放疗早期对口腔颌面部肿瘤患者免疫功能影响的临床研究

目的 :通过对患者放疗后早期与放疗前的血液,进行免疫细胞检测及外周血基因表达谱芯片检测,分析放疗早期对机体免疫功能的影响。方法:22例口腔颌面肿瘤患者,检测放疗前1周和后1周的血液中,CD3+T、CD4+T、CD8+T、B、NK细胞数量及比例变化。通过表达谱芯片检测筛选放疗前后血液标本的差异基因,用R语言及DAVID数据库行功能分类和信号传导通路相关性分析。结果:患者淋巴细胞及其亚类数目在放疗后明显下降。表达谱芯片筛选出368个免疫炎症相关基因。涉及的信号通路主要是T细胞受体信号通路、黏附分子、NK介导细胞毒效应等相关通路。结论:放疗能部分激活CD8+T及NK细胞介导的杀伤免疫,同时抑制T细胞和NK细胞的分化及其信号传导通路。     

牙周炎组织中miRNA表达谱差异筛选及功能预测分析

目的 筛选牙周炎患者组织中的差异表达miRNA,探讨其生物学功能以及参与的信号通路。方法 通过对微阵列数据库GSE54710中的158例牙周炎患者和40例健康人的牙龈组织中的基因芯片数据进行生物信息学分析,筛选差异表达miRNA,并预测参与的生物学功能和信号通路。采用SPSS 19.0软件包对数据进行统计学分析。结果 5种miRNAs（hsa-miR-451、hsa-miR-223、hsa-miR-486-5p、hsa-miR-3917、hsa-miR-671-5p）显著上调,4种miRNAs（hsa-miR-203、hsa-miR-210、hsa-miR-1246、hsa-miR-1260 ）显著下调。其中,hsa-miR-1260的靶基因584个,hsa-miR-451的靶基因139个。KEGG通路富集分析显示,hsa -miR-1260靶基因显著富集到TGF-beta等12条信号通路,hsa-miR-451靶基因显著富集到17条信号通路。结论 得到牙周炎组织中miRNAs的表达谱,牙周炎诱导的hsa-miR-1260和hsa-miR-451可能在牙周炎的生理病理学中起到关键作用。     

Treg 细胞介导的免疫应答在牙周炎小鼠中的研究

目的:研究牙周炎小鼠中调节性T(Treg)细胞介导的免疫应答情况。方法:将8只7周龄C57BL/6雌性小鼠随机分为2组(n=4)。通过口腔涂抹牙龈卟啉单胞菌(P.gingivalis)建立牙周炎模型,对照组口腔涂抹羧甲基纤维素。涂抹结束后第4周处死小鼠,流式细胞仪检测CD4+叉头翼螺旋转录分子(Foxp3)+细胞;ELISA检测转化生长因子(TGF)-β1和白细胞介素(IL)-10的表达。结果:牙周炎小鼠牙龈、颈部淋巴结和外周血中CD4+Foxp3+细胞在CD4+T细胞中的比例降低(P<0.01)。牙周炎小鼠TGF-β1在牙龈组织中的表达以及IL-10在牙龈、颈部淋巴结和血清中的表达增高(P<0.05)。结论:在牙周炎的发病过程中,Treg细胞介导的免疫应答减弱,牙龈、颈部淋巴结和外周血可能是牙周炎中Treg细胞主要的细胞来源。     

辅助性T17细胞在牙周炎小鼠中的免疫状态研究

目的 研究辅助性T（Th）17细胞在牙周炎小鼠中的免疫状态。方法 将7周龄C57BL/6雌性小鼠随机分为牙周炎组和对照组,每组4只。牙周炎组采用口腔涂抹牙龈卟啉单胞菌（P. gingivalis）的方法建立牙周炎动物模型。对照组涂抹PBS液。在涂抹结束后的第4周取材,流式细胞仪检测CD4+维甲酸相关核孤儿受体（ROR）γτ+（Th17）细胞;酶联免疫吸附（ELISA）检测Th17细胞相关的细胞因子白细胞介素（IL）-17A的蛋白表达。结果 牙周炎小鼠牙龈组织、颈部淋巴结和外周血中CD4+ RORγτ+（Th17）细胞在总CD4+ T细胞中的比例和细胞数量显著高于对照组（P<0.01）。与对照组相比,牙周炎组IL-17A的表达增加（P<0.05）。结论 在牙周炎的发生发展中,Th17细胞介导的细胞免疫应答增强,牙龈组织、颈部淋巴结和外周血可能是Th17细胞介导免疫应答的主要场所。     

牙周炎与急性心肌梗死串扰基因的生物信息学分析

目的 基于生物信息学方式探究牙周炎与急性心肌梗死之间潜在的串扰基因、共享的信号通路,揭示两疾病之间潜在的病理生理学关系。方法 从GEO数据库中下载牙周炎基因芯片数据集GSE16134与急性心肌梗死基因芯片数据集GSE66360,利用limma包筛选出两疾病的差异表达基因,取交集后获得串扰基因。对串扰基因进行基因本体论和京都基因与基因组百科全书富集分析;通过STRING数据库建立串扰基因的蛋白质互作网络,利用Cytoscape软件中的Cytohubba插件筛选关键基因（hub基因）;绘制受试者工作特征曲线,检验hub基因诊断价值。结果 筛选出41个串扰基因,主要富集在对细菌源分子的反应,对脂多糖的反应,中性粒细胞迁移等生物学功能,以及脂质与动脉粥样硬化,NF-κB,百日咳等信号通路;PPI网络中筛选了10个相互作用程度较高的hub基因,分别为：IL-1β,CXCL8,FCGR3B,PECAM1,MMP9,CD14,CXCL1,HCK,LYN,CSF2RB。其中IL-1β与CXCL8在两疾病中均具有良好的诊断价值。结论 IL-1β与CXCL8是牙周炎与心肌梗死之间重要的串扰基因,可能在牙周...     

牙周炎宿主转化生长因子-β^+调节性B细胞变化特点

目的:探讨牙周炎时宿主不同组织内转化生长因子β(transforming growth factor-β,TGF-β)+调节性B细胞的变化特点,比较健康个体与牙周炎模型之间的差异,分析其潜在的作用机制,为进一步揭示牙周炎病理机制提供理论支持。方法:采用SD大鼠建立实验性牙周炎病理模型。采用流式细胞术分析各组不同组织内TGF-β^+调节性B细胞占总淋巴细胞的比例,采用实时荧光定量聚合酶链反应(Real-time polymerase chain reaction,Real-time PCR)检测各样本中B细胞发育相关因子B细胞活化因子(B-cellactivatingfactor,BAFF)、增殖诱导配体(aproliferation-inducingligand,APRIL)及白细胞介素-33(interleukin-33,IL-33)的表达水平。结果:与健康组比较,牙周炎大鼠牙龈中TGF-β^+调节性B细胞比例显著上调(P<0.05);而在外周血中两者比较差异无统计学意义(P>0.05)。在牙龈中,牙周炎大鼠各检测基因的相对表达水平与健康组比较差异显著(P<0.05);在外周血中,牙周炎大鼠与健康组各检测基因的相对表达水平比较差异无统计学意义(P>0.05)。结论:牙周炎时宿主牙龈中TGF-β^+调节性B细胞上调,这与局部BAFF、APRIL及IL-33等细胞因子表达水平呈正相关。     

坏死性凋亡对牙周炎免疫微环境的影响机制初探

目的：探索坏死性凋亡对牙周炎免疫微环境的影响及机制初探。方法：筛选出牙周炎中差异表达的坏死性凋亡相关基因，通过机器学习算法计算其中的核心基因并构建诊断模型。分别分析牙周炎与健康组中的免疫细胞相对丰度，并计算其与坏死性凋亡相关基因的相关性。收集人健康牙龈与炎症牙龈组织，并提取其RNA，通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction,RT-qPCR)验证坏死性凋亡核心基因的组间表达。结果：共有10个坏死性凋亡相关基因差异表达，其中核心基因共有8个，炎症组织相对于健康组织，浆细胞、B细胞、中性粒细胞与NK细胞含量升高，而巨噬细胞、休眠树突状细胞含量下降。其中，混合系激酶域样伪激酶（mixed lineage kinase domain like pseudokinase,MLKL)、黑色素瘤缺乏因子2 (absent in melanoma 2,AIM2)与浆细胞表达量呈显著正相关。结论：坏死性凋亡对牙周炎免疫微环境存在影响。     

老年牙周炎牙龈组织Fas/Apo-1(CD95)抗原表达的免疫组织化学研究

目的 :观察慢性老年牙周炎牙龈组织中Fas/Apo - 1(CD95 )抗原表达和分布情况。方法 :采用免疫组织化学染色方法对 10例慢性老年牙周炎患者、10例慢性成人牙周炎患者、10例青少年牙周炎患者和 10例健康老年人牙龈组织中Fas/Apo - 1(CD95 )抗原阳性表达和分布情况进行了观察和比较。结果 :在慢性老年牙周炎组完整的牙龈鳞状上皮间桥、核周胞浆Fas/Apo - 1(CD95 )抗原阳性表达和结缔组织中淋巴细胞Fas/Apo - 1(CD95 )抗原阳性表达为 4组中最强 ;健康老年人组上皮角化层Fas/Apo - 1(CD95 )抗原阳性表达为 4组中最强 ;慢性成人牙周炎组和青少年牙周炎组结缔组织中淋巴细胞和吞噬细胞Fas/Apo - 1(CD95 )抗原阳性表达强于健康老年人组 ;慢性老年牙周炎组上皮细胞和上皮细胞的细胞间桥Fas/Apo - 1(CD95 )抗原阳性表达例数明显高于慢性成人牙周炎组和青少年牙周炎组 (P <0 .0 5 ) ;健康老年人组结缔组织中淋巴细胞和吞噬细胞Fas/Apo - 1(CD95 )抗原阳性表达例数明显低于其它 3组 (P <0 .0 5 )。结论 :由于感染性炎症和衰老的影响 ,①牙周炎患者和健康老年人牙龈组织中鳞状上皮凋亡细胞发生部位与身体其他器官鳞状上皮细胞凋亡发生部位不同 ;②在慢性老年牙周炎患者牙龈组织中上皮细胞和炎性细胞对细胞凋亡易感性     

慢性牙周炎牙龈组织细胞凋亡及凋亡相关蛋白Caspase-3的表达

目的:研究慢性牙周炎牙龈组织中细胞凋亡的发生情况和Caspase-3蛋白的表达,探讨其在慢性牙周炎病变发生中的意义。方法:应用脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记法(TUNEL法)、免疫组织化学方法检测21例慢性牙周炎牙龈组织和21例健康牙龈组织中的细胞凋亡指数(apoptosis index,AI)及Caspase-3蛋白的表达。结果:慢性牙周炎组牙龈组织中细胞凋亡指数明显高于正常对照组(P<0.05)。与正常组相比,慢性牙周炎组牙龈组织中细胞caspase-3表达明显增强,两组间有显著性差异(P<0.05)。结论:慢性牙周炎病人牙龈组织细胞发生凋亡,且通过激活细胞凋亡信号传导途径中的Caspase-3而导致慢性牙周炎发生。     

牙龈卟啉单胞菌脂多糖影响单核细胞表达Th17细胞分化相关因子的研究

目的:探讨牙龈卟啉单胞菌脂多糖对人外周血CD14+单核细胞表达Th17细胞分化相关细胞因子IL-1β、IL-6、IL-23和TGF-β的影响。方法:采用脂多糖提取试剂盒提取牙龈卟啉单胞菌标准株ATCC33277和高毒力株W83脂多糖;免疫磁珠法分选健康志愿者外周血CD14+单核细胞。以大肠杆菌脂多糖为对照,1μg/ml牙龈卟啉单胞菌脂多糖处理单核细胞,分别培养12、24、36h后收集细胞和上清液,实时定量PCR及ELISA法检测IL-1β、IL-6、IL-23、TGF-βm R NA和蛋白水平的表达。结果:各型脂多糖均可显著上调单核细胞四种细胞因子m RNA及蛋白水平的表达;上调作用具有时间效应,在12h达到高峰。并且三种菌株脂多糖对不同细胞因子表达的影响存在一定差异。结论:牙龈卟啉单胞菌脂多糖可显著上调CD14+单核细胞表达IL-1β,IL-6,IL-23,TGF-β,可能与牙周炎症环境中Th 17细胞的分化相关。     

口腔鳞状细胞癌预后相关基因标志物的分析

［摘要］ 目的 通过生物信息分析途径对口腔鳞状细胞癌患者与正常人群的差异基因进行分析,从分子水平探讨关键基因以及参与的信号通路,初步探索口腔鳞状细胞癌发生、发展的基因标志物。方法 从公共数据库基因表达数据库（GEO）中下载口腔鳞状细胞癌的相关芯片数据（GSE3524和GSE6631）,筛选出口腔鳞状细胞癌组织和对照组织表达有显著差异的基因。并对其功能及预后进行分析。结果 共筛选出129个差异表达基因,其中表达上调45个,下调84个,对其进行基因本体、京都基因与基因组百科全书和蛋白互作网络分析,发现分泌型焦磷酸蛋白（secreted phosphoprotein 1, SPP1）、金属基质蛋白酶（matrix metalloproteinase 1,MMP1）、丝氨酸蛋白酶抑制剂（serpin family E member 1,SERPINE1）、纤溶酶原激活剂（plasminogen activator urokinase,PLAU）处于基因核心节点位置。同时,根据这些关键基因表达水平的高低对口腔鳞状细胞癌患者进行分组,高表达组患者生存时间均低于低表达组,差异有统计学意义（PSPP1=0.045、PMMP1=0.046、PSERPINE1=0.0024和PPLAU=0.00049）。结论 基因组学分析方法筛选出的关键基因和信号通路有助于研究口腔鳞状细胞癌发生、发展的机制,也进一步为治疗靶点及预后分子标志物的选择提供了依据。     

Immunohistochemical localization of Toll‐like receptors 1–10 in periodontitis

Background/aim: In periodontitis, bacteria and pathogen‐associated molecular patterns are sensed by Toll‐like receptors (TLRs), which initiate intracellular signaling cascades that may lead to host inflammation. In this study, the expression and distribution of TLRs (TLR‐1 to TLR‐10) were immunohistochemically detected in gingival epithelium and connective tissue. Methods: Immunohistochemistry was used for the localization of TLRs in gingival tissue samples from 10 patients with chronic periodontitis and 10 healthy controls; these samples were obtained during routine periodontal flap operations and during extraction operations performed for retained wisdom teeth, respectively. For the evaluation, epithelial layers were stratified to basal, spinous, and superficial layers, and the percentages of TLR‐positive cells were determined. Results: Both healthy and periodontitis gingival tissues expressed all TLRs except TLR‐10. In patients with periodontitis, epithelial cells showed increased TLR expression towards the basal layer. Healthy controls showed more variable cellular TLR expression and distribution between the layers of epithelium. In the connective tissue, consistently higher TLR expression was found within the periodontitis group compared to the healthy group. Conclusions: For the first time, the cellular expression and distribution of TLR‐1 to TLR‐10 have been studied in periodontitis, indicating that TLR‐1 to TLR‐9 are differentially expressed both in connective tissue and epithelial layers. Except for TLR‐7 and TLR‐8, all the other TLRs showed statistically significant differences between patients with periodontitis and healthy controls, suggesting their involvement in the pathogenesis of periodontitis.     

Immunohistochemical localization of Toll-like receptors 1-10 in periodontitis

Background/aim:  In periodontitis, bacteria and pathogen-associated molecular patterns are sensed by Toll-like receptors (TLRs), which initiate intracellular signaling cascades that may lead to host inflammation. In this study, the expression and distribution of TLRs (TLR-1 to TLR-10) were immunohistochemically detected in gingival epithelium and connective tissue.
Methods:  Immunohistochemistry was used for the localization of TLRs in gingival tissue samples from 10 patients with chronic periodontitis and 10 healthy controls; these samples were obtained during routine periodontal flap operations and during extraction operations performed for retained wisdom teeth, respectively. For the evaluation, epithelial layers were stratified to basal, spinous, and superficial layers, and the percentages of TLR-positive cells were determined.
Results:  Both healthy and periodontitis gingival tissues expressed all TLRs except TLR-10. In patients with periodontitis, epithelial cells showed increased TLR expression towards the basal layer. Healthy controls showed more variable cellular TLR expression and distribution between the layers of epithelium. In the connective tissue, consistently higher TLR expression was found within the periodontitis group compared to the healthy group.
Conclusions:  For the first time, the cellular expression and distribution of TLR-1 to TLR-10 have been studied in periodontitis, indicating that TLR-1 to TLR-9 are differentially expressed both in connective tissue and epithelial layers. Except for TLR-7 and TLR-8, all the other TLRs showed statistically significant differences between patients with periodontitis and healthy controls, suggesting their involvement in the pathogenesis of periodontitis.     

根尖周炎动物模型中CD14、TLR4的表达

目的研究大鼠根尖周炎炎症组织中脂多糖炎症信号受体CD14、Toll样受体4（Toll．1ikereceptor4，TLR4）的表达特点，探讨根尖周炎中脂多糖的信号转导途径。方法建立大鼠磨牙内毒素根尖周炎模型，采用免疫组化染色观察根尖周炎炎症组织中CD14、TLR4的表达情况，并计算CD14、TLR4的阳性细胞率。结果正常根尖周组织中未发现CD14和TLR4免疫阳性细胞，根尖周炎症组织中CD14和TLR4表达阳性，CD14、TLR4阳性细胞率差异无统计学意义（P〉0．05）。结论与正常根尖周组织相比，炎症根尖周组织中CD14和TLR4的表达显著增强，CD14和TLR4的表达量差异无统计学意义，提示脂多糖可能通过CD14、TLR4信号受体在根尖周炎症中发挥作用。     

基于RNA-Seq分析挖掘唾液腺多形性腺瘤核心基因及通路

目的：筛选多形性腺瘤（pleomorphic adenoma,PA）与瘤旁唾液腺腺体间的差异表达基因,挖掘PA形成过程中的核心基因及通路。方法：采用RNA-Seq技术,检测5例PA患者的配对肿瘤与瘤旁唾液腺腺体,筛选2组间差异基因。运用蛋白互作数据库STRING分析预测差异基因所编码蛋白间的相互作用。筛选互作网络中的核心模块及核心基因,分析核心模块中激活的信号通路,预测上述模块与PLAG1可能的相互作用关系。RT-PCR验证核心基因在20例PA患者的配对肿瘤组织及瘤旁唾液腺中的表达。采用SPSS 20.0软件包对数据进行统计学分析。结果：共测得3810个差异基因,其中2021个下调,1789个上调。核心模块中存在PI3K-AKT信号通路、ER受体信号通路、Rap1信号通路、cGMP-PKG信号通路的激活。PLAG1可能通过PHLPP1、LRRK2、β-catenin蛋白与核心模块发生作用。RT-PCR显示,核心基因在20例PA样本及其瘤旁唾液腺组织中的差异趋势与测序结果一致,且具有统计学意义（P<0.0001）。结论：核心基因ADCY3、 ADCY5、 ADORA1、 APLN、 APP、 C5、 CCL28、 DRD2、 FPR3、 GABBR1可能在PA发生、发展过程中起着重要作用,可为预防PA复发及预后标志物筛选提供思路。PLAG1可能通过PHLPP1、LRRK2、β-catenin间接激活PI3K-AKT信号通路、ER受体信号通路、Rap1信号通路、cGMP-PKG信号通路,从而诱发PA形成。     

Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis-affected gingival tissues

Abe D, Kubota T, Morozumi T, Shimizu T, Nakasone N, Itagaki M, Yoshie H. Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis‐affected gingival tissues. J Periodont Res 2011; 46: 345–353. © 2011 John Wiley & Sons A/S Background and Objective: Gene expression is related to the pathogenesis of periodontitis and plays a crucial role in local tissue destruction and disease susceptibility. The aims of the present study were to identify the expression of specific genes and biological pathways in periodontitis‐affected gingival tissue using microarray and quantitative real‐time RT‐PCR analyses. Material and Methods: Healthy and periodontitis‐affected gingival tissues were taken from three patients with severe chronic periodontitis. Total RNAs from six gingival tissue samples were used for microarray analyses. Data‐mining analyses, such as comparisons, gene ontology and pathway analyses, were performed and biological pathways with a significant role in periodontitis were identified. In addition, quantitative real‐time RT‐PCR analysis was performed on samples obtained from 14 patients with chronic periodontitis and from 14 healthy individuals in order to confirm the results of the pathway analysis. Results: Comparison analyses found 15 up‐regulated and 13 down‐regulated genes (all of which showed a change of more than twofold in expression levels) in periodontitis‐affected gingival tissues. Pathway analysis identified 15 up‐regulated biological pathways, including leukocyte transendothelial migration, and five down‐regulated pathways, including cell communication. Quantitative real‐time RT‐PCR verified that five genes in the leukocyte transendothelial migration pathway were significantly up‐regulated, and four genes in the cell communication pathway were significantly down‐regulated, which was consistent with pathway analysis. Conclusion: We identified up‐regulated genes (ITGB‐2, MMP‐2, CXCL‐12, CXCR‐4 and Rac‐2) and down‐regulated genes (connexin, DSG‐1, DSC‐1 and nestin) in periodontitis‐affected gingival tissues; these genes may be related to the stimulation of leukocyte transendothelial migration and to the the impairment of cell‐to‐cell communication in periodontitis.     

Presence of Porphyromonas gingivalis and plasma cell dominance in gingival tissues with periodontitis

Oral Diseases (2010) 16 , 375–381 Objective: Porphyromonas gingivalis can invade and survive within its host epithelial cells. The aim of this study was to test our hypothesis that persistent presence of intracellular periodontal pathogens in gingival tissue causes the chronic inflammation and that an inappropriate immune response is a risk factor for periodontitis. Methods: Together with the presence of P. gingivalis, the distribution of B cells, plasma cells, and CD4⁺, CD8⁺, and FOXP3⁺ regulatory T cells was evaluated in gingival tissues from healthy (n = 7) and periodontitis (n = 8) sites by in situ hybridization and immunohistochemistry, respectively. Results: Porphyromonas gingivalis was detected in proximity to inflammatory infiltrates in three and seven biopsies from the healthy and periodontitis sites, respectively. Compared with healthy sites, periodontal lesions contained a significantly increased number of each immune cell studied with a relative dominance of plasma cells over T cells. Conclusions: Persistent bacterial invasion of gingival tissues in combination with a plasma cell‐dominant immune response may be involved in the pathogenesis of periodontitis.     

快速进展性牙周炎牙龈组织中单核/巨噬细胞浸润与Mcp-1表达

目的 研究快速进展性牙周炎牙龈组织中的单核／巨噬细胞浸润与单核细胞趋化蛋白１（ｍｏｎｏｃｙｔｅ ｃｈｅｍｏａｔｔｒａｃｔａｎｔ ｐｒｏｔｅｉｎ－１,ＭＣＰ－１）表达有无异常变化。方法 采用免疫组织化学ＳＰ染色法,取１４例快速进展性牙周炎、６例慢性牙周炎和６例健康牙龈组织,分别用抗ＣＤ１４、ＣＤ６８单克隆抗体检测牙龈组织中的单核／巨噬细胞,用ＭＣＰ－１多克隆抗测于龈组织中ＭＣＰ－１的表达。结果 快速