintenance of tissue integrity and during inflammatory/immune responses. We postulated that different characteristics of PDLF and GF are defined by the differential expression of specific genes. To test this, we investigated the possible variance of gene expression profile between cultured PDLF and GF, using DNA microarray technology. One hundred sixty-three genes were found differentially expressed by at least three-fold between PDLF and GF. Genes encoding transmembrane proteins and cytoskeleton-related proteins tended to be up-regulated in PDLF, whereas genes encoding cell-cycle regulation proteins and metabolism-related proteins tended to be up-regulated in GF. We concluded that PDLF and GF appear to display different gene expression patterns that may reflect intrinsic functional differences of the two cell populations and may well coordinate with their tissue-specific activities.     

Subtractive cloning: new genes for studying inflammatory disorders

Understanding of the biology of interaction between pathogens and host is the central question in studying inflammatory disorders. Subtractive DNA cloning is one of the most efficient and comprehensive methods available for identifying eukaryotic genes regulated under specific physiological conditions, including inflammation and host response. Here we explore the utility of subtractive DNA cloning and describe suppression subtractive hybridization (SSH), a polymerase chain reaction (PCR)-based DNA subtraction method that has been developed and evolved in our labs over several years. The SSH method possesses a number of advantages as compared to other subtractive cloning techniques, making it one of the most adventitious methods for cloning differentially expressed genes. Besides isolation of differentially expressed eukaryotic mRNAs, subtractive DNA cloning can be used to identify genes that are differentially expressed between diverse bacterial species. These genes can be of great interest, as some may encode strain-specific traits such as drug resistance, or bacterial surface proteins involved in determining the virulence of a particular strain. Other genes may be useful as markers for epidemiological or evolutionary studies. To demonstrate the potential of the SSH technique, we describe here the comprehensive characterization of 2 SSH subtracted libraries constructed in our laboratories. One library was created using eukaryotic cDNA subtraction and is specific for mRNAs up-regulated in CD25 positive cells from mouse lymph nodes as compared to CD25 negative cells. The second subtracted library is specific for a methicillin-resistant Staphylococcus aureus bacterial strain, but not in a methicillin-sensitive strain. The bacterial genomes of these 2 strains have been completely sequenced and this second library provides an excellent reference for testing the ability of SSH to recover all strain-specific gene content. The analysis of these 2 subtracted libraries serves as the basis for a discussion of the strength and limitations of the SSH technique. We will also compare and contrast subtractive DNA cloning to other current technologies used to isolate differentially expressed genes.     

Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis

Scheres N, Laine ML, de Vries TJ, Everts V, van Winkelhoff AJ. Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01229.x © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth‐supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. Material and Methods: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real‐time PCR, and protein expression was analyzed using ELISA. Results: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)‐1β, IL‐6, IL‐8, tumor necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cell expressed and secreted (RANTES). Macrophage colony‐stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL‐6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. Conclusion: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.     

Isolation and partial sequencing of potentially odontoblast-specific/enriched rat cDNA clones obtained by suppression subtractive hybridization

Odontoblasts, which are responsible for dentine formation, are known to synthesize unique gene products such as dentine sialophosphoprotein. To further identify and clone novel odontoblast-specific genes, a suppression subtractive hybridization technique was used here. Differentially or predominantly expressed cDNAs in odontoblasts of rat incisors were obtained by subtracting the common cDNAs expressed in odontoblasts, osteoblasts and pulp cells. Clones were then partially sequenced and analysed for nucleotide sequence homology by the basic local alignment search tool program. From a total of 1290 clones analysed, 538 odontoblast-enriched clones were identified in the subtracted cDNA library. Out of 538 clones, 498 clones (92.6%) demonstrated high identity with genes in the GenBank database. In contrast, 31 clones (5.7%) showed low sequence identity with known genes, among which 18 clones (3.3%) were observed more than once, thereby possibly representing odontoblast-specific/enriched genes. The majority (390 clones; 72.5%) of the clones with high homology to known genes were found to be the rat/mouse dentine sialophosphate by dot-blot analysis (326 clones) and sequencing (64 clones). The second highest enrichment (39 clones) was for phosphate-regulating gene with homology to endopeptidase on the X-chromosome, which codes for a neutral endopeptidase. After suppression subtractive hybridization, several cDNAs that are commonly present in osteoblasts and odontoblasts appeared unsuppressed. Therefore, a rat odontoblast-specific/enriched subtraction cDNA library has been created from which a number of potentially novel genes for odontoblasts could be identified.