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。     

伴放线放线杆菌诱导T淋巴细胞通过Fas-FasL途径的细胞凋亡的研究

目的 :检测与Aa诱导T淋巴细胞凋亡有关的Fas(CD95 )介导的细胞凋亡途径。方法 :选取 10名全身及牙周组织健康受试者 ,分离外周血单核细胞 (PBMC) ,Aa体外刺激PBMC ,用特殊的单克隆抗体 (Fas、FasL)标记 ,并进行流式细胞仪检测。结果 :Fas和FasL的表达量明显上调。实验组Fas:2 4h - 2 7.2 2 %± 4 .10 ,96h - 6 8.2 6 %± 3.14 ;FasL :2 4h 6 .18%± 1.37,96h - 2 2 .6 4 %± 2 .82。用抗Fas单克隆抗体阻滞Fas-FasL相互作用导致明显的T细胞凋亡减少 ,百分比为 2 2 .72 %± 3.5 4 ,未加抗体的为 5 1.4 7%± 3.75。 (P <0 .0 0 0 5 ,但残余的细胞凋亡活动比阴性对照仍高。结论 :Aa诱导T淋巴细胞主要通过Fas -FasL途径凋亡 ,在细胞凋亡的分子机制上得到了数据证明 ,且有时间依赖性     

白介素10对伴放线放线杆菌内毒素诱导兔肺巨噬细胞凋亡的影响

目的：探讨白介素10（IL-10）对伴放线放线杆菌内毒素（Aa—LPS）体外诱导兔肺巨噬细胞凋亡作用的影响。方法：经兔气管肺泡灌洗获得肺泡巨噬细胞,随机分为空白对照组、Aa—LPS组、Aa—LPS＋IL-10组。按实验分组加入Aa—LPS（1汕g／mL）、IL-lo（o．1p．g／mL）,24h后裂解细胞,荧光定量PCR法检测促凋亡基因bax、p53和caspase-3的表达。结果：Aa—LPS组bax、caspase-3的表达较空白对照组明显升高（P〈0．05）,p53的表达与空白对照组比较差异无统计学意义（P〉0．05）。Aa—LPS＋IL-10组bax、p53、caspase．3的表达较Aa—LPS组降低（P〈0．05）。结论：Aa—LPS体外对肺巨噬细胞有促凋亡作用,IL-10可抑制Aa—LPS的促凋亡作用,其机制可能与细胞凋亡的线粒体途径有关。     

B淋巴细胞对牙周致病菌免疫反应及骨细收因子RANKL表达的影响

目的:通过评价B淋巴细胞与牙周致病菌——伴放线放线杆菌(Aa)发生免疫反应过程中核因子-КB受体活化配体(RANKL)的表达,探讨B淋巴细胞是否参与Aa引起的牙周骨吸收。方法:采用RT-PCR方法测定大鼠脾细胞中第1和7d细胞因子的mRNA转录水平,采用流式细胞技术测定大鼠脾细胞中B细胞表达RANKL IgG阳性细胞的百分数,应用TRAP法评价B淋巴细胞对破骨细胞分化的诱导潜力。实验结果采用SPSS 10.0软件包进行分析。结果:在无Aa抗原刺激的条件下,大鼠脾细胞培养1d和7d的TNF-α及IL-4表达水平均升高,IL-10、RANKL的表达水平无明显变化;加入Aa组培养1d时,TNF-α的表达显著增加,7d后,IL-4、IL-10及RANKL的mRNA转录水平显著增加;B细胞的百分数以及表达RANKL的IgG阳性细胞百分数与不加Aa组相比显著增加;Aa免疫组加入Aa培养后,表达RANKL的IgG阳性细胞百分数增加显著高于非免疫组。Aa免疫组的B淋巴细胞与RAW 264.7细胞共同培养后,TRAP染色阳性细胞显著增加(P0.01);加入人OPG-Fc培养,可显著抑制TRAP阳性细胞的形成(P0.05)。结论:B淋巴细胞经特异性抗原Aa活化后,通过上调RANKL的表达,增加对破骨细胞分化的诱导潜力,参与牙周骨吸收。     

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。     

PPARγ激动剂对T细胞增殖及破骨细胞形成的影响

目的:观察过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptor-γ,PPARγ)激动剂15d-PGJ2对小鼠T细胞增殖、T细胞分泌的细胞因子表达及破骨样细胞形成的影响,并探讨其可能的作用机制。方法:体外分离伴放线放线杆菌(A.actinomycetetemcomitans,Aa)免疫的BALB/c小鼠颈部淋巴细胞,提取T细胞进行体外扩增,分别加入浓度为0、1×10-8、1×10-7,1×10-6,1×10-5mol/L的15d-PGJ2进行干预。培养3d后,3H-Tdr掺入法测定T细胞的增殖反应;ELISA法测定细胞上清中核因子-κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)、TNF-α和IL-10的表达水平;取扩增的T细胞上清与RAW 264.7细胞共同培养后,抗酒石酸酸性磷酸酶染色(tartrate resistant acid phosphatase,TRAP)测定破骨样细胞的形成。采用SPSS 11.0软件包对数据进行统计学分析。结果:以1×10-5mol/L 15d-PGJ2处理的小鼠T细胞3d后,与对照组相比,T细胞增殖显著受抑;上清中RANKL、TNF-α的表达水平下降,IL-10无显著变化;TRAP染色阳性细胞数减少,具有统计学意义(P<0.05),且呈浓度依赖性。结论:PPARγ激动剂15d-PGJ2可抑制T细胞增殖,减少炎性因子分泌,降低破骨样细胞的形成,提示PPARγ配体在抑制T细胞诱导的骨吸收方面发挥积极作用。     

伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的体外研究

目的研究伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的作用。方法选取10名全身及牙周组织健康受试者,分离外周血淋巴细胞,在有／无伴放线放线杆菌情况下培养0—96h,用荧光探针（AnnexinV—FITC、PI、CD69-TC7）进行标记,并进行流式细胞仪检测。结果全淋巴细胞加伴放线放线杆菌组AnnexinV＋／PI-细胞百分数在48h、72h、96h分别为13．42±2．88、22．74±2．18、46．92±4．28,全淋巴细胞组AnnexinV＋／PI-细胞百分数在48h、72h、96h分别为8．46±2．53、6．36±2．36、9．36±2．67,2组间存在明显差异（P〈0．01）。CD69加淋巴细胞加伴放线放线杆菌组和CD69＋淋巴细胞组AnnexinV＋／PI-细胞百分数除48h外的4个时间点上都无明显差异（P〉0．05）。CD69＋淋巴细胞加伴放线放线杆菌组AnnexinV＋／PI-细胞百分数在各个时间点上都明显高于全淋巴细胞加伴放线放线杆菌组（P〈0．01）。结论伴放线放线杆菌能够诱导人外周血淋巴细胞活化,并且能够通过活化促进其凋亡。     

维生素A对牙周致病菌免疫小鼠免疫应答调节的作用

目的:探讨维生素A缺乏对特异性牙周致病菌-伴放线放线杆菌(A.actinomycetetemcomitans,Aa)引起的小鼠免疫应答作用的影响.方法:整个实验过程采用无维生素A饮食(vitamine A-depleted diet,VAD)或常规维生素A饮食(vitamine A-sufficient regular diet,RD)喂养BALB/c鼠.2周后,免疫Aa建立免疫动物模型,6周后处死小鼠,ELISA法测定血清中的抗Aa特异性抗体总IgG、IgM及IgG亚类抗体滴度及细胞上清中细胞因子的浓度,3H-Tdr掺入法测定T细胞增殖反应.实验结果采用SPSS 11.5软件包进行统计学分析.结果:Aa免疫的总IgG和IgM抗体水平明显升高,非免疫组则不能产生抗体.Aa免疫+VAD组与Aa免疫+RD组相比,总IgG水平显著升高(P<0.05);IgG2a的抗体水平明显增加,而IgG1亚型的抗体水平却明显降低,差异显著(P<0.05).Aa免疫组可诱导机体产生较强的特异性T细胞免疫反应,而Aa免疫+VAD组T细胞增殖反应明显高于Aa免疫+RD组,具有统计学差异(P<0.05);细胞上清中RANKL、IFN-γ及TNF-α的表达增加,IL-10的表达降低(P<0.05).结论:饮食中维生素A缺乏,可增加Aa免疫鼠引起的免疫炎症反应,提示充足的维生素A是维持机体健康的重要因素.     

Tca8113-4-1BBL细胞联合CD28单抗在口腔鳞癌免疫治疗中的作用

目的:探讨转4-1BBL肿瘤细胞联合抗CD28单抗体外诱导抗肿瘤活性的能力.方法:通过脂质体法将重组载体pEGFP/neo-h4-1BBL转染人舌鳞癌细胞Tca8113,经G418(400μg/mL)筛选及有限稀释后,获得稳定高表达克隆,分别用RT-PCR和Western印迹检测转染细胞中h4-1BBLmRNA和蛋白的表达.将转染与未转染4-1BBL基因的Tea8113细胞用丝裂霉素C(MMC)处理后,制成肿瘤细胞瘤苗.联合抗CD28单克隆抗体与经体外抗CD3mAb诱导的人外周血T淋巴细胞共同培养,测定T细胞增殖、CTL杀伤活性及产生细胞因子(IL-2和IFN-γ)的能力.实验数据以SPSS12.0软件包进行方差分析.结果:h4-1BBL基因真核表达载体在Tca8113细胞中获得稳定表达.转染h4-1BBL基因的Tca8113细胞联合抗CD28单抗能显著刺激T细胞活化、增殖(P(0.01),促进IL-2、IFN-γ分泌(P<0.01),并能有效地诱导CTL的特异性杀伤活性(P<0.01).结论:转4-1BBL肿瘤细胞联合抗CD28抗体能显著增强肿瘤细胞的免疫原性,诱导T细胞产生有效的抗肿瘤免疫应答.     

口腔扁平苔藓患者Th1／Th2失衡表达的研究

目的：观察口腔扁平苔藓（oral liclaen planus,OLP）患者外周血中Th1、Th2型细胞转录因子T-het和GATA-3以及Thl、Th2型细胞因子的表达,进一步探索OLP的发病机制。方法：通过密度梯度离心法分离20例充血糜烂型,16例光滑型OLP病例和19例正常对照组人群的外周血单个核细胞,逆转录-聚合酶链反应（RT—PCR）法检测各组外周血单个核细胞中T—betmRNA和GATA-3 mRNA的表达;ELISA法检测各组血清中干扰素-γ（interferon gamma,IFN-1）和白细胞介素-4（interleukin-4,IL-4）的表达。结果：充血糜烂型和光滑型OLP患者中T—betmRNA、IFN-γ的表达均低于正常对照组,差异有统计学意义（P〈0．01）;而充血糜烂型和光滑型OEP患者中GATA-3 mRNA、IL-4的表达均高于正常对照组,差异有统计学意义（P〈0．01）。T—bet mRNA和GATA-3 mRNA的表达在充血糜烂型及光滑型OLP患者组间的差异有统计学意义（P〈0．01）;而IFN-γ和IL-4的表达在充血糜烂型及光滑型OLP患者组间的差异无统计学意义（P〉0．05）。结论：Th1／Th2失衡表达与OLP发病机制密切相关,为临床治疗OLP提供参考。     

PCR-based identification of Treponema maltophilum,T amylovorum,T medium,and T lecithinolyticum in primary root canal infections

Objective: Molecular genetic methods have significantly contributed to the knowledge about the microbiota associated with infected root canals. Albeit spirochetes have been commonly observed in primary root canal infections, only recently they have been identified. The purpose of the present study was to investigate the occurrence of four treponemes—Treponema maltophilum, Treponema lecithinolyticum, Treponema amylovorum, and Treponema medium—in cases of primary endodontic infections associated with different forms of periradicular diseases through a 16S rDNA-based nested PCR assay. Design: Samples were taken from thirty-one infected root canals associated with either asymptomatic or symptomatic apical periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers, followed by a second round of amplification using the first PCR products to detect a specific fragment of the 16S rDNA of each target Treponema species. Results: All cases were positive for the universal bacterial primers, indicating that samples contained bacterial DNA. Of the four target species, T. maltophilum was the most prevalent, being detected in 39% of the cases (33% of the asymptomatic cases and 50% of the symptomatic cases). T. lecithinolyticum was the next more prevalent among the species tested, being found in 26% of the samples (33% of asymptomatic cases and 10% of the symptomatic cases). T. amylovorum was found in 7% of the cases (5% of the asymptomatic cases and 10% of the symptomatic cases), while T. medium was in 13% of the cases (14% of the asymptomatic cases and 10% of the symptomatic cases). None of the species tested was significantly associated with clinical symptoms. Conclusions: This was possibly the hitherto first study to report the occurrence of T. lecithinolyticum, T. amylovorum, and T. medium in infections of endodontic origin. Overall, findings suggested that these oral treponemes, particularly T. maltophilum and T. lecithinolyticum, can be involved in the pathogenesis of periradicular diseases.     

Alveolar bone loss in T helper 1/T helper 2 cytokine-deficient mice

BACKGROUND AND OBJECTIVES: The role of cytokines in bone loss is important in the context of periodontitis, where inflammation-induced bone destruction is a major manifestation. Numerous cytokines have been implicated as mediators of bone resorption. The purpose of this study was to observe the impact of targeted gene deletion of T helper 1 (Th1) and T helper 2 (Th2) cytokines on naturally occurring alveolar bone loss in genetically modified mice. MATERIAL AND METHODS: Alveolar bone loss was measured histomorphometrically in interleukin-4, interleukin-10, interleukin-12p40, interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) knockout mice at 6, 16 and 30 wk of age. RESULTS: Both Th1 (interleukin-12p40, IFN-gamma, TNF) and Th2 (interleukin-10, interleukin-4) knockout mice exhibited significantly more alveolar bone loss than their respective wild-type control mice (p<0.001). Interleukin-10-/- and interleukin-12p40-/- mice exhibited a three-fold increase in alveolar bone loss at 30 wk of age, whereas bone loss in IFN-gamma-/-, TNF-/- and interleukin-4-/- mice was 1.5- to two-fold higher compared with wild-type control mice. CONCLUSION: The results of the present study indicate that both Th1 and Th2 cytokines play an important role in maintaining alveolar bone homeostasis. The kinetics of alveolar bone loss seen in cytokine gene knockout mice indicates that bone loss is age dependent and late in onset.     

Despite large-scale T cell activation, only a minor subset of T cells responding in vitro to Actinobacillus actinomycetemcomitans differentiate into effector T cells

Recent studies in our laboratory have demonstrated that Actinobacillus actinomycetemcomitans has a potent T cell stimulatory effect, activating more than half of all T cells. However, since the fate of these activated T cells was not known, the present study sought to determine whether all of these T cells differentiate into effector cells. To that end, the intracellular expression of T cell cytokines (IL-2, IFN-gamma, IL-4 and IL-10) in response to A. actinomycetemcomitans was determined by flow cytometry. Results demonstrated a time-dependent increase in the expression of the cytokines, most reaching peak levels at 24-48 h. At 48 h, the proportion of T cells expressing each of the cytokines were as follows: IL-2 (1.7%+/-0.3), IFN-gamma (1.8%+/-0.5), IL-4 (1.0%+/-0.2) and IL-10 (1.5%+/-0.5). These data indicated that only 2-5% of all T cells stimulated with A. actinomycetemcomitans expressed any T cell cytokines. The finding of large-scale T cell activation in the absence of cytokine expression suggests that the activation of T cells in response to A. actinomycetemcomitans is incomplete. To investigate this phenomenon, peripheral blood mononuclear cells (PBMC) were cultured with A. actinomycetemcomitans for 24 h followed by sorting of the activated (CD69+) cells by immunomagnetic separation and restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Results demonstrated that nearly 90% of the T cells were unresponsive to further restimulation. A possible explanation for this unresponsiveness is the induction of clonal anergy among the responding T cells. To determine possible preferential effects of the stimulation on specific cytokines, the expression of each cytokine among T cells responding to A. actinomycetemcomitans was compared to the maximum levels achieved by PMA + ionomycin stimulation. Results showed that number of IL-2+ and IFN-gamma+ T cells observed in response to A. actinomycetemcomitans were between 2% and 7% of those seen in response to PMA + ionomycin. Conversely, the proportions of T cells expressing IL-4 or IL-10 were between 35% and 90% of those following stimulation with PMA + ionomycin. Hence, A. actinomycetemcomitans appears to more preferentially induce T cells expressing IL-4 and IL-10. Collectively, these data suggest that the in vitro stimulation of T cells with A. actinomycetemcomitans leads to partial activation, i.e. only a minor subset of T cells responding to A. actinomycetemcomitans differentiate into effector cells, while a significant proportion become unresponsive to restimulation, suggesting clonal anergy.     

Clinical behavior of T1–T2 squamous cell carcinoma of the oral cavity

ObjectivesThe main aim of the present study is to analyze the differences in the clinical behavior of pT1 and pT2 oral squamous cell carcinoma of the oral cavity and the importance of tumor thickness in these groups of patients.MethodsA retrospective analysis was conducted using the records of patients diagnosed with pT1 and pT2 oral squamous cell carcinoma between 2006 and 2015 to identify significant differences between these two groups of patients. Several pathological features such as T-stage, N-stage, tumor thickness, surgical margins, and locoregional failure were analyzed.Results194 patients were included in this study. Tumor thickness >0.4 cm was significantly related with nodal involvement and overall survival (p < 0.001). T and N stage, tumor thickness, extracapsular spread and surgical margins were associated with poorer outcomes in terms of overall survival (p < 0.001).ConclusionTumor thickness represents an extremely important prognostic factor and to include depth of invasion (DOI) in the staging of oral squamous cell carcinoma will help in the choice of better treatment strategies and to improve overall survival.