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。     

益生菌鼠李糖乳杆菌通过下调促炎性Th17改善实验性牙周炎

目的 探讨益生菌鼠李糖乳杆菌（Lactobacillus rhamnosus GG, LGG）能否及如何经由肠道免疫影响实验性牙周炎。方法 丝线结扎小鼠上颌第二磨牙构建实验性牙周炎（PD）模型，灌胃新鲜LGG至小鼠体内。实验分为PD对照组和PD+LGG实验组，Micro-CT扫描重建上颌牙槽骨三维结构，流式检测下颌下淋巴结（submandibular lymph node cells, SLCs）和脾脏淋巴（splenic lymphocyte, SPLs）中IL-17A+Th17和CD25+Foxp3+Treg的百分比，RT-qPCR和ELISA分别检测牙龈和血清中IL-17A、IL-6、TNF-α、IL-1β及转录因子RORγt、FoxP3的表达。结果 灌胃LGG显著减缓牙周炎牙槽骨吸收，PD+LGG组SLCs和SPLs中促炎性Th17细胞百分比均明显下降，免疫抑制性Treg的百分比在两组之间未见统计学差异，同时PD+LGG组牙龈和血清内IL-17A、IL-6、TNF-α、IL-1β、RORγt的表达均明显下调。结论 应用益生菌LGG有利于改善实验性牙周炎，其机制可能在于下调牙周局...     

根尖牙乳头干细胞外泌体对舍格伦综合征小鼠治疗效果的实验研究

目的    探索根尖牙乳头干细胞来源的外泌体（exosomes derived from stem cells from apical papilla,SCAP-Exo）对舍格伦综合征（Sjögren syndrome,SS）小鼠的治疗效果。方法    酶消化法提取根尖牙乳头干细胞,超速离心法提取SCAP-Exo,并应用透射电镜、纳米颗粒跟踪技术及Western Blot进行鉴定。选择10周龄雌性健康ICR小鼠6只作为对照组;选择10周龄雌性NOD小鼠12只随机分为NOD组和SCAP-Exo组,每组6只。饲养2、4周后,SCAP-Exo组小鼠尾静脉注射SCAP-Exo,其他组小鼠尾静脉注射PBS。于饲养6周后检测各组小鼠唾液流率。随后收集小鼠外周血,流式细胞术检测外周血中辅助性T细胞17（T helper 17,Th17）/CD4+ T细胞比例,ELISA检测血清中白细胞介素-17A（interleukin-17A,IL-17A）表达水平。最终处死各组小鼠,通过HE染色观察下颌下腺中淋巴细胞浸润水平。结果    透射电镜下可见SCAP-Exo呈杯状的囊泡结构;纳米颗粒跟踪技术分析SCAP-Exo直径为30 ~ 150 nm;外泌体特异性标记蛋白CD9、Alix呈阳性表达,不表达细胞内质网特异性蛋白Calnexin。3组小鼠唾液流率、外周血Th17/CD4+ T细胞比例及IL-17A表达水平总的比较,差异均有统计学意义（F值分别为59.169、293.217、189.583,均P < 0.05）。其中,与对照组相比,NOD组小鼠唾液流率明显降低、外周血Th17/CD4+ T细胞比例及IL-17A表达水平明显升高;而相较于NOD组,SCAP-Exo组小鼠唾液流率明显增加、外周血Th17/CD4+ T细胞比例及IL-17A表达水平明显降低;差异均有统计学意义（均P < 0.05）。相较于NOD组,SCAP-Exo组小鼠下颌下腺淋巴细胞浸润水平显著降低,仅存在轻度散在的淋巴细胞浸润或出现极个别的淋巴细胞浸润灶。结论    SCAP-Exo能有效治疗SS小鼠,可能与其调节Th17细胞转化有关。     

牙周炎不同阶段Th17/Treg细胞平衡变化初探

目的 建立实验性大鼠牙周炎模型,观察外周血及牙周组织Th17/Treg细胞平衡在牙周炎不同阶段的变化以及牙槽骨高度变化,为探寻用于研究牙周炎正畸牙齿移动的动物模型提供参考。方法 32只SD雄性大鼠随机分为3个实验组(牙周炎A组、牙周炎B组和牙周炎C组,每组8只)和1个对照组(8只)。实验组采用牙颈部丝线结扎并局部注射LPS的方法建立实验性牙周炎模型后,牙周炎A组处死,采用流式细胞仪检测外周血Th17细胞、Treg细胞比例变化。免疫组化检测牙周组织ROR-γt、Foxp3表达。实时定量PCR检测牙龈组织ROR-γt mRNA、Foxp3 mRNA的表达。牙周炎B组和C组去除局部刺激并行牙周洁治,分别于术后第7、14天进行牙周检查,并行上述流式细胞等检测。结果 牙周炎A、B及C组外周血Th17细胞、Treg细胞、Th17/Treg细胞比例及牙龈组织ROR-γt mRNA、Foxp3 mRNA的表达均较对照组明显升高( P <0.05)。牙周炎B组和C组Th17细胞、Th17/Treg细胞比例及牙龈组织ROR-γt mRNA的表达较牙周炎A组明显降低( P <0.05),但Treg细胞及Foxp3 mRNA表达较牙周炎A组明显升高( P <0.05)。牙周炎B组和C组相比,Th17细胞、Treg细胞及Th17/Treg细胞比例差异无统计学意义( P >0.05)。牙周炎A组、B组、C组牙槽嵴顶距釉牙骨质界距离均较对照组明显增加( P <0.05),但三组间差异无统计学意义( P >0.05)。结论 Th17、Treg细胞及Th17/Treg细胞平衡参与不同阶段牙周炎的免疫调控。局部炎症因素去除后,大鼠牙周炎病变稳定,可以尝试作为研究牙周炎正畸牙齿移动的动物模型。     

慢性牙周炎伴冠心病患者Th17/Treg细胞平衡的临床意义

目的探讨慢性牙周炎伴冠心病患者外周血中辅助性T细胞17（Th17）、调节性T细胞（Treg）及Th17/Treg细胞平衡表达水平及临床意义。 方法本研究采用2 × 2析因设计,根据冠状动脉造影结果及牙周炎诊断标准,收集2019年3—12月就诊于新疆维吾尔自治区人民医院心内科及门诊口腔科患者113例研究对象,其中健康对照组28例、单纯慢性牙周炎组26例、单纯冠心病组27例和冠心病合并慢性牙周炎组32例。流式细胞术检测外周血Th17、Treg细胞比例,计算Th17/Treg细胞比值。采用方差分析比较各组细胞比例,2 × 2析因分析探讨牙周炎和冠心病对Th17/Treg细胞平衡的交互作用。 结果与健康对照组、慢性牙周炎组及冠心病组相比,冠心病合并慢性牙周炎组Th17细胞比例[（1.4 ± 0.6）%]显著升高,差异有统计学意义（F = 18.40,P<0.001）。与健康对照组[（8.73 ± 2.70）%]相比,冠心病合并慢性牙周炎组Treg细胞比例[（6.26 ± 2.51）%]显著降低,差异有统计学意义（F = 6.29,P = 0.001）。Th17/Treg比值在牙周炎伴冠心病组[0.221（0.317）]与健康对照组[0.055（0.054）]和慢性牙周炎组[0.119（0.115）]比较显著升高,差异有统计学意义（χ² = 40.25,P<0.001）,存在Th17/Treg失衡;析因分析表明,牙周炎与冠心病对Treg细胞水平存在交互效应（F = 3.91,P = 0.041）。 结论牙周炎伴冠心病患者存在Th17/Treg比例失衡,且向Th17细胞偏移,表明Th17/Treg细胞平衡可能参与牙周炎伴冠心病的发生、发展。     

实验性牙周炎大鼠体内外周血及牙周组织中Th17/Treg的检测

目的初步探讨实验性牙周炎大鼠外周血及炎症牙周组织中辅助性T细胞17（Th17）和调节性T细胞（Treg）的表达。 方法采用丝线结扎并牙龈卟啉单胞菌菌液涂抹法建立SD大鼠牙周炎模型,采集外周血、龈沟液以及颌骨样本。体视显微镜及苏木精-伊红染色法观察牙周组织病理改变,流式细胞术检测外周血Th17细胞与Treg细胞的比例,酶联免疫吸附法检测外周血及龈沟液中白细胞介素17（IL-17）、IL-10、转化生长因子β（TGF-β）表达水平,实时荧光定量聚合酶链反应（PCR）法检测牙周组织中RORγt、Foxp3 mRNA的表达。应用SPSS 22.0软件独立样本t检验及方差分析对数据进行统计学分析。 结果实验大鼠牙周组织病理改变符合牙周炎病理改变特征,牙槽骨吸收明显。实验组外周血Th17（F=30.88,P=0.00）、Treg细胞（F=147.03,P=0.00）比例以及Th17/Treg比率较对照组均显著上升（F=219.53,P=0.00）;实验组牙周组织中RORγt（F=35.61,P=0.04）和Foxp3 mRNA（F=216.60,P=0.01）表达较对照组表达显著上升;RORγt/Foxp3 mRNA比率较对照组升高,但差异无统计学意义（F=0.63,P=0.44）;实验组外周血血清中IL-10浓度较对照组显著上升（F=6.41,P=0.02）,而IL-17（F=0.08,P=0.78）、TGF-β（F=3.48,P=0.06）浓度与对照组差异无统计学意义;实验组龈沟液中IL-17（F=9.03,P=0.01）较对照组显著上升;IL-10（F=5.85,P=0.08）及TGF-β含量（F=9.28,P=0.06）含量较对照组升高,但差异无统计学意义。 结论实验性牙周炎大鼠外周血及牙周炎症组织中Th17与Treg表达均显著上调,可能与牙周炎症组织病理改变以及相关全身系统性疾病存在相关性。     

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。     

慢性牙周炎病人基础治疗前后Th17型细胞因子及RORC的表达变化

目的:研究慢性牙周炎(CP)病人牙周基础治疗前后龈沟液(GCF)中的Th17型细胞因子及其外周血特异性转录因子RORC的表达变化规律。方法:选择CP病人30例,使用ELISA检测牙周基础治疗前后GCF中Th17型细胞因子(IL-17、IL-21)的水平变化,并利用荧光定量PCR检测牙周基础治疗前后外周血CD4+T细胞中特异性转录因子RORC的表达水平。结果:牙周基础治疗后龈沟液中的Th17型细胞因子IL-17、IL-21水平以及外周血CD4+T细胞中特异性转录因子RORC表达水平均较治疗前显著下降,差异均有统计学意义(P<0.05)。结论:Th17型细胞因子在慢性牙周炎发生发展中发挥重要的致炎作用。     

牙周炎患者外周血Th1、Th2、Th17表达及与牙龈组织IL-17、IFN-γ水平的相关性研究

目的 观察牙周炎患者外周血辅助性T1(Th1)、辅助性T2(Th2)、辅助性T17(Th17)和牙龈组织白细胞介素(IL-17)、干扰素-γ(IFN-γ)表达水平,分析其相关性.方法 选择我院口腔科2017年7月～2019年12月97例牙周炎患者作为研究对象,根据炎症严重程度分别纳入轻度组(n=29)、中度组(n=35...     

慢性牙周炎患者IL-17mRNA的表达及意义

目的：观察TL-17mRNA基因在慢性牙刷炎和健康牙龈组织中的表达水平变化,探讨TL-17在慢性牙周炎发生发展巾的作用。方法：选择慢性牙周炎患者23例,正常对照组15例,记录才周临床指标,采用Real—time PCR方法定量检测TL-17mRNA在牙龈组织中的表达。结果：慢性牙周炎组TL-17mRNA相对表达晕（0．00147＋0．00055）显著高于正常牙龈组（O．00047±0．00019）（P〈0．01）,并且慢性牙周炎组TL-17mRNA表达水平与牙龈指数（r=0．58,P〈0．01）、牙周袋探诊深度（r=0．57,P〈0．01）、附着丧失水平（r=0．49,P〈0．05）呈正相关。结论：Th17相关细胞凶子IL—17呵能住慢性牙周炎发病机制中发挥致炎作用。     

The presence of IL-17+/FOXP3+ double-positive cells in periodontitis

Increasing evidence suggests that distinct inflammatory cytokines convert forkhead box protein P3 (FOXP3(+)) regulatory T-cells (Tregs) into IL-17-producing cells (Th17 cells) in vitro. However, this functional plasticity has not been examined in the pathogenesis of periodontal disease. In this study, we analyzed the IL-17A(+)FOXP3(+) cells present in periodontitis lesions to determine the association between Treg conversion and the pathogenesis of periodontitis. The immunohistochemical analysis of gingival tissues demonstrated that the numbers of Th17 cells (IL-17A(+)FOXP3(-)) and Tregs (IL-17A(-)FOXP3(+)) were greater in periodontitis lesions than in gingivitis lesions. We further identified a small number of IL-17A(+)FOXP3(+) cells in periodontitis lesions but not in gingivitis lesions. The flow cytometry analysis of CD4(+) T-cell lines established from gingival tissues and the peripheral blood of periodontitis patients showed that the proportion of Tregs was reduced and the proportion of IL-17A(+)FOXP3(+) cells among all FOXP3(+) cells was elevated in gingival tissue T-cell lines relative to the proportions in peripheral blood T-cell lines. Our findings indicate that Treg-Th17 conversion may occur in periodontitis lesions. Further studies addressing the role of Treg conversion during inflammatory responses against periodontopathic bacteria are needed.     

Different responses in B cells induced by Porphyromonas gingivalis and Fusobacterium nucleatum.

A phenotypic study had shown that gingival B cells respond differently to two periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum. Further investigation now shows a reduction in the percentage of Ki-67 + T cells in cultures of gingival and peripheral blood mononuclear cells stimulated with P. gingivalis for 3 and 6 days, respectively, but no suppression of Ki-67 expression in B cells in response to either P. gingivalis or F. nucleatum. Depletion studies of cultures of peripheral blood mononuclear cells showed that in the absence of CD4 cells, the percentage of CD19+ and CD20+ B cells stimulated with P. gingivalis increased after 6 days whereas depletion of CD8 cells resulted in a rise in the percentage of F. nucleatum- and P. gingivalis-stimulated B cells, although this was not significant in the case of P. gingivalis. Specific antibody to P. gingivalis and F. nucleatum was found in culture supernatants of gingival but not of peripheral blood mononuclear cells, indicating a possible higher frequency of antigen-specific B cells in periodontal lesions. IgG was the predominant isotype in both gingival and control peripheral blood cultures, followed closely by IgA in gingival cultures. F. nucleatum stimulated higher levels of Ig in cultures of peripheral blood mononuclear cells than P. gingivalis or cells cultured in medium only, whereas in gingival cell cultures, stimulation by P. gingivalis appeared to result in higher levels of IgG. Also Ig was present at day 3 in gingival cultures, whereas in the blood cell cultures, Ig was only detected at day 6, further suggesting a degree of activation of of gingival B cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

壳寡糖对牙周炎大鼠牙槽骨吸收及Th17/Treg平衡和OPG/RANKL/RANK通路的影响

目的: 探讨壳寡糖对牙周炎大鼠牙槽骨吸收及Th17/Treg平衡和OPG/RANKL/RANK通路的影响。方法: 建立牙周炎大鼠模型,随机分为模型组、壳寡糖低剂量组、壳寡糖中剂量组、壳寡糖高剂量组和甲硝唑组,每组12只,另取12只作为对照组。分组处理后,评估牙龈指数、牙槽骨吸收值;H-E染色观察牙周组织病理形态学变化;流式细胞术检测外周血中Th17/Treg细胞比值;酶联免疫吸附试验（ELISA）检测各组大鼠血清中IL-17、TGF-β、RANKL、OPG水平,实时荧光定量PCR（qRT-PCR）检测各组大鼠牙周组织OPG、RANKL mRNA表达水平。采用SPSS 24.0软件包对数据进行统计学分析。结果: 与对照组相比,模型组大鼠牙周组织呈现牙周膜纤维束断裂、排列紊乱,毛细血管扩张、增生,炎症细胞浸润等病理损伤;牙龈指数、牙槽骨吸收值、Th17/Treg比值、血清RANKL及IL-17水平、牙周组织RANKL mRNA水平显著升高（P<0.05）,血清OPG及TGF-β水平、牙周组织OPG mRNA水平显著降低（P<0.05）。与模型组相比,壳寡糖低、中、高剂量组和甲硝唑组大鼠牙周组织病理损伤减轻;牙龈指数、牙槽骨吸收值、Th17/Treg比值、血清RANKL及IL-17水平、牙周组织RANKL mRNA水平显著降低（P<0.05）,血清OPG及TGF-β水平、牙周组织OPG mRNA水平显著升高（P<0.05）,且壳寡糖各组呈剂量依赖性,壳寡糖高剂量组与甲硝唑组相比,差异无统计学意义（P>0.05）。结论: 壳寡糖可促使Th17/Treg平衡恢复正常,上调OPG表达,下调RANKL表达,抑制牙周炎大鼠牙槽骨吸收,改善其临床症状。     

Establishment of peripheral blood and gingival T lymphocyte clones responsive to Porphyromonas gingivalis

T cells are central to the immune response to infection and studies have indicated a local immunoregulatory imbalance may exist in human periodontal disease. Since Porphyromonas gingivalis is generally recognized as a major periodontopathogen, the aim of this study was to establish T cell lines and clones specific to P. gingivalis from the gingival tissues and peripheral blood of P. gingivalis — infected subjects. Two subjects were selected from two groups of individuals (one from each group) established on the basis of P. gingivalis in their plaque and the presence of serum antibodies which react with P. gingivalis antigens. The two groups differed however in their clinical susceptibility (adult periodontitis) or resistance (gingivitis) to periodontal breakdown. The mean ages ± standard error of the mean of the two groups were 47.9 ± 2.2 and 49.6 ± 3.7, respectively, so that resistance in the gingivitis group was related to the age of the subjects. T cell lines and clones were established from the peripheral blood of one patient from each of the two groups and also from the gingival tissues of the same periodontitis subject. This study has demonstrated the capability of establishing P. gingivalis -specific T cell lines and clones from P. gingivalis -infected subjects and FACS analysis of the T cell receptor variable regions demonstrated that the clones were indeed monoclonal. The CD4:CD8 ratios of the peripheral blood-derived T cell lines were 1.2 and 0.4 for the gingivitis-derived line and the periodontitis-derived line, respectively, thus supporting the clinical differences displayed by the two subjects.     

Interleukin 1, interleukin 6 and transforming growth factor-β production by human gingival mononuclear cells following stimulation with Porphyromonas gingivalis and Fusobacterium nucleatum

Gingival mononuclear cell production of interleukin 1 (IL-1), interleukin 6 (IL-6) and transforming growth factor-β(TGF-β) after stimulation with the putative periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum was investigated. Using an ELISA method, gingival mononuclear cells extracted from 18 adult periodontitis subjects were found to be producing IL-1. However, IL-1 activity could only be detected in 5 out of these 18 cases when tested using a thymocyte proliferation bio-assay, suggesting the presence of IL-1 inhibitors. Depletion of monocytes from peripheral blood cultures resulted in a significant decrease in IL-1 activity following P. gingivalis stimulation while there was no effect in the level of IL-1 activity following stimulation with F. nucleatum. This suggests that P. gingivalis and F. nucleatum stimulate different cell types to produce IL-1. Like IL-1. IL-6 production by gingival mononuclear cells was significantly greater than that produced by the control peripheral blood mononuclear cells. Following P. gingivalis and F. nucleatum stimulation, higher levels of IL-6 could be detected; however, both organisms stimulated similar levels. Intracytoplasmic immunofluorescence staining demonstrated a lower percent TGF-β⁺ cells in bacterial stimulated peripheral blood mononuclear cell cultures compared with cells in medium alone. In the gingival mononuclear cell cultures, the percentage TGF-β⁺ cells peaked at day 1 in F. nucleatum -stimulated, whereas in P. gingivalis -stimulated cultures the peak TGF-β⁺ cells occurred at day 3, again suggesting stimulation of different cell subsets. These results ilustrate that different periodontopathic bacteria may stimulate different cell types to produce cytokines which may have synergistic or antagonistic effects.