扁平苔藓患者血清及唾液中IL-1O的表达及相关性研究

目的：探讨口腔扁平苔藓（OLP）患者唾液及血清中白细胞介素-10 （IL-10）的表达情况及其相关性,以进一步了解OLP患者唾液代替血液作为生物学样本来研究OLP中IL-10的可行性.方法：实验分3组：OLP糜烂组15例,OLP非糜烂组15例,正常对照组15例.采用ELISA法分别检测3组对象血清和唾液中IL-10的含量,对结果进行统计分析.结果：OLP糜烂组血清中IL-10水平为（30.11±3.02） pg/mL,明显高于非糜烂组（19.03±3.33） pg/mL和正常对照组（16.43±2.19） pg/mL,差异有统计学意义（P ＜0.05）;OLP糜烂组唾液中IL-10平均水平为（10.05 ±3.12） pg/mL明显高于非糜烂组（5.50±1.1） pg/mL和正常对照组（4.47±1.7） pg/mL,差异有统计学意义（P＜0.05）;两实验组血清和唾液中IL-10水平成正相关,（r=0.731,P＜0.01）.结论：实验组血清和唾液中IL-10水平高度相关,可以通过检查唾液替代检查血液来研究OLP中IL-10的水平.     

母体唾液及龈沟液细胞因子水平与早产低体重新生儿关系的病例对照研究

目的 验证牙周炎是否为早产低体重新生儿(preterm low birth weight,PLBW)母亲体内低度炎症的来源之一,初步探讨PLBW与炎症因子之间的关系.方法 以83例PLBW的母亲(PLBW组)及44名健康新生儿(normal birth weight,NBW)的母亲(NBW组)为研究对象,留取受试对象的静息全唾液、龈沟液,应用酶联免疫吸附试验检测唾液和龈沟液中白细胞介素(IL)-1β和IL-6水平,比较两组间唾液和龈沟液中细胞因子水平的差异,并分析细胞因子与分娩孕周、新生儿体重之间的相关性.结果 唾液IL-1β水平在PLBW组[(78.32±11.81)ng/L]显著高于NBW组[(39.66±11.89)ng/L],差异有统计学意义(P<0.05);分娩孕周与唾液IL-6之间呈显著的负相关关系(r=-0.274,P<0.01);新生儿体重与龈沟液IL-1β(r=-0.231,P<0.01)、唾液IL-6(r=-0.424,P<0.01)之间呈显著的负相关关系.结论 唾液和龈沟液中炎性因子水平越高,分娩孕周越短、新生儿体重越轻,母体牙周炎程度与PLBW之间可能存在一定程度的相关关系.     

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。     

高迁移率族蛋白B1对牙周膜成纤维细胞表达细胞因子影响的研究

目的观察高迁移率族蛋白B1（HMGB1）对人牙周膜成纤维细胞表达白细胞介素-6（IL-6）、破骨细胞核因子κB受体活化因子（RANKL）、骨保护因子（OPG）的影响,初步探讨HMGB1在牙周疾病的作用。方法采用原代组织块培养法,培养人牙周膜成纤维细胞,用第4~6代的细胞进行实验。分别用10、30、100 ng·mL-1质量浓度的HMGB1孵育牙周膜成纤维细胞24 h后,RT-PCR检测IL-6、RANKL、OPG的mRNA表达;Western blot法检测RANKL、OPG的蛋白表达。均以0 ng·mL-1质量浓度组为对照,所得数据用单因素方差分析处理。结果HMGB1在10、30、100 ng·mL-1质量浓度时,细胞中的RANKL/OPG mRNA的比值增高（P<0.05）,100 ng·mL-1质量浓度时细胞中的IL-6 mRNA的表达量增高（P<0.05）。Western blot检测结果显示10 ng·mL-1质量浓度组的RANKL/OPG的比值有明显增高。结论一定浓度的HMGB1可使牙周膜细胞中的RANKL/OPG比值增高,还会诱导炎症因子IL-6 mRNA表达上调。提示HMGB1可能会在牙周炎的发病以及炎症进展中发挥作用。     

口腔扁平苔藓患者血清IFN-γ、IL-10和IL-17表达分析

目的观察口腔扁平苔藓(OLP)患者血清中Th型细胞因子IFN-γ、IL-10和IL-17表达,初步探讨其在OLP发生发展中的作用。方法选择2011—2014年在上海市口腔病防治院口腔内科就诊的OLP患者15例,健康志愿者15例,采集静脉血,酶联免疫吸附试验(ELISA)检测血清中IFN-γ、IL-10和IL-17含量。结果实验组、对照组OLP患者血清中Th1型细胞因子IFN-γ的含量无显著差异(P>0.05),Th2型细胞因子IL-10含量为(43.3±4.6)、(29.0±5.1)pg/m L,实验组高于对照组(P<0.05)。IFN-γ与IL-10的比值(即Th1/Th2)为0.418、0.712,实验组较对照组的比值减小。两组Th17型细胞因子IL-17含量为(23.6±2.7)、(16.8±3.3)pg/m L,实验组高于正常对照组(P<0.05)。结论 Th型相关细胞因子失衡与OLP的发生发展密切相关。     

不同fimA基因型牙龈卟啉单胞菌刺激上皮细胞表达细胞因子的比较

目的比较不同fimA基因型牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）刺激下口腔上皮细胞表达细胞因子的水平。方法P.gingivalis ATCC33277（IfimA）,W83（ⅣfimA）,47A-1（ⅣfimA）分别与KB细胞ATCCCCL17共同孵育6h,在3h和6h收集细胞和培养上清液。逆转录-聚合酶链式反应检测KB细胞IL-8,IL-6,IL-1βmRNA的表达,酶联免疫反应检测培养上清液中IL-8,IL-6,IL-1β的变化。结果3h时,ⅣfimA组IL-6蛋白水平低于IfimA组（P〈0.05）,6h时,ⅣfimA组IL-6和IL-8蛋白水平均低于ⅠfimA组（P〈0.05）;IL-8,IL-6mRNA和蛋白水平表达不一致,提示存在转录后水平的调节。3h,6h时,IL-1β对P.gingvalis刺激不敏感,mRNA和蛋白表达水平都很低。结论P.gingivalis fimA基因型与上皮细胞表达细胞因子的水平相关,提示P.gingivalis致病性与其fimA基因型相关。     

程序性死亡分子1及其配体在口腔鳞状细胞癌患者外周血中的表达及临床意义

目的 检测口腔鳞状细胞癌患者外周血中程序性死亡分子1（PD-1）及其配体（PD-L1）的表达水平,探讨其生物学及临床意义。方法 选取82例口腔鳞状细胞癌患者（口腔鳞癌组）和25例健康对照者（对照组）为研究对象,收集研究对象的外周血,采用流式细胞术检测外周血T淋巴细胞表面PD-1、PD-L1的表达及T淋巴细胞亚群计数,采用酶联免疫吸附方法检测血清中可溶性PD-1（sPD-1）和可溶性PD-L1（sPD-L1）的表达水平。采用SPSS 17.0软件对数据进行检验和相关性分析。结果 口腔鳞癌组外周血CD8+ T淋巴细胞百分数明显高于对照组（P<0.05）,而CD3+、CD4+T淋巴细胞百分数及CD4+/CD8+T亚群百分数比值均低于对照组（P<0.05）。口腔鳞癌组外周血CD4+、CD8+ T淋巴细胞表面PD-1、PD-L1的阳性表达率均高于对照组（P<0.01）。口腔鳞癌组血清sPD-L1水平高于对照组（P<0.05）,而sPD-1水平二者差异无统计学意义（P>0.05）。sPD-L1的表达与临床分期、肿瘤分化程度及淋巴结转移状态相关（P<0.05）,与性别、年龄、肿瘤部位及大小无关。结论 口腔鳞状细胞癌患者外周血T淋巴细胞亚群存在不同程度的免疫功能抑制,CD4+和CD8+ T淋巴细胞表面PD-1及PD-L1表达显著升高。异常升高的sPD-L1可能与口腔鳞状细胞癌的发生发展有关。     

X-性染色体连锁凋亡抑制蛋白与口腔鳞癌细胞Tca8113化疗耐药的关系

目的研究X-性染色体连锁凋亡抑制蛋白（XIAP）在口腔鳞癌细胞Tca8113中的表达水平,并探讨XIAP表达与Tca8113细胞对化疗药物耐药性之间的关系。方法用平阳霉素（PYM）间歇性加药,逐步递增剂量,采用甲基噻唑基四唑（MTT）法检测药物处理前后细胞对PYM的敏感性,逆转录聚合酶链反应（RT-PCR）分别检测XIAP在药物处理前后的各Tca8113细胞组中的表达变化,并探讨XIAP与细胞耐药性的关系。结果Tca8113细胞在PYM间断作用下产生耐受,Tca8113-1-10组、Tca8113-10-10组耐药细胞对PYM的半数有效浓度（IC50）分别为（12.758±0.030）、（18.986±0.150）μg·mL-1。Tca8113-1-20、Tca8113-10-20组耐药细胞对PYM的IC50分别为（26.302±0.072）、（35.294±0.115）μg·mL-1。XAIP的表达水平与细胞对PYM的耐药有相关性（P<0.01）。结论XIAP在口腔鳞癌中的表达水平升高,可能与鳞癌的化疗耐药性有关,这可作为口腔鳞癌基因治疗的一个新靶点。     

维甲酸对小鼠T细胞增殖及骨破坏因子RANKL表达的研究

目的 探讨全反式维甲酸对体外培养的小鼠T淋巴细胞的增殖及核因子-КB受体活化因子配体(RANKL)表达的调节作用及相关机制。方法 体外分离Aa感染的BALB/C小鼠颈T淋巴细胞,分别加入浓度为0、1×10-8、1×10-7、1×10-6、1×10-5mol/L的全反式维甲酸,培养3 d后,取100μl上清保存在-70℃冰箱中,以备sRANKL及IL-10等细胞因子的测定;另加入100μl含有3Hthymidine(0.5μCi/孔)RPMI培养液继续培养18 h,进行T细胞3H增殖率测定。结果 维甲酸处理组与非处理组相比,T细胞3H增殖率下降;上清中RANKL的表达水平下降,IL-10的表达水平增强(P<0.05),二者具有负相关性(r=-0.774,P=0.001),且呈剂量依赖性。结论 维甲酸可通过上调IL-10的表达并下调T细胞上的RANKL的表达水平,抑制T细胞的免疫功能,且呈剂量依赖性。     

牙龈卟啉单胞菌表面相关物质刺激淋巴细胞分化效应T细胞的研究

目的探讨牙龈卟啉单胞菌表面相关物质（SAM）刺激淋巴细胞活化后效应T细胞的表达及意义。方法选取10名全身及牙周组织健康受试者静脉血,分离外周血单核细胞（PBMC）,体外刺激PBMC：实验组加入SAM冻干产物（浓度为25μg/mL）;阳性对照组加入刺激抗原PMA（25μg/mL）＋ionomycin（1μg/mL）;阴性对照组不加任何刺激抗原。流式细胞仪检测T细胞内细胞因子IL-2、IFN-γ、IL-4、IL-10的表达。结果 SAM刺激淋巴细胞后上述四种细胞因子呈低水平表达;SAM诱导IL-4、IL-10表达的能力相对较强。结论 SAM刺激可造成T淋巴细胞免疫缺陷,导致IL-2、IFN-γ等功能障碍,激发TH2细胞活性,可导致机体免疫功能受损和牙周组织的继发性损伤。     

Detection of human herpesvirus 6 in patients with oral chronic graft-vs-host disease following allogeneic progenitor cell transplantation

INTRODUCTION: Chronic graft-vs-host disease (cGVHD) is a major cause of morbidity in long-term survivors of allogeneic hematopoietic progenitor cell transplantation. Herpesviruses are involved in the occurrence and progression of various oral diseases. AIM: The aim of this study was to investigate the role of human herpesvirus 6 (HHV6) in patients with oral manifestations of cGVHD. MATERIALS AND METHODS: Peripheral blood and oral fluids (whole saliva, gingival crevicular fluid and parotid gland saliva) from 19 cGVHD patients, and 28 blood donors were examined for HHV6. Oral tissue samples were collected from 12 cGVHD patients and 12 healthy individuals. Nested polymerase chain reaction was employed to identify the HHV6. RESULTS AND CONCLUSION: The virus was detected in whole saliva in 13 cGVHD patients (68%) and in 19 blood donors (67%). HHV6 was not identified in any of the gingival crevicular fluid and parotid gland saliva samples in cGVHD patients. In the control group 14.3% of both, four gingival crevicular fluid and four parotid gland saliva samples were positive. Two oral tissue samples of cGVHD patients were positive for HHV6. These results indicate that patients with oral manifestations of cGVHD and healthy individuals present high and similar incidence of HHV6 in blood and oral fluids. These data do not support the importance of HHV6 in oral lesions of cGVHD.     

糜烂型OLP患者唾液Th1、Th2、Th17相关细胞因子及β防御素的表达

目的：检测Th1、Th2、Th17相关细胞因子和HBD－2,－3在糜烂型口腔扁平苔藓（EOLP）患者唾液中的表达,探讨其在EOLP发生发展中的作用。方法：分别纳入EOLP患者30例及按年龄和性别匹配的健康者20例作为实验组和对照组,采用流式微球技术（Cytometric Bead Array,CBA）检测唾液中IL－1β、TNF－α、IL－6、IL－17、IFN－γ、IL－4、IL－10的表达水平,并采用酶联免疫吸附测试法（ELISA）检测唾液中HBD－2和HBD－3。结果：EOLP患者唾液中IL－1β、IL－6、HBD－2、HBD－3水平显著高于对照组（P＜0．05）,TNF－α、IL－17、IFN－γ、IL－4、IL－10与对照组比较差异无显著性（P＞0．05）。患者唾液HBD－2的表达与IFN－γ呈正相关、与IL－17呈负相关,HBD－3的表达与IL－17和IL－10均呈负相关。结论：唾液中IL－1β、IL－6、HBD－2和HBD－3的高表达可能与EOLP的发生发展密切相关。     

Cytokine profiles in parotid saliva from HIV-1-infected individuals: changes associated with opportunistic infections in the oral cavity

The purpose of this study was to quantitate levels of cytokines in parotid saliva of subjects infected with human immunodeficiency virus-1 (HIV-1) and to determine if the cytokine profiles differ in subjects with an oral opportunistic infection, i.e., candidiasis or oral hairy leukoplakia. Parotid saliva samples were obtained from HIV-infected individuals with or without candidiasis or oral hairy leukoplakia and from healthy controls and were assessed by ELISA for levels of interleukin (IL)-1, IL-2, IL-4, IL-5, IL-10, transforming growth factor-beta, tumor necrosis factor-alpha and interferon (IFN)-gamma. Saliva from HIV-infected subjects with oral candidiasis had significantly higher levels of IFN-gamma than that seen in HIV-infected individuals with no oral disease and significantly higher levels of IL-2, IL-5 and IFN-gamma than saliva of healthy controls. No significant difference was seen in cytokine levels in saliva from HIV-infected subjects with no oral infections and healthy controls. The HIV-infected subjects with oral hairy leukoplakia displayed significantly higher levels of both IL-1 alpha and IFN-gamma compared with the HIV and no oral disease group and a higher level of IFN-gamma than seen in saliva from the healthy control group. In comparing cytokine levels from both HIV and oral disease groups, significant differences were detected in levels of IL-5 and IL-10. These results indicate that the profile of salivary cytokines is altered as a result of the oral opportunistic infection candidiasis or oral hairy leukoplakia and also by concurrent HIV infection.     

Comparison of salivary cytokine levels in oral cancer patients and healthy subjects

In order to find informative salivary biomarkers specific to oral cancer we examined expression of 4 kinds of cytokine in saliva. Levels of interleukins (IL-1beta, -6, -8) and osteopontin were measured by ELISA using whole saliva samples collected from 19 patients with oral cancer (9 men, 10 women; mean age, 60.9 years) and 20 healthy persons (15 men, 5 women; mean age, 32 years). Expression of the 4 cytokines was higher in patients with oral cancer than in healthy controls. The difference was significant in IL-6, in particular. The results suggest that saliva offers a potential target for a screening test aimed at detection of precancerous lesions.     

s-IgA and cytokine levels in whole saliva of Sjögren's syndrome patients before and after oral pilocarpine hydrochloride administration: a pilot study

Previous investigations have found elevated levels of s-IgA in the parotid saliva and normal levels in submandibular saliva of patients with Sjögren's syndrome (SS). Fox et al. also found elevated levels of cytokines (i.e., IL-2 and IL-6) in serum, salivary epithelial cells and parotid saliva of patients with SS. The oral administration of pilocarpine hydrochloride stimulates whole and parotid salivary flow. The purpose of this study was to determine the levels of s-IgA and IL-2 and IL-6 in whole saliva before and after administration of pilocarpine hydrochloride in SS subjects. Ten definitively diagnosed SS subjects were enrolled in the study, as were ten controls (C). The mean age was 57.2 years and all subjects were female. Whole unstimulated saliva (WUS) was collected by standard techniques for 5 min, after which the volume and flow rate were determined (mean WUS: SS = 0.047 vs C = 0.480 ml/min). Samples were centrifuged and the immunoglobulin analysis performed on the supernatants by immunoreactivity in a double-sandwich technique as previously described by Rudney et al. Cytokine analysis was performed similarly utilizing commercially available kits from R&;D Systems. The results as analyzed by pairwise t-tests revealed comparable levels of s-IgA in the saliva of the SS patients, as compared to controls at baseline (means±SEM: SS-IgA = 348.1±82.0 vs C-IgA = 284.0±65.1 μg/ml; NS ). Whole salivary flow was significantly increased (328%) in the SS subject group 60 min after the administration of 5 mg pilocarpine hydrochloride (means±SEM: 0.0472±0.017 vs 0.1546±0.054 ml/min; P<0.01). There was no significant change in the concentration of s-IgA in the SS subject group following the pilocarpine dose (means±SEM: SS-IgA = 439.9±121.2 μl/ml; P = NS). There were elevated levels of IL-2 in the saliva of four out of the ten and IL-6 in two out of the ten SS patients, as compared to controls (means±SEM: SS-IL-2 = 127.8±11.4 vs C-IL-2 = 30.8±1.6 pg/ml and SS-IL-6 = 41.4±7.1 vs C-11.6 ± 2.8 pg/ml). There was also a significant decrease in the concentration of IL-2 in the same four out of ten SS subjects following the pilocarpine dose (means±SEM: SS-IL-2 = 32.4±10.3; P<0.01). These preliminary results indicate that s-IgA levels do not change with increased salivary flow following the administration of pilocarpine hydrochloride in patients with Sjögren's syndrome. While cytokines are elevated in the whole saliva of some SS patients, a decrease in IL-2 concentration may occur with increased salivary flow.     

人类疱疹病毒6型与口腔鳞癌发生的相关性研究及临床意义

目的研究人类疱疹病毒6型(HHV-6)感染与口腔鳞癌发生的相关性及其临床意义。方法运用巢式PCR技术,检测82例口腔鳞癌、25例癌前病变及40例正常对照人群唾液、血液、组织中HHV-6感染情况。结果正常对照组、癌前病变组、鳞癌组HHV-6在唾液中的检出率分别为45.0%、56.0%、72.0%,在血液中的检出率分别为22.5%、48.0%、57.3%,总阳性率为47.5%、56.0%、80.5%,趋势差异均具统计学意义(P<0.05);3组HHV-6检出率唾液普遍高于血液,在正常组和鳞癌组中差异具统计学意义(P<0.05)。鳞癌组癌组织中HHV-6检出率(78.95%)显著大于癌旁正常组织(10.53%),具统计学意义(P<0.01);随着鳞癌分化程度的降低,HHV-6的阳性率有下降的趋势,但无统计学差异(P>0.05)。结论唾液腺是HHV-6长期潜伏和增殖的部位,唾液是其主要的传播媒介;HHV-6的激活/再感染发生在黏膜病变的早期,其感染与口腔鳞癌的发生存在相关性,可能直接参与了细胞癌变的过程。     

Cytokine profiles in parotid saliva from HIV‐1‐infected individuals: changes associated with opportunistic infections in the oral cavity

The purpose of this study was to quantitate levels of cytokines in parotid saliva of subjects infected with human immunodeficiency virus‐1 (HIV‐1) and to determine if the cytokine profiles differ in subjects with an oral opportunistic infection, i.e., candidiasis or oral hairy leukoplakia. Parotid saliva samples were obtained from HIV‐infected individuals with or without candidiasis or oral hairy leukoplakia and from healthy controls and were assessed by ELISA for levels of interleukin (IL)‐1, IL‐2, IL‐4, IL‐5, IL‐10, transforming growth factor‐β, tumor necrosis factor‐α and interferon (IFN)‐γ. Saliva from HIV‐infected subjects with oral candidiasis had significantly higher levels of IFN‐γ than that seen in HIV‐infected individuals with no oral disease and significantly higher levels of IL‐2, IL‐5 and IFN‐γ than saliva of healthy controls. No significant difference was seen in cytokine levels in saliva from HIV‐infected subjects with no oral infections and healthy controls. The HIV‐infected subjects with oral hairy leukoplakia displayed significantly higher levels of both IL‐1α and IFN‐γ compared with the HIV and no oral disease group and a higher level of IFN‐γ than seen in saliva from the healthy control group. In comparing cytokine levels from both HIV and oral disease groups, significant differences were detected in levels of IL‐5 and IL‐10. These results indicate that the profile of salivary cytokines is altered as a result of the oral opportunistic infection candidiasis or oral hairy leukoplakia and also by concurrent HIV infection.     

Correlation between salivary IL-1beta levels and periodontal clinical status

OBJECTIVE: To assess the concentration of the proinflammatory cytokine IL-1beta in saliva of periodontally diseased and healthy patients and their relationship with the periodontal status. DESIGN: Unstimulated whole saliva samples from patients with chronic periodontitis (n=30), aggressive periodontitis (n=18) and healthy controls (n=18) were obtained for the study. The periodontal status of each subject was assessed by criteria based on probing depth, clinical attachment loss and the extent/severity of periodontal breakdown. The levels of IL-1beta were measured in saliva samples with a high sensitivity enzyme-linked immunosorbent assay (ELISA). RESULTS: Although no significant difference (P=0.624) was found for salivary IL-1beta levels between periodontitis groups, they were significantly greater (P<0.01) than those detected for healthy controls. Furthermore, Spearman correlation analysis showed statistically significant correlations (P<0.01) between data from salivary IL-1beta levels and clinical measurements. CONCLUSION: The findings of the present study reemphasize the importance of whole saliva as sampling method in terms of immunological purposes in periodontal disease and suggest that the elevated IL-1beta concentration may be one of the host-response components associated to the clinical manifestations of periodontal disease.