遗传性牙龈纤维瘤病（附2例报告）

目的通过探讨遗传性牙龈纤维瘤病（HGF）的临床特点及治疗方法,增进对本病的认识,从而提高诊断治疗水平。方法先证者法收集两个HGF家系全部成员资料,观察不同家系及同一家系不同个体的临床表型和发病特点,绘制系谱图,分析可能的遗传方式。对两名先证者采用手术治疗。结果两家系发病患者均符合非综合征型HGF特征。发病患者不同个体间的表现度不同。两家系均符合常染色体显性遗传特征。经随访,手术患者治疗效果良好。结论 HGF遗传方式以常染色体显性遗传为主,且同一家系的不同受累个体其增生程度轻重不一,极具差异,具有高度遗传异质性。手术是治疗该病的有效的方法。     

常染色体显性遗传性牙龈纤维瘤病的临床和遗传学特点分析

目的：探讨遗传性牙龈纤维瘤病(HGF)的临床表型和遗传学特点。方法：先证者法收集5个HGF家系并进行问卷和口腔检查，观察不同家系及同一家系不同个体的临床表型和发病特点，分析可能的遗传方式，绘制系谱图。结果：所有家系符合常染色体显性非综合征型HGF特征，发病年龄在牙齿萌出期，患者均有典型的牙龈增生，但不同个体其增生范围和严重程度有明显差异。龈切术可极大地恢复口腔功能和颜面外形，但部分病例在术后有复发倾向。结论：收集的5个家系均为非综合征型常染色体显性遗传HGF，且疾病外显率高，表现度变异大。     

遗传性牙龈纤维瘤病中国家系中SOS1基因突变的研究

目的 筛查中国遗传性牙龈纤维瘤病(hereditary gingival fibromatosis,HGF)家系中SOS1基因,为寻找HGF致病基因突变提供线索.方法 收集到两个中国非综合征性HGF家系,患者表现出典型而且一致的临床表型.采取受试对象的外周血,提取基因组DNA.针对SOS1基因的23个外显子序列设计引物,PCR扩增纯化后进行Sanger测序.结果 两个中国HGF家系的患者均未携带已知SOS1基因的插入突变(c.3248-3249insC),在SOS1基因的外显子区,以及外显子与内含子交接的剪接区,均未发现其他的突变位点.结论 SOS1基因不是HGF中国家系的致病基因,中国HGF具有不同的遗传背景,存在其他潜在的致病基因和突变.应该利用全外显子组测序等方法,在中国HGF家系中寻找新的致病基因.     

永生化人成牙本质细胞样细胞系的转化特征

目的:明确永生化人成牙本质细胞样细胞系hTERT -hOd- l是否具有转化特征。方法:复苏已建立的永生化人成牙本质细胞样细胞系hTERT- hOd -l,从致瘤性、血清依赖性、悬浮生长能力和接触抑制性等方面观察细胞的转化特征。结果:细胞无裸鼠致瘤性,在软琼脂内不能生长,血清依赖性无显著降低,仍具有接触抑制性。结论:hTERT- hOd- l基本上为正常细胞而无明显转化特征。     

一个非综合征型多数牙缺失家系的临床及遗传学特征分析

目的:探讨一个非综合征型多数牙缺失家系的临床表型及遗传学特点.方法:对家系内部分患者及正常成员进行口腔专科检查和家系调查,总结分析其临床特征,并绘制系谱图以明确其遗传方式.结果:(1)该家系符合常染色体显性遗传模式,外显率较高;(2)患者牙列发育异常表现在牙齿数目、形态、位置及(牙合)关系等方面,先天缺牙以第二前磨牙及第三磨牙较为常见;(3)家系内不同个体的临床表型存在差异. 结论:该家系中,先天性缺牙呈常染色体显性遗传模式,外显率较高,表型差异较大.其临床特征以第二前磨牙和第三磨牙先天缺失较为多见.     

冻融前后人牙髓等成纤维细胞超微结构和染色体数目的比较

本研究对人牙髓牙周牙龈成纤维细胞冻存和复苏后超微结构和染色体数目进行了观察比较,结果表明:冻融前后三种细胞70%以上染色体数目为23对,冻融后三种细胞线粒体等细胞器有部分老化,溶酶体数目增多.     

外胚叶发育不全一家系的基因型分析

目的:分析外胚叶发育不全家系的表型特点和遗传特征,并对家系基因型进行分析。方法:收集1个外胚叶发育不全家系,采用临床检查和家系调查的方法,调查并记录先证者及家系成员的病史和体格检查资料。对家系成员EDAR基因开放阅读框内外显子编码区及外显子-内含子接头区核苷酸序列进行分析。结果:收集到的家系为常染色体显性遗传,患者临床表现典型,家系内表现度差异小。家系成员EDAR基因开放阅读框内未检测到基因突变。结论:本研究收集的外胚叶发育不全家系临床症状明显,致病基因排除EDAR基因。     

牙本质发育异常家系调查和表型分析

目的:调查和分析中国人牙本质发育异常家系及临床表型,进一步明确其诊断和分型。方法:采用先证者查证法调查和收集中国人牙本质发育异常家系,绘制系谱图,确定遗传方式,根据临床表现和X线征象特点对各家系受累个体进行表型分析。结果:共收集4个中国人牙本质发育异常家系,家系Ⅰ、Ⅱ、Ⅲ为常染色体显性遗传的Ⅱ型牙本质发育不全,家系Ⅳ先证者符合Ⅱ型牙本质发育不全诊断;临床表型在各家系及同一家系不同个体间存在异同。结论:独立发生于牙本质的各型遗传性牙本质发育异常存在共有表型,可作为同一类疾病研究。     

涎腺淋巴上皮癌的发生

摘要涎腺淋巴上皮癌是原发于涎腺的罕见的恶性肿瘤之一,其发生与EB病毒感染关系密切,本文着重就涎腺淋巴上皮癌发生学研究现状及EB病毒在该瘤发生中可能的作用及作用机理方面的研究进展进行了综述。     

不同冻存方法对骨髓基质细胞生物学活性的影响

目的:比较不同冻存方法对骨髓基质细胞(BMSc)增殖分化能力的影响。方法:从Beagle犬股骨抽取骨髓,培养,传代,至第4代时将其以不同细胞浓度(1×107/L、1×108/L、1×109/L)、不同浓度冻存保护剂DMSO(5%、10%、15%)、不同降温方式以及消化或原位等方法进行冻存,细胞复苏后传至第7代,观察冻存对其增殖分化能力的影响。结果:各种方法冻存的BMSc均保持了较高的增殖、分化能力。高细胞浓度、10%DMSO的冻存保护剂有利于细胞活性的保存(P<0.05),3种降温方式对BMSc的生物特性的影响没有显著差别。-80℃原位冻存时BMSc仍保存了较高的增殖、分化能力。结论:较高的冻存细胞浓度、选用含10%DMSO的冻存保护剂、适宜的降温方式有利于冻存。-80℃原位冻存可作为BMSc短期的保存方法。     

人牙龈成纤维细胞原代培养方法的比较研究

目的：建立和评价人牙龈成纤维细胞原代培养方法,并观察其生物学特性。方法：分别用组织块法、2种改良酶消组织块法培养人牙龈成纤维细胞（HGF）,用形态学、免疫荧光鉴定细胞来源。通过活细胞观察,MTT比色实验研究细胞体外生物特性。比较3种培养方法培养HGF的效果。结果：细胞抗波形丝蛋白染色阳性,抗角蛋白染色阴性,符合人牙龈成纤维细胞的形态学特征和生物学特性。组织块法、改良酶消组织块法（翻瓶法）、改良酶消组织块法（盖玻片法）的细胞培养成功率分别为26.7%、54%、60%。组织块法和2种改良酶消组织块法间的成功率差异有显著性（P〈0.01）,2种改良酶消组织块法间的成功率差异无显著性（P〉0.05）。结论：本实验建立的细胞系为人牙龈成纤维细胞。2种改良酶消化组织块法可显著提高人牙龈成纤维细胞原代培养成功率。     

Proliferation of fibroblasts cultured from normal gingiva and hereditary gingival fibromatosis is dependent on fatty acid synthase activity

BACKGROUND: Fatty acid synthase (FAS) is the enzyme that synthesizes palmitate from malonyl-CoA and acetyl-CoA. Recent studies have shown that FAS is overexpressed in human cancers and that its activity is necessary for cell proliferation. Hereditary gingival fibromatosis (HGF) is a genetic disease manifested as a progressive enlargement of the gingiva. The pathogenesis of this condition is not understood; however, a proliferative advantage of HGF fibroblasts in comparison with cells from normal gingiva (NG) has been described. The aim of this study was to investigate the role of FAS in NG and HGF fibroblast proliferation. METHODS: NG and HGF fibroblasts had their proliferative potential assessed by automated cell counting and immunocytochemistry against Ki-67 or proliferating cell nuclear antigen (PCNA). The production of FAS, androgen receptor (AR), and ErbB2 was analyzed by Western blot and the pattern of FAS expression studied by immunocytochemistry. FAS activity was blocked by the specific inhibitor cerulenin. RESULTS: Higher proliferation rates were found in fibroblasts isolated from HGF than from NG. HGF fibroblasts with greater proliferative potential produced more FAS and AR than the cell lines with lower growth rates, and all studied cell lines produced similar amounts of the ErbB2 protein. In addition, the FAS inhibitor cerulenin was able to significantly reduce the proliferation of both NG and HGF cells. CONCLUSIONS: These results show that FAS is expressed by gingival fibroblasts and that highly proliferative HGF cells produced more FAS and AR than the other fibroblast cell lines. Moreover, FAS inhibition significantly reduced both NG and HGF fibroblast growth, suggesting a role for the androgen-driven fatty acid biosynthesis in their proliferation.     

Immunohistochemical detection of hepatocyte growth factor, transforming growth factor-β and their receptors in epithelial odontogenic tumors

BACKGROUND: Tumors derived from odontogenic epithelium exhibit considerable variation and are classified into several benign and malignant entities. To clarify the role of growth factors in oncogenesis, cytodifferentiation and progression of epithelial odontogenic tumors, expression of hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta) and their receptors were analyzed in these tumors as well as in tooth germs. METHODS: Specimens of five tooth germs, 34 ameloblastomas, three calcifying epithelial odontogenic tumors (CEOTs), two clear cell odontogenic tumors (CCOTs), five adenomatoid odontogenic tumors (AOTs), six calcifying odontogenic cysts (COCs) and six malignant ameloblastomas were examined immunohistochemically with the use of antibodies against HGF, TGF-beta and their receptors. RESULTS: In tooth germs and epithelial odontogenic tumors, immunoreactivity for HGF and TGF-beta was detected in both epithelial and mesenchymal cells, while expression of their receptors was found only in epithelial cells. In tooth germs and main types of ameloblastomas, HGF and TGF-beta reactivity was marked in epithelial cells near the basement membrane, and their receptors were diffusely positive in most epithelial cells. In subtypes of ameloblastomas, reduced expression of HGF, c-Met and TGF-beta and increased reactivity for TGF-beta receptors were detected in keratinizing cells in acanthomatous ameloblastomas, and granular cells in granular cell ameloblastomas demonstrated little or no expression of HGF, TGF-beta or their receptors. As compared with main types of ameloblastomas, basal cell ameloblastomas showed high HGF reactivity, and desmoplastic ameloblastomas exhibited elevated reactivity for TGF-beta and its receptors. Neoplastic cells in CEOTs, AOTs and COCs showed reactivity for HGF, TGF-beta and their receptors. Elevated HGF and TGF-beta reactivity was found in pseudoglandular cells in AOTs, and high expression of their receptors was noted in ghost cells in COCs. Metastasizing ameloblastomas showed similar expression patterns of HGF, TGF-beta and their receptors to those of benign ameloblastomas, while CCOTs and ameloblastic carcinomas had increased HGF expression and low reactivity for TGF-beta and its receptors as compared with benign ameloblastomas. CONCLUSIONS: Immunohistochemical localization of HGF, TGF-beta and their receptors in tooth germs and epithelial odontogenic tumors supports the hypothesis that HGF and TGF-beta act on epithelial cells via paracrine and autocrine mechanisms. Altered expression of the agents in these epithelial odontogenic tumors, especially subtypes of ameloblastomas, AOTs and COCs, suggests that HGF and TGF-beta signaling might affect differentiation of neoplastic odontogenic epithelial cells. Activated HGF/c-Met pathway and reduced TGF-beta signaling in CCOTs and ameloblastic carcinomas may be associated with the malignant potential of these epithelial odontogenic tumors.     

Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth

Sakuraba H, Fujiwara N, Sasaki‐Oikawa A, Sakano M, Tabata Y, Otsu K, Ishizeki K, Harada H. Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth. J Periodont Res 2012; 47: 81–88. © 2011 John Wiley & Sons A/S Background and Objective: It is well known that tooth root formation is initiated by the development of Hertwig’s epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial–mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. Material and Methods: The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF‐soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5′‐bromo‐2′‐deoxyuridine (BrdU) assay in an organ‐culture system. Results: HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ‐culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down‐regulated when antibody against HGF receptor was present in the culture medium. Conclusion: Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development.     

Migration induced by epidermal and hepatocyte growth factors in oral squamous carcinoma cells in vitro: role of MEK/ERK, p38 and PI-3 kinase/Akt

J Oral Pathol Med (2012) 41 : 547–558 Background: Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti‐tumour signal inhibitors on the migratory activity. Methods: Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time‐lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting. Results: In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose‐dependent activation of cell migration. Addition of the EGFR‐specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF‐induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI‐3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF‐induced migration was particularly sensitive to PI‐3 K‐inhibition, while in C12 cells, both HGF‐ and EGF‐induced migration were highly sensitive to p38‐blockade. Conclusion: The results demonstrate that the MEK/ERK, p38 and PI‐3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.     

牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞生物学性能的影响

目的:观察牙齿外伤钛固定夹板(titanium trauma splint,TTS)浸提液对人牙龈成纤维细胞(human gingival fibroblasts,HGF)生物学性能的影响,为临床应用提供理论依据。方法:分离、培养鉴定人牙龈成纤维细胞,将牙齿外伤钛固定夹板浸提液加入人牙龈成纤维细胞培养基中,通过倒置显微镜观察细胞形态学改变、MTT法细胞毒性实验和细胞凋亡实验,观察牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞生物学性能的影响。结果:牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞的形态无明显改变,其增殖和凋亡的影响较正常人牙龈成纤维细胞相比无明显细胞毒性,不引起细胞凋亡。结论:牙齿外伤钛固定夹板具有良好的生物安全性,有一定的临床应用前景。     

Exogenous hepatocyte growth factor inhibits myoblast differentiation by inducing myf5 expression and suppressing myoD expression in an organ culture system of embryonic mouse tongue

We examined the effects of exogenous hepatocyte growth factor (HGF) on the differentiation and proliferation of tongue myoblasts by using an organ culture system of tongue obtained from mouse embryos at embryonic day (E) 13. Exogenous HGF induced reductions in the quantities of muscle creatine kinase and myogenin mRNAs and in the number of fast myosin heavy chain-positive myoblasts and myotubes, suggesting that HGF suppressed the differentiation of myoblasts in the cultured E13 tongues. Exogenous HGF induced no significant changes in the percentage of proliferating cell nuclear antigen (PCNA)-positive cell nuclei to total cell nuclei (labeling index) in the muscle portion of the cultured E13 tongue, suggesting that HGF did not affect the proliferation of myoblasts. Exogenous HGF induced the expression of myf5 mRNA but inhibited the expression of myoD mRNA. Since mouse tongue myoblasts are reported to complete proliferation by E13, it appears that exogenous HGF arrests myoblasts in the cell cycle and does not allow them to enter the differentiation process. This is achieved by controlling the expression of myf5 and myoD mRNAs, thus inhibiting the differentiation of tongue myoblasts.     

Effect of periodontal dressings on human gingiva fibroblasts in vitro

In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC #1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings.     

Transforming growth factor-beta1 autocrine stimulation regulates fibroblast proliferation in hereditary gingival fibromatosis.

BACKGROUND: Hereditary gingival fibromatosis (HGF) is a rare oral disease characterized by a slow and progressive enlargement of both the maxilla and mandible gingiva. Increased proliferation, elevated synthesis of extracellular matrix, particularly collagen, and reduced levels of matrix metalloproteinases seem to contribute to the pathogenesis of gingival overgrowth in HGF patients. Transforming growth factor-beta1 (TGF-beta1) is an important cytokine thought to play a major role in fibrotic disorders such as HGF due to its ability to stimulate the synthesis and reduce the degradation of extracellular matrix. In HGF fibroblasts, TGF-beta1 autocrine stimulation reduces expression and production of matrix metalloproteinases. However, the role of TGF-beta1 in fibroblast growth modulation has not been established in this disease. METHODS: The aim of this study was to confirm the increased proliferation rate of HGF fibroblast cell lines and to explore a possible autocrine role of TGF-beta1 as a cell growth stimulator by blocking production of this endogenous cytokine using 2 well-established systems: antisense oligonucleotides and neutralizing antibodies. RESULTS: Four different cellular proliferation assays, bromodeoxyuridine labeling, argyrophilic nucleolar organizing region staining, proliferating cell nuclear antigen, and mitotic indexes, confirmed that fibroblasts from HGF proliferate significantly faster than those from normal gingiva. Antisense oligonucleotides reduced TGF-beta1 production as demonstrated by capture enzyme-linked immunosorbent assay, whereas TGF-beta1 expression levels were not significantly modified. Blocking TGF-beta1 synthesis with oligonucleotides or its activity with specific antibodies resulted in a decreased magnitude of HGF fibroblast proliferation. CONCLUSION: These results are consistent with the existence of an autocrine role of TGF-beta1 as a stimulator of HGF fibroblast proliferation.