哺乳动物的雷帕霉素靶蛋白及其底物在口腔鳞状细胞癌中的表达

目的　探讨哺乳动物的雷帕霉素靶蛋白(mTOR)及其底物对口腔鳞状细胞癌生长的影响。方法　提取 RNA,通过RT-PCR和Western印迹分析观察mTOR及其底物p70 S6激酶(p70 S6k)的α1、α2、β1、β2不同亚型及起始因子4E结合蛋白1(4EBP1)在口腔鳞状细胞癌中的表达。结果　RT-PCR与Western印迹的结果一致。随着肿瘤恶性度的提高,mTOR、p70 S6K的表达明显增加,而4EBP1的表达则下降。结论　mTOR及其底物表达水平的强弱与口腔鳞状细胞癌的分化程度密切相关,它可能成为未来癌症治疗的重要靶向蛋白。     

p53 gene status and expression of p53, MDM2, and p14ARF proteins in ameloblastomas

BACKGROUND: To clarify the roles of the p53-MDM2-p14(ARF) cell cycle regulation system in oncogenesis and cytodifferentiation of odontogenic tumors, p53 gene status and expression of p53, MDM2, and p14(ARF) proteins was analyzed in ameloblastomas as well as tooth germs. METHODS: Paraffin sections of 16 tooth germs and 46 benign and 5 malignant ameloblastomas were examined immunohistochemically for the expression of p53, MDM2, and p14(ARF) proteins. Frozen tissue samples of 10 benign ameloblastomas and 1 malignant (metastasizing) ameloblastoma were analyzed by direct DNA sequencing to detect p53 gene alteration. RESULTS: Immunohistochemical reactivity for p53 was detected in 2 of 13 tooth germs, 13 of 29 ameloblastomas, and 5 of 5 malignant ameloblastomas, and the expression ratio of p53 in tooth germs was significantly lower than those in benign and malignant ameloblastomas. Direct DNA sequencing showed no alteration of p53 gene exons 5-8 in any sample of 10 benign ameloblastomas and 1 metastasizing ameloblastoma. Expression of MDM2 and p14(ARF) was detected in all samples of normal and neoplastic odontogenic epithelium, and the expression ratios in tooth germs tended to be lower than those in benign and malignant ameloblastomas. In ameloblastomas, expression of p53, MDM2, and p14(ARF) was significantly higher in plexiform cases than in follicular cases. Markedly decreased reactivity for p53, MDM2, and p14(ARF) was detected in keratinizing and granular cells in ameloblastoma subtypes. Basal cell ameloblastoma showed slightly higher reactivity for p53, MDM2, and p14(ARF) as compared with other subtypes. CONCLUSION: Elevated expression of p53, MDM2, and p14(ARF) in benign and malignant ameloblastomas suggests that alteration of the p53-MDM2-p14(ARF) cascade is involved in oncogenesis and/of malignant transformation of odontogenic epithelium. p53 gene status implied that p53 mutation might play a minor role in neoplastic changes of odontogenic epithelium. Immunoreactivity for p53, MDM2, and p14(ARF) in ameloblastoma variants suggests that these factors might be associated with tissue structuring and cytodifferentiation of ameloblastomas.     

Distribution of blood group nantigens A, B and H in ameloblastomas

Abstract – The histologic distribution of blood group antigens A, B and H was examined in ameloblasromas and normal mucosa from 15 patients. The blood group antigens were demonstreated with a semiquantitative immunofluorescence staining method. Blood group antigens A and B were absent in the semiquantitative immunofluorescence staining method. Blood group antigens A and B were absent in the ameloblastomas, whereas the spinous cells of the oral mucosal epithelium showed a positive reaction in correspondence with the patient's blood group. Blood group antigen H was demonstrated in 14 of 15 from these patients blood group antigen H in all cases was located to the cell membranes of the spinous cells. The present findings suggest that a difference in ability to synthesize blood group antigen H and the blood group antigens A and B exists in the amelobiastoma.     

PI3K/AKT/mTOR/p70S6K通路在人舌鳞癌细胞耐药中的作用

目的:通过抑制舌鳞癌细胞中PI3K/AKT/mTOR/p70S6K信号通路的表达,检测细胞凋亡与自噬的变化,探讨舌鳞癌耐药的机制。方法:以舌鳞癌细胞Cal27与舌鳞癌耐顺铂细胞Cal27/CDDP为研究对象。分别用PI3K/AKT抑制剂LY294002、mTOR抑制剂Rapamycin、p70S6K抑制剂LY2584702作用该通路各个环节。Western blot检测通路抑制剂作用后相关蛋白的变化。Cyto-ID荧光染色检测自噬体的形成。流式细胞术检测细胞凋亡水平。结果:Western blot结果显示加入抑制剂后舌鳞癌细胞Beclin1表达分别高于对照组,LC3II与LC3I以及Bax与Bcl-2的比值均升高,该通路的p-AKT、p-mTOR、p-p70S6K等蛋白均有下降(P<0.05)。流式结果显示加入抑制剂后的舌鳞癌细胞凋亡率分别较对照组升高(P<0.05)。Cyto-ID荧光染色后结果显示加入抑制剂后的舌鳞癌细胞自噬小体的数目明显高于对照组(P<0.05)。对比两组细胞显示加入抑制剂后的Cal27细胞发生凋亡与自噬的水平高于Cal27/CDDP(P<0.05)。结论:抑制PI3K/AKT/mTOR/p70S6K信号通路可诱导舌鳞癌细胞Cal27与舌鳞癌耐顺铂细胞Cal27/CDDP发生凋亡与自噬,激活的PI3K/AKT/mTOR/p70S6K通路抑制细胞凋亡与自噬是Cal27/CDDP细胞产生顺铂耐药的原因之一。     

丝/苏氨酸蛋白激酶/哺乳动物雷帕霉素靶蛋白/p70 S6K信号通路在口腔鳞癌中的表达及临床意义

目的 探讨丝/苏氨酸蛋白激酶（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）/p70 S6K信号通路在口腔鳞癌组织中的表达情况,为口腔鳞癌的早期诊断和治疗提供参考。方法 收集口腔鳞癌标本51例,癌旁黏膜组织10例,正常口腔黏膜10例。采用免疫组织化学SP法检测口腔鳞癌、癌旁黏膜组织及正常口腔黏膜中p-Akt、p-mTOR及p70 S6K的表达情况,分析三者相互之间表达的相关性。结果 p-Akt、p-mTOR及p70 S6K在口腔鳞癌组中的表达显著高于正常口腔黏膜组和癌旁黏膜组的表达。p-Akt、p-mTOR及p70 S6K在口腔鳞癌的表达与患者的年龄、性别及临床分期无相关性,但与口腔鳞癌的分化程度及有无淋巴结转移有相关性。p-Akt、p-mTOR及p70 S6K在口腔鳞癌的表达相互之间具有较强的正相关关系。结论 Akt/mTOR/p70 S6K信号通路分子在口腔鳞癌中表达活跃,提示可能与口腔鳞癌的发生发展具有重要的相关性。     

Expression of bone morphogenetic proteins and their associated molecules in ameloblastomas and adenomatoid odontogenic tumors

OBJECTIVE: To further clarify the roles of regulators of embryonic development, bone morphogenetic protein (BMPs) and their associated molecules, in oncogenesis and cytodifferentiation of odontogenic tumors, the expression of these regulator molecules were analyzed in epithelial odontogenic tumors as well as in tooth germs. MATERIALS AND METHODS: Tooth germs, ameloblastomas, adenomatoid odontogenic tumors, and malignant ameloblastomas were examined by RT-PCR and immunohistochemistry for detection of BMP-2, -4, -7, BMP receptors I and II (BMPR-I, BMPR-II), core-binding factor alpha1 (CBFA1), and osterix. RESULTS: mRNA expression of BMPs, BMPRs, CBFA1, and osterix was detected in all odontogenic tissues. Immunohistochemical reactivity for BMPs, BMPRs, and CBFA1 was detected in both epithelial and mesenchymal cells of tooth germs and epithelial odontogenic tumors. BMPs and BMPRs were evidently expressed in odontogenic epithelial cells in tooth germs and epithelial odontogenic tumors. Acanthomatous ameloblastomas showed increased BMP-7 reactivity in keratinizing cells. Nuclear CBFA1 expression was detected scatteredly in odontogenic epithelial cells in normal and neoplastic odontogenic tissues, as well as in some mesenchymal cells in tooth germs and in some stromal cells in epithelial odontogenic tumors. Ameloblastic carcinomas showed low reactivity for BMPs, BMPRs, and CBFA1. CONCLUSION: BMPs and their associated molecules might play a role in cytodifferentiation of normal and neoplastic odontogenic epithelium via epithelial-mesenchymal interactions.     

正畸力作用下兔牙周组织中雷帕霉素靶蛋白和核糖体蛋白S6激酶的表达

目的探讨正畸牙移动过程中雷帕霉素靶蛋白（mTOR）和核糖体蛋白S6激酶（p70 S6K）在牙周组织改建中的作用。方法选用24只日本大耳白兔建立正畸牙移动的动物模型,将实验动物上颌右侧戴矫治器,作为实验侧;左侧未戴矫治器,作为对照侧。分别在戴矫治器后3、5、7、14 d各处死6只实验动物。采用苏木精-伊红染色、逆转录聚合酶链反应（RT-PCR）及Western blot免疫印迹分析方法观察牙周组织形态学变化及mTOR和p70 S6K表达的变化。结果组织形态学观察可见,实验侧牙周组织较对照侧有明显改建,牙槽骨由致密变得疏松,细胞的排列由有序变得紊乱,牙槽骨的骨壁由平整变得凹凸不平,出现了破骨细胞及骨陷窝。RT-PCR结果显示,加力3 d后牙周组织中p70 S6K mRNA表达增强,7 d后牙周组织中p70 S6K mRNA明显增强,随后缓慢下降,与对照侧相比,其差异有统计学意义（P＜0.01）。Western blot免疫印迹分析与RT-PCR结果一致。结论在正畸牙移动过程中,兔牙周组织中mTOR和p70 S6K的表达明显增强,提示mTOR和p70 S6K参与牙周组织改建,并在牙周组织改建中起重要作用。     

口腔鳞状细胞癌中WWP2、PTEN和p70S6K的表达及相关性研究

目的探讨WWP2、第十号染色体缺失与张力蛋白同源的磷酸酶基因（PTEN）和p70核糖体蛋白S6激酶（p70S6K）蛋白在口腔鳞状细胞癌（OSCC）组织中的差异表达及相关性,以期为其早期防治及生物治疗提供参考。 方法应用免疫组化检测12例正常口腔黏膜、20例白斑及54例OSCC组织中WWP2、PTEN和p70S6K蛋白的表达和相关性,并分析其与OSCC组中患者临床病理参数之间的关系。实验数据采用SPSS 16.0统计软件进行Kruskal Wallis test、χ²检验和Fisher′s精确检验,WWP2、PTEN和p70S6K的表达两两进行Spearman等级相关分析。 结果WWP2、PTEN和p70S6K在正常对照组、口腔白斑组及OSCC组中的强表达率分别为33.33%、40%和68.52%;91.67%、85%和48.15%以及25%、45%和75.93%,与其他组相比,以上三种蛋白的表达差异均有统计学意义（P<0.05）。相关性分析表明WWP2与PTEN之间呈负相关（r = -0.236,P = 0.028）。PTEN与p70S6K之间呈负相关（r = -0.301,P = 0.005）。WWP2与p70S6K之间呈正相关关系（r = 0.315,P = 0.003）。OSCC组织中WWP2与OSCC的临床分期有相关性,p70S6K与OSCC的分化程度和有无淋巴结转移有相关性（P<0.05）。 结论WWP2、PTEN和p70S6K在口腔黏膜癌变过程的各阶段的组织中差异表达,提示可能在OSCC的发生、发展过程中起重要作用。     

小檗碱对牙髓干细胞MAPK/ERK信号通路及成牙本质向分化的影响研究

目的 探讨小檗碱对牙髓干细胞(dental pulp stem cells,DPSCs)成牙本质向分化及p38丝裂原活化蛋白激酶(MAPK)/细胞外调节蛋白激酶(ERK)信号通路的影响.方法 采用酶消化法提取DPSCs,将培养鉴定的DPSCs传代培养至第3～5代,用于以下实验.实验分为对照组(正常培养DPSCs)、矿化...     

Expression of osteonectin/secreted protein acidic and rich in cysteine and matrix metalloproteinases in ameloblastoma

J Oral Pathol Med (2010) 39 : 242–249 Background: Ameloblastoma is the most common clinically‐significant epithelial odontogenic tumor, and is considered a benign but locally‐aggressive tumor of the craniofacial region. Osteonectin/secreted protein acidic and rich in cysteine (SPARC) is induced in response to a number of biological processes such as tumor growth and metastasis, whereas matrix metalloproteinases (MMPs) degrade the extracellular matrix and participate in various biological processes including tumor invasion and metastasis. We hypothesize that SPARC acts with MMPs for the local invasiveness of ameloblastoma. The aim of this study was to examine the association of SPARC with MMP‐1, MMP‐2, and MMP‐9 in ameloblastoma. Method: Immunohistochemical expression of SPARC, MMP‐1, MMP‐2, and MMP‐9 as well as co‐expression of SPARC and MMP‐9 were examined in a cohort of 23 cases of ameloblastoma. Results: SPARC, MMP‐1, ‐2, and ‐9 were detected in the cytoplasm of the ameloblastic‐like columnar cells and stellate‐reticulum‐like cells as well as in the stromal tissues of fibroblasts and endothelial cells of our cohort of ameloblastoma patients. Furthermore, co‐expression of SPARC and MMP‐9 were found in 23 cases of ameloblastoma. This may be the first study to demonstrate that the expression level of SPARC was statistically correlated with MMP‐9 but not with MMP‐1 or ‐2 in ameloblastoma. Conclusion: Our results suggest a putative association between SPARC and MMPs (especially MMP‐9) in ameloblastoma to regulate tumor invasion.     

抑癌基因p16在成釉细胞瘤及牙源性角化囊肿中表达的研究

目的 研究成釉细胞瘤（Ameloblastoma,AB）中 p16蛋白及 p16 mRNA表达,探讨其生物学意义。方法 应用免疫组化方法（SP）检测40例 AB及牙源性角化囊肿（Odontogenic keratocyst,OKC）7例,基底细胞癌（Basal cell carcinoma,BCC）8例 p16蛋白的表达,同时用原位杂交法检测 p16 mRNA在20例 AB中表达。结果 40例AB,7例 OKC,8例 BCC,p16蛋白缺失表达分别为70％,85.7％,100％。p16mRNA缺失率80.5％,与蛋白缺失表达一致率53.8％。结论 （1）AB与 OKC的发生与p16蛋白缺失有关。（2）AB中原发与复发比,原发与恶变比 p16蛋白缺失率不同（P<0.05）。（3）AB及 OKC,BCC三种不同病变经统计学处理 P>0.05,末见明显相关性。（4）在 AB中p16蛋白与 p16 mRNA表达 P<0.05,具有一定相关性。     

Expression of endothelial monocyte-activating polypeptide II in the rat dental follicle and its potential role in tooth eruption

Endothelial monocyte-activating polypeptide II (EMAP-II) is an inflammatory cytokine with chemotactic activity. Because the dental follicle (DF) recruits mononuclear cells (osteoclast precursors) to promote the osteoclastogenesis needed for tooth eruption, it was the aim of this study to determine if EMAP-II contributes to this recruitment. Using a DNA microarray, EMAP-II was found to be highly expressed in vivo in the DFs of day 1 to day 11 postnatal rats, with its expression elevated on days 1 and 3. Use of a short interfering RNA (siRNA) to knock down EMAP-II expression resulted in a reduction in the expression of colony-stimulating factor-1 ( CSF-1) and monocyte chemoattractant protein-1 ( MCP-1) in the DF cells. Addition of EMAP-II protein to the DF cells partially restored the expression of CSF-1 and MCP-1. In chemotaxis assays using either conditioned medium of the DF cells with anti-(EMAP-II) immunoglobulin G added or conditioned medium of DF cells with EMAP-II knocked down by siRNA, migration indexes of bone marrow mononuclear cells were significantly reduced. These results suggest that EMAP-II is another chemotactic molecule in the dental follicle involved in the recruitment of mononuclear cells, and that EMAP-II may exert its chemotactic function directly by recruiting mononuclear cells and indirectly by enhancing the expression of other chemotactic molecules (CSF-1 and MCP-1).     

Hippo/YAP信号通路与细胞增殖相关信号通路交叉作用的研究进展

Hippo/YAP信号通路首次被发现于果蝇中,在哺乳动物中具有高度保守的特性,可通过直接或间接地调节细胞增殖、程序性细胞死亡以起到平衡机体内环境稳态、维持器官大小的作用.YAP作为Hippo信号通路中的一个转录共激活因子,与磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)、Wnt/β-联蛋白、细胞外信号调节激酶(ERK) 1/2等通路中相关分子相互作用,使得Hippo通路与其他增殖调控相关信号通路形成“交通”,构成一个复杂的信号通路网,共同调控细胞的增殖.本文就目前对Hippo信号通路中共激活因子YAP与增殖调控信号通路间的交叉影响及其机制的研究进展作一综述.     

Demonstration of a K+ -stimulated and ouabain-sensitive p-nitrophenyl phosphatase activity in enamel- and dentin-forming tissues in the rat

abstract — Nitrophenyl phosphate hydrolysis was studied at neutral pH with tissue preparations of the rat secretory and maturation enamel organs and denial pulp. By introduction of inhibitors to nonspecific alkaline phosphatase activity and stimulants to the K⁺stiinulated and ouabain-sensitive p -nitrophenyl phosphatase activity, the latter-enzyme activity could be demonstrated. This enzyme activity is generally held to be representative of the enzyme sodium- and potassium-stimulated adenosine triphosphatase. The K⁺-stimulated activity was magnesium dependent and highly sensitive to fluoride. It was inhibited completely by 3 mM fluoride in the incubation medium and about 1 mM produced hall the maximum inhibition. The K⁺-independent enzyme activity was inhibited 50–60% by fluoride in concentrations between 3 and 15 mM. The high fluoride sensitivity of the K⁺-stimulated activity may perhaps help to explain the vulnerability of dental tissues to fluoride.     

Adenosine regulates the production of interleukin-6 by human gingival fibroblasts via cyclic AMP/protein kinase A pathway

Adenosine has been reported to alter a variety functions of the cells that participate in inflammatory responses. However, the effect(s) of adenosine on human gingival fibroblasts (HGF), one of the immunomodulator cells in inflamed periodontal lesions, remains to be established. In this study, we examined the influence of adenosine on the production of interleukin (IL)‐6 by HGF. Ligation of adenosine receptors with adenosine or its related analogue, 2‐chloroadenosine (2‐CADO), increased IL‐6 production by HGF without any other stimuli. In addition, adenosine and 2‐CADO enhanced the cyclic AMP (cAMP) level in HGF as did prostaglandin E₁(PGE₁) and forskolin. Interestingly, these cAMP‐arising reagents and the permeable cAMP analogue, dibutyryl cAMP (dbtcAMP), also increased IL‐6 production by HGF. These results suggest that cAMP is involved in adenosine‐ induced IL‐6 production by HGF. Adenosine‐induced IL‐6 production was suppressed by protein kinase A (PKA) inhibitor, H89, indicating that cAMP/PKA pathway is involved in the induction. Moreover, the experiments using antagonists specific for adenosine receptor subtypes revealed that the adenosine‐induced IL‐6 production by HGF was, at least in part, mediated by the adenosine A2b receptor. These results provide new evidence for the possible effects of adenosine or its related analogue as an immunomodulator in inflammatory periodontal lesions.     

p53 and p21WAF1/CIP1 overexpression at the invasive front of lower lip squamous cell carcinoma

BACKGROUND: Lower lip squamous cell carcinoma (LLSCC) is an oral cancer that has distinct epidemiology and etiopathogenesis. Although risk factors for this neoplasia are acknowledged, few studies have investigated the molecular basis of its development and behavior. METHODS: Expression of p53 and p21(WAF1/CIP1) was examined by immunohistochemistry of archived tissue from 21 specimens of LLSCC. Differences in this expression between the whole tumor (WT) and the invasive front (IF) as well as correlation between p53 and p21(WAF1/CIP1) expression were analyzed. RESULTS: p53 and p21(WAF1/CIP1) were overexpressed at the IF of LLSCC. The expression of both proteins was higher at IF than at WT. No correlation was observed between p53 and p21(WAF1/CIP1) expression. CONCLUSIONS: Our results indicate that p53 and p21(WAF1/CIP1) overexpression is important in LLSCC pathogenesis, reinforce that IF is the most important area for tumor behavior, and support that p53-independent mechanisms should be involved in the expression of p21(WAF1/CIP1).