Mutational analysis of candidate genes in 24 amelogenesis imperfecta families

Amelogenesis imperfecta (AI) is a heterogeneous group of inherited defects in dental enamel formation. The malformed enamel can be unusually thin, soft, rough and stained. The strict definition of AI includes only those cases where enamel defects occur in the absence of other symptoms. Currently, there are seven candidate genes for AI: amelogenin, enamelin, ameloblastin, tuftelin, distal-less homeobox 3, enamelysin, and kallikrein 4. To identify sequence variations in AI candidate genes in patients with isolated enamel defects, and to deduce the likely effect of each sequence variation on protein expression and structure, families with isolated enamel defects were recruited. The coding exons and nearby intron sequences were amplified for each of the AI candidate genes by using genomic DNA from the proband as template. The amplification products for the proband were sequenced. Then, other family members were tested to determine their genotype with respect to each sequence variation. All subjects received an oral examination, and intraoral photographs and dental radiographs were obtained. Out of 24 families with isolated enamel defects, only six disease-causing mutations were identified in the AI candidate genes. This finding suggests that many additional genes potentially contribute to the etiology of AI.     

Induction of dental pulp fibroblast matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 gene expression by interleukin-1alpha and tumor necrosis factor-alpha through a prostaglandin-dependent pathway

Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the degradation of extracellular matrix in many inflammatory diseases. Little is known regarding the expression of these mediators in dental pulp fibroblasts. The effects of proinflammatory cytokines (interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha)) and prostaglandin E2 (PGE2) on pulp fibroblast MMP-1 and TIMP-1 gene expression were investigated. Northern hybridization showed that IL-1alpha and TNF-alpha induced significant MMP-1 gene expression, with only little effect on TIMP-1 gene. Exogenous PGE2, however, upregulated TIMP-1 mRNA synthesis but not MMP-1. Concomitant addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 suppressed MMP-1 mRNA production, compared with the groups treated with IL-1alpha or TNF-alpha alone. In contrast, PGE2 enhanced the upregulatory effects of TIMP-1 mRNA by IL-1alpha or TNF-alpha. Furthermore, cytokine stimulation of MMP-1 and TIMP-1 gene expressions can be enhanced or blocked by indomethacin, respectively, and reversed by exogenous PGE2. These results suggested that cytokine-stimulated MMP-1 and TIMP-1 gene expression in dental pulp fibroblasts was mediated, at least in part, by a prostaglandin-dependent pathway. The differential regulation of IL-1alpha or TNF-alpha-induced MMP-1 and TIMP-1 mRNA synthesis, as well as the direct upregulation of TIMP-1 gene expression by PGE2, also implied that prostaglandin may serve as a protective mechanism from excessive tissue breakdown during pulpitis.     

Platelet-derived growth factor-B gene delivery sustains gingival fibroblast signal transduction

Background and Objective: Platelet‐derived growth factor‐BB is a potent mediator of tooth‐supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet‐derived growth factor‐BB application is its short half‐life in vivo. Gene therapy has shown strong promise for the long‐term delivery of platelet‐derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet‐derived growth factor‐B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad‐PDGF‐B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell‐surface proteins associated with cellular signaling. Material and Methods: HGFs from human subjects were treated by adenoviral PDGF‐B, PDGF‐1308 (a dominant negative mutant of PDGF) and recombinant human platelet‐derived growth factor‐BB, and then incubated in serum‐free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF‐B was measured by RT‐PCR and Western blot. Cell proliferation was evaluated by [methyl‐³H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF‐B gene transfer. The expression of α and β PDGFR and Akt were measured by Western blot analysis. Results: Sustained in vitro expression of PDGF‐B in HGFs by Ad‐PDGF‐B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF‐BB and Ad‐PDGF‐1308, Ad‐PDGF‐B maintained cell growth in serum‐free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF‐B also prolonged downregulation of the growth arrest specific gene (gas) PDGFαR. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long‐term cell signaling effect as a result of the over‐expression of Ad‐PDGF‐B, compared with pulse recombinant human platelet‐derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad‐PDGF‐B, for at least up to 96 h. Conclusion: These findings further demonstrate that gene delivery of PDGF‐B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet‐derived growth factor‐BB protein. These data on platelet‐derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.     

口腔鳞癌手术后中药治疗远期疗效观察

作者曾对２３８例口腔鳞癌术后应用中药“参阳”方的疗效进行前瞻性的研究,本文继续对已生存４～８年的５６例病员门诊随访结果进行分析．结果是生存情况：７年生存期原治疗组高于原对照组１２．５％,原治疗组３０例均健在,而原对照组中的２６例,２例带瘤生存,１例死亡;Ｔ细胞亚群测定中,复发死亡者的ＣＤ８值均升高,ＣＤ４／ＣＤ８比值均下降;中医辨证中,正常者及肾气虚者的例数以治疗组为多,而阴虚与气阴两虚者,治疗组低于对照组;由上提示：经“参阳”方治疗者有较好的远期疗效．     

The distribution of nitric oxide synthetase in Vcx and its relation with the expression of FOS induced by teeth movement in rats]

It was studied the central role of nitric oxide(NO) during experimental teeth movement and the relation between nitric oxide synthetase (NOS) positive neurons and FOS like immunoreactivity (FLN) with the NADPH-diaphorase histochemistry and immunocytochemical reaction method. Results indicated that NOS positive neurons and FLN showed typical distribution in Vcx and there was some overlap between them. It suggests that NO is involved in the central modulation of the stimulating message of teeth movement, and which further explains the central modulation mechanism of experimental teeth movement in rats.     

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.     

Persistent endodontic lesion due to complex cementodentinal tears in a maxillary central incisor--a case report

The cementodentinal tear is rarely detected by noninvasive procedures owing to its clinical picture simulating a root fracture or a periodontal or endodontic lesion. We present a case of complex cementodentinal tears in a 79-year-old woman who presented a repeated swelling at the labial mucosa of the left maxillary central incisor for 6 months. Periapical radiographs demonstrated a vertical radiolucent fracture line extending from the root apex along the mesial aspect of the root to near the middle portion of the root of the left maxillary central incisor. Because endodontic re-treatment failed to cure the disease, periapical surgery was performed, and 2 fractured U-shaped root fragments around the apical root surface were removed. Histologic examination showed that the 2 fractured root fragments were composed mainly of the dentin covered by a thin layer of the cementum and overlying periodontal ligament tissue, suggesting cementodentinal tears. A swelling recurred 8 months after the initial operation. Therefore, a second periapical surgery was performed. Although no obvious fracture line was observed around the root surface, the second surgery did not cure the disease, either. A persistent small swelling was noted at the alveolar mucosa of the affected tooth during the follow-up. We conclude that although a cementodentinal tear can be detected by a careful radiographic examination, its clinical outcome is not predictable by surgical removal only.     

CSF-1对体外培养人牙囊细胞OPG表达的影响

目的研究集落刺激因子(CSF-1)对体外培养的人牙囊细胞骨保护素(OPG)表达的影响。方法原代培养人牙囊细胞,传代至第3代,制备细胞爬片,进行OPG免疫组化染色;3~6代人牙囊细胞长至80%时,用胰酶消化,离心,重悬,制成单细胞悬液,接种到细胞培养皿,细胞长满至80%时,培养基中加入25ng/ml的CSF-1,孵育0.5、1、3、6h,分别收集上清液,ELISA法检测OPG蛋白分泌量的变化,提取总RNA进行RT-PCR,检测OPG mRNA的表达变化。结果正常人牙囊细胞OPG蛋白表达阳性;25ng/ml的CSF-1可降低人牙囊细胞OPG的表达,共同孵育1h,OPG表达降至最低(P<0.05)。结论牙囊细胞表达OPG,同时在牙齿萌出的特定时期,CSF-1通过下调人牙囊细胞OPG,减弱对破骨细胞形成的抑制作用,促进破骨细胞分化成熟,调节牙齿萌出。     

p53基因抑制涎腺腺样囊性癌细胞端粒酶及增殖活性

目的研究外源性野生型p53基因对涎腺腺样囊性癌细胞的抑制作用。方法构建携带野生型p53基因的腺病毒表达载体，以脂质体法转染涎腺腺样囊性癌SACC-83细胞，RT-PCR检测p53基因表达；采用TRAP—PCR—ELISA法检测转染细胞端粒酶活性，荧光素酶分析法检测人端粒酶逆转录酶基因（hTERT）肩动子的转录；流式细胞术、软琼脂集落实验及裸鼠成瘤实验观察细胞生物特性的变化。结果外源性野生型p53基因导入使p53基因在涎腺腺样囊性癌细胞SACC-83中表达增强，其端粒酶活性降低、hTERT启动子转录抑制；转染细胞出现G1期阻滞，软琼脂集落形成率减少，裸鼠成瘤能力降低。结论腺病毒载体介导的外源性野生型p53基因可以抑制涎腺腺样囊性癌细胞端粒酶活性及细胞恶性表型。     

Analysis of postoperative symptoms following surgical endodontic treatment.

OBJECTIVE: The purpose of this prospective study was to analyze postoperative pain and swelling of patients undergoing surgical endodontic treatment using a strict protocol incorporating measures to control postoperative symptoms. METHOD AND MATERIALS: The study consisted of 82 patients referred for surgical endodontic treatment. All surgical procedures were performed using a microsurgical technique employing strict protocol. All patients were premedicated with a single dose of oral dexamethasone (8 mg) preoperatively and two single doses (4 mg) 1 and 2 days postoperatively. Antibiotics were prescribed selectively only when severe symptoms were present due to infection. Patients were administered chlorhexidine mouthwash twice daily starting 3 days before the operation and an additional 7 days postoperatively starting the day after surgery. Cold compresses were applied on the skin at the site of surgery intermittently every 15 minutes during the operative day. Pain and swelling were recorded pre- and postoperatively, and the influence of different variables on postoperative sequelae were analyzed. RESULTS: One day postoperatively, 76.4% of the patients were completely pain free, less than 4% had moderate pain, and 64.7% did not report any swelling. The preoperative symptoms significantly influenced the pain experience post-surgery. CONCLUSION: There was a low incidence of postoperative pain and swelling following endodontic surgical treatment according to protocol with measures to control postoperative signs and symptoms. Patients with preoperative pain were more likely to have postoperative pain.