口腔粘膜癌前病变和鳞癌组织细胞增殖和凋亡的原位观察

目的研究自口腔正常粘膜上皮向异常增生和鳞癌组织演变过程中细胞增殖和凋亡的变化,阐明口腔癌发病机制.方法采用免疫组织化学S-P法和原位末端标记法检测10例正常口腔粘膜上皮、48例异常增生上皮和42例鳞癌组织中增殖细胞数和凋亡数.结果异常增生上皮细胞增殖和凋亡数均明显高于正常组(P＜0.05),但随着增生程度的加重,伴随着细胞增殖能力逐级增强,细胞凋亡数无明显提高(P＞0.05).鳞癌组织中,随着组织学分级的增加,增殖数明显增多,凋亡数逐级减少.结论癌前病变中,细胞凋亡速度增加跟不上增殖速度,导致向鳞癌转变.癌细胞增殖不断加强,凋亡不断下降,两者共同作用的结果导致鳞癌的发展.     

Cell proliferation and apoptosis in ameloblastomas and keratocystic odontogenic tumors

A high proliferative activity of the odontogenic epithelium in ameloblastoma (AM) and keratocystic odontogenic tumor (KOT) has been demonstrated. However, no previous study has simultaneously evaluated cell proliferation and apoptotic indexes in AM and KOT, comparing both lesions. The aim of this study was to assess and compare cell proliferation and apoptotic rates between these two tumors. Specimens of 11 solid AM and 11 sporadic KOT were evaluated. The proliferation index (PI) was assessed by immunohistochemical detection of Ki-67 and the apoptotic index (AI) by methyl green-pyronine and in situ DNA nick end-labelling methods. KOT presented a higher PI than AM (p<0.05). No statistically significant difference was found in the AI between AM and KOT. PI and AI were higher in the peripheral cells of AM and respectively in the suprabasal and superficial layers of KOT. In conclusion, KOT showed a higher cell proliferation than AM and the AI was similar between these tumors. These findings reinforce the classification of KOT as an odontogenic tumor and should contribute to its aggressive clinical behavior.     

Cell proliferation and apoptosis in the fusion of human primary and secondary palates

The markers of cell proliferation (Ki-67) and apoptosis [caspase-3, TdT-mediated biotin-dUTP nick-end labelling (TUNEL)] and the expression of syndecan-1 and heat shock protein 70 (Hsp70) were analyzed immunohistochemically in 11 developing human palates, from developmental weeks 6 to 10. During fusion of the primary palate, the proportion of proliferating cells decreased from 42 to 32% and the proportion of apoptotic cells decreased from 11 to 7% in the medial-edge epithelium. At later stages, the proportions of both types of cells decreased in the ectomesenchyme, except for proliferating cells in its non-condensing part. At developmental weeks 9-10, the epithelial seam in the secondary palate comprised 28% proliferative cells and 5% apoptotic cells. While condensing ectomesenchyme contained more apoptotic cells than proliferating cells, the opposite was observed for the non-condensing ectomesenchyme. Co-expression of syndecan-1 and Hsp70 was detected in cells budding from the epithelial seam. Our study indicates similar principles for human primary palate and secondary palate fusion, and parallel persistence of proliferation and apoptotic activity. While proliferation enables growth and fusion of different palatal primordia, apoptosis results in the removal of of large numbers epithelial cells at the fusion point. The disintegration of seam remnants seems to be executed through the processes of change in protein content and cell migration, probably leading to cell death as their final outcome.     

Cell proliferation, apoptosis and apoptosis-related factors in odontogenic keratocysts and in dentigerous cysts

BACKGROUND: The purpose of this study was to elucidate why odontogenic keratocysts (OKC) can form cystic lesions but not tumor masses, notwithstanding their prominent proliferative activity. METHODS: We investigated cellular proliferation, cell death, and expression of apoptosis-related proteins in the lining cells of OKCs and of dentigerous cysts (DGCs). RESULTS: TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were observed in the surface layers of OKCs and of DGCs. However, no TUNEL-positive cells were seen in the basal or intermediate layers of both cysts. Ki67-positive ratio in the intermediate layer was the highest in OKCs. The p53-positive ratio of the intermediate layer was highest in OKCs. Bcl-2-positive cells were discernible exclusively in the basal layer of OKCs. CONCLUSIONS: These results suggest that cellular proliferation and death is regulated in association with apoptosis-related proteins in the lining epithelia of OKCs, and subsequently those cysts are seen as cystic lesions but not as tumor masses.     

环孢素对小鼠牙龈上皮细胞增殖和凋亡的影响

目的观察环孢素(CsA)对小鼠牙龈上皮细胞增殖和凋亡的影响,探讨环孢素导致龈增生的机制。方法CsA胃饲小鼠8d后注射5-溴-2-尿嘧啶核苷(BrdU),在不同时间取小鼠牙龈及腭黏膜组织作BrdU染色,光镜下观察其上皮中阳性细胞的数目、分布以及完全代谢的周期,并与对照组比较。结果实验组小鼠的牙龈上皮中阳性细胞数与对照组比没有差异,但其生长周期比对照组长。其腭黏膜上皮细胞生长周期较牙龈黏膜上皮细胞无明显延长,阳性细胞数与对照组比较无差异。结论CsA可抑制小鼠牙龈上皮细胞的凋亡,但对细胞增殖没有影响。     

Cell synthesis, proliferation and apoptosis in human dental periapical lesions analysed by in situ hybridisation and immunohistochemistry

OBJECTIVE: The role of structural and host defensive cells in periapical lesions has been assessed previously by morphometric and immunohistochemical studies. The aim of this study was to investigate the function of periapical cells by employing molecular techniques to estimate the cell synthetic activity, proliferation and apoptosis in these lesionS. We specifically sought answers to the following questionS. Which cells of the periapical lesions are quiescent or actively synthesising proteins? Do immune cells proliferate in this region in the same way as epithelial cells proliferate? Furthermore do cells in periapical lesions undergo apoptosis, and if so which cells exhibit this programmed cell death? MATERIALS: Twenty-five periapical tissue samples (15 granulomas and 10 radicular cysts) were assessed. Poly-adenosine (poly (A)) RNA and ribosomal RNA (rRNA) bearing cells in formalin-fixed/paraffin-embedded periapical tissues were analysed by in situ hybridization (ISH) using digoxigenin-labelled oligo d (T) and 28S rRNA probes respectively in order to estimate cell synthetic activity. Furthermore, S-phase proliferating and cycling cells were examined by ISH using a histone probe and Ki-67 immunostaining so as to assess cellular proliferation. Mononuclear cells were further differentiated by immunohistochemistry (IHC) as T cells, B cells and macrophageS. Apoptotic cells were determined by in situ end-labelling methodology for detecting fragmented DNA.RESULTS: Poly (A) RNA (mostly messenger RNA) and 28S rRNA-expressing cells were detected in all sampleS. Plasma cells exhibited strongest staining for the two probes, with slight to moderate staining found in the epithelium, fibroblasts, macrophages, endothelial cells and lymphocytes, whereas almost all polymorphonuclear leucocytes (PMN) were negative for these probeS. A few histone mRNA-expressing cells were detected in basal and suprabasal epithelial cells and mononuclear cells in 15/25 cases but their reactivity was weak. Ki-67 positive cells were found in all samples and their numbers were generally higher than histone mRNA positive cellS. Apoptotic cells were detected in 23/25 cases and the majority of apoptotic cells were PMN which were engulfed by large cytophagocytic macrophages. CONCLUSION: This study indicates that in dental periapical lesions, apoptosis occurs predominantly in PMN. It is evident that most cells apart from PMN are exhibiting synthetic activity but only epithelial cells undergo proliferation which implies that immune cells must proliferate at distant lymph nodes and travel to the periapical lesion rather than proliferating within the lesion. These results suggest considerable advantages in estimating gene expression within cells in addition to the immunohi-stochemical detection of cells to determine cell activity at inflamed siteS. Clearly, functional cell synthetic activity, resolution and clearance systems operate in peri- apical cystic and granuloma lesions.     

Cell proliferation and apoptosis in the primary enamel knot measured by flow cytometry of laser microdissected samples

Laser capture microdissection (LCM) uniquely allows the selection of specific cell populations from histological sections. These selected cells are then catapulted into a test tube without any contamination from surrounding tissues. During the last ten years, many significant results have been achieved, particularly at the level of DNA and RNA where amplification techniques are available. However, where amplification procedures are difficult, the benefits of LCM diminish.To overcome such difficulties, a novel approach, combining laser capture microdissection and flow cytometry, has been tested here for detection of apoptosis and proliferation in tissue bound cell populations without any amplification steps. The mouse cap stage molar tooth germ was used as a model. At the centre of the inner enamel epithelium, the primary enamel knot is a clearly defined apoptotic population with minimal proliferation, flanked by the highly proliferative cervical loops on each side. Thus within the tooth germ epithelium at this stage, two distinct populations of cells are found side by side. These populations were selected by laser capture microdissection and then analysed by flow cytometry for apoptosis and proliferation. Flow cytometric results correlated well with immunohistochemical findings, demonstrating the success and sensitivity of this combined procedure.     

Transgenic rescue of enamel phenotype in Ambn null mice

Ameloblastin null mice fail to make an enamel layer, but the defects could be due to an absence of functional ameloblastin or to the secretion of a potentially toxic mutant ameloblastin. We hypothesized that the enamel phenotype could be rescued by the transgenic expression of normal ameloblastin in Ambn mutant mice. We established and analyzed 5 transgenic lines that expressed ameloblastin from the amelogenin (AmelX) promoter and identified transgenic lines that express virtually no transgene, slightly less than normal (Tg+), somewhat higher than normal (Tg++), and much higher than normal (Tg+++) levels of ameloblastin. All lines expressing detectable levels of ameloblastin at least partially recovered the enamel phenotype. When ameloblastin expression was only somewhat higher than normal, the enamel covering the molars and incisors was of normal thickness, had clearly defined rod and interrod enamel, and held up well in function. We conclude that ameloblastin is essential for dental enamel formation.     

Altered ion-responsive gene expression in Mmp20 null mice

During enamel maturation, hydroxyapatite crystallites expand in volume, releasing protons that acidify the developing enamel. This acidity is neutralized by the buffering activity of carbonic anhydrases and ion transporters. Less hydroxyapatite forms in matrix metalloproteinase-20 null (Mmp20(-/-)) mouse incisors, because enamel thickness is reduced by approximately 50%. We therefore asked if ion regulation was altered in Mmp20(-/-) mouse enamel. Staining of wild-type and Mmp20(-/-) incisors with pH indicators demonstrated that wild-type mice had pronounced changes in enamel pH as development progressed. These pH changes were greatly attenuated in Mmp20(-/-) mice. Expression of 4 ion-regulatory genes (Atp2b4, Slc4a2, Car6, Cftr) was significantly decreased in enamel organs from Mmp20(-/-) mice. Notably, expression of secreted carbonic anhydrase (Car6) was reduced to almost undetectable levels in the null enamel organ. In contrast, Odam and Klk4 expression was unaffected. We concluded that a feedback mechanism regulates ion-responsive gene expression during enamel development.     

大鼠切牙发育中釉蛋白的免疫荧光定位

目的:观察釉蛋白在大鼠切牙发育不同时期的表达和分布情况.方法:采用免疫荧光法观察釉蛋白在大鼠切牙发育不同时期的表达与分布.结果:在成釉器增殖期、分化早期,未见釉蛋白表达;在分化晚期,前成釉细胞有弱阳性表达;在成熟早期,成釉细胞和成牙本质细胞均阳性表达;在成熟中期,成釉细胞及其基质呈强阳性表达,而成牙本质细胞阳性表达;在成熟晚期,成釉细胞和成牙本质细胞弱阳性表达.在成釉器各期,釉蛋白在外釉细胞、星网状细胞、牙乳头细胞均呈阴性表达.结论:釉蛋白参与釉基质和牙本质基质的形成,可能与牙发育中基质形成的信息传递有关.     

Immunocytochemical localization of enamelin proteins in developing bovine teeth

Enamelins were localized at both the light and electron microscopic level using an antienamelin monoclonal antibody and indirect immunogold methods. Bovine fetal incisors (crown-rump length 17-30 cm) were preserved in Karnovsky's fixative and embedded in Epon. For light microscopy, 2 microM thick sections were immunostained by the indirect method using the monoclonal antibody and goat anti-mouse IgG linked to 5 nM gold particles, followed by silver enhancement to increase the sensitivity of the method. For electron microscopy, thin sections were immunostained (indirect) with the antienamelin monoclonal antibody and goat anti-mouse IgG linked to 5 or 15 nM gold. Control samples were treated with an unrelated monoclonal antibody. The localization of enamelins was confined in the light microscopic sections to the extracellular enamel matrix. No gold staining was observed in the ameloblasts or other enamel organ cells even though the gold-silver technique is extremely sensitive. Ultrastructurally, enamelin was localized in the enamel extracellular matrix and associated ameloblasts. Both the crystal-containing and granular matrix were positively stained, with most gold particles being closely associated with the crystals. Counting of gold particles indicated more than 4 times as many amelogeninas enamelin-reactive antigenic sites in similar regions. Decalcification did not increase immunostaining with the anti-enamelin antibody in the extracellular matrix. Within ameloblasts, the gold particles were associated with secretory granules and Golgi complexes. Thus it appears that enamelins are synthesized in ameloblasts and secreted into the extracellular matrix in a similar manner to amelogenins and are preferentially associated with matrix hydroxyapatite crystals. Transient levels of enamelins within the ameloblasts are apparently too low to be detected by light microscopy.     

缺氧对成骨细胞增殖及凋亡的影响

目的：通过在缺氧条件下体外培养成骨细胞,探讨缺氧对成骨细胞增殖及凋亡的影响。方法：采用酶消化法传代培养乳SD大鼠颅盖骨细胞并建立缺氧模型。用台盼蓝染色法分别计数培养1、3、5、7天的细胞数;用流式细胞仪检测对照组与缺氧1、3、5天的细胞凋亡率。结果：缺氧组的细胞数少于正常氧组;对照组与缺氧1、3、5天的细胞凋亡率逐渐增高。结论：缺氧抑制成骨细胞的增殖,同时促进成骨细胞的凋亡。     

小鼠牙胚发育过程中釉原蛋白和釉蛋白的表达特征

目的 观察釉原蛋白和釉蛋白在小鼠牙胚发育中的表达特征,进一步揭示釉原蛋白和釉蛋白的生物学特性.方法 分别制备出生后第1、3、7和14天小鼠下颌第一磨牙牙胚切片,采用免疫组织化学和反转录聚合酶链反应法检测釉原蛋白和釉蛋白的组织学定位和基因转录表达水平.结果 釉原蛋白表达于分泌期成釉细胞的胞质和釉质基质全层,在新生小鼠牙胚成牙本质细胞中有一过性表达;釉蛋白表达于分泌期釉质基质中,在成釉质细胞突边缘和釉质基质深层呈强阳性表达,釉原蛋白和釉蛋白在矿化成熟的釉质中均未见表达.釉原蛋白和釉蛋白在出生后第7天mRNA表达量的相对值分别为0.813±0.085和0.799±0.064,显著高于其他时间的表达水平(P＜0.05).结论 釉原蛋白和釉蛋白主要由分泌期成釉细胞合成并分泌到釉质基质中,其表达具有高度的时空特异性,提示它们在釉质形成和生物矿化中有重要的作用.     

A comparison of enamelin and amelogenin expression in developing mouse molars

Amelogenin and enamelin are structural proteins in the enamel matrix of developing teeth. The temporal and spatial patterns of enamelin expression in developing mouse molars have not been characterized, while controversy remains with respect to amelogenin expression by odontoblasts and cementoblasts. Here we report the results of in situ hybridization analyses of amelogenin and enamelin expression in mouse molars from postnatal days 1, 2, 3, 7, 9, 14, and 21. Amelogenin and enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onsets of amelogenin and enamelin expression were approximately synchronous with the initial accumulation of predentin matrix. Both proteins were expressed by ameloblasts throughout the secretory, transition, and early maturation stages. Enamelin expression terminated in maturation stage ameloblasts on day 9, while amelogenin expression is still detected in maturation stage ameloblasts on day 14. No amelogenin expression was observed in day 21 mouse molars. Amelogenin and enamelin RNA messages were restricted to ameloblasts. No expression was observed in pulp, bone, or along the developing root. We conclude that amelogenin and enamelin are enamel-specific and do not directly participate in the formation of dentin or cementum in developing mouse molars.     

Cloning and characterization of the mouse and human enamelin genes

Enamelin is likely to be essential for proper dental enamel formation. It is secreted by ameloblasts throughout the secretory stage and can readily be isolated from the enamel matrix of developing teeth. The gene encoding human enamelin is located on the long arm of chromosome 4, in a region previously linked to an autosomal-dominant form of amelogenesis imperfecta (AI). To gain information on the structure of the enamelin gene and to facilitate the future assessment of the role of enamelin in normal and diseased enamel formation, we have cloned and characterized the mouse and human enamelin genes. Both genes are about 25 kilobases long. The enamelin gene has 10 exons interrupted by 9 introns. Translation initiates in exon 3 and terminates in exon 10. All of the intron/exon junctions within the mouse and human enamelin coding regions are between codons, so there are no partial codons in any exon, and deletion of one or more coding exons by alternative RNA splicing would not shift the downstream reading frame.     

Enamel proteins and proteases in Mmp20 and Klk4 null and double-null mice

Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are thought to be necessary to clear proteins from the enamel matrix of developing teeth. We characterized Mmp20 and Klk4 null mice to better understand their roles in matrix degradation and removal. Histological examination showed retained organic matrix in Mmp20, Klk4, and Mmp20/Klk4 double-null mouse enamel matrix, but not in the wild-type. X-gal histostaining of Mmp20 null mice heterozygous for the Klk4 knockout/lacZ knockin showed that Klk4 is expressed normally in the Mmp20 null background. This finding was corroborated by zymogram and western blotting, which discovered a 40-kDa protease induced in the maturation stage of Mmp20 null mice. Proteins were extracted from secretory-stage or maturation-stage maxillary first molars from wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mice and were analyzed by SDS-PAGE and western blotting. Only intact amelogenins and ameloblastin were observed in secretory-stage enamel of Mmp20 null mice, whereas the secretory-stage matrix from Klk4 null mice was identical to the matrix from wild-type mice. More residual matrix was observed in the double-null mice compared with either of the single-null mice. These results support the importance of MMP20 during the secretory stage and of KLK4 during the maturation stage and show there is only limited functional redundancy for these enzymes.     

HEMA reduces cell proliferation and induces apoptosis in vitro.

OBJECTIVES: Methacrylate monomers have been identified in aqueous extracts of freshly cured compomers. Both cells in the pulpal cavity and various cells of the oral mucosa can potentially be exposed to these leachables. Short-term exposure to dental monomers at relatively high concentrations induces adverse biological effects in vitro. The mechanisms involved have not been fully elucidated although involvement of various signaling pathways including ROS formation, activation of MAP-kinases and caspases has been suggested. The aim of this study was to investigate potential cellular responses following long-term exposure to relatively low and potentially more clinical relevant HEMA concentrations. METHODS: A submandibular gland cell line was exposed to HEMA (20-600 microM) for up to 72h. The impact on cell proliferation, apoptosis, and possible underlying mechanisms was assessed by flow cytometry, microscopy and western blotting. RESULTS: Exposure to HEMA (600 microM) resulted in reduced cell proliferation after 24h and increased apoptosis after 60h. Further, we observed ATM dependent phosphorylation of p53, advocating an initial DNA damage in the HEMA exposed cells. SIGNIFICANCE: In conclusion, we show that exposure to relatively low concentration of HEMA for a prolonged time result in cell death, possibly as a consequence of DNA damage.