非肥胖型糖尿病小鼠自发性涎腺炎的发生与发展

目的 观察雌性非肥胖型糖尿病（NOD）小鼠自发性涎腺炎的发生发展过程。方法 选择5、10、15、20周龄雌性NOD小鼠各6只。测定刺激全唾液流率（STFR）、施墨试验、唾液总蛋白、常规HE切片及电镜超微结构观察涎腺组织的改变。以BALB／c小鼠作对照。结果 10、15、20周NOD小鼠STFR、唾液总蛋白均明显低于对照组（P〈0．05），相应的下颌下腺分泌颗粒减少。10周雌性NOD小鼠涎腺炎发病率为4／6，15、20周均为6／6。淋巴细胞浸润主要见于下颌下腺，舌下腺极少，腮腺未见。10周时NOD小鼠已有淋巴细胞浸润灶形成。15周显著增多而且面积增加。STFR与淋巴细胞浸润灶数呈负相关。结论 雌性NOD小鼠5～10周淋巴细胞开始浸润下颌下腺，刺激唾液和蛋白分泌降低。不同腺体受累情况不同。     

Toll样受体7、8、9在原发性舍格伦综合征中的表达

目的:检测原发性舍格伦综合征(primary Sjogren's syndrome,pSS)患者血细胞中Toll样受体(TLR)7、8、9的表达水平,并检测TLR7、9在pSS患者腮腺组织中的表达.方法:采用实时定量PCR,对37例pSS患者和24例对照组患者血细胞中TLR7、8、9的水平进行检测,分析2组之间的差异.应用免疫荧光法和免疫组化分别检测TLR7和TLR9在pSS患者腮腺组织中的表达.应用SAS6.12软件包,对数据进行t检验.结果:实时定量PCR显示,pSS组的TLR7、9的mRNA表达量显著高于对照组,2组之间有显著差异(P<0.05),pSS组的TLR7是对照组的2.17倍,pSS组的TLR9是对照组的2.33倍.而TLR8在2组之间无显著差异(p>0.05).免疫荧光和免疫组化发现,TLR7、9在pSS组患者腮腺组织中的导管上皮细胞、淋巴细胞和上皮岛均为阳性;而对照组腮腺组织中仅导管上皮细胞为阳性.结论:TLR7和TLR9在原发性舍格伦综合征患者中存在表达异常.     

Activation of innate immunity accelerates sialoadenitis in a mouse model for Sjögren's syndrome-like disease

Oral Diseases (2011) 17 , 801–807 Objective: Sjögren’s syndrome is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the salivary and lacrimal glands. This study was undertaken to investigate the effects of innate immunity activation on sialoadenitis in a mouse strain genetically susceptible for development of SS‐like disease. Methods: Female New Zealand Black X New Zealand White F1 mice were repeatedly treated with toll‐like 3 receptor agonist poly(I:C). Submandibular glands were investigated at different time points for sialoadenitis by immunohistochemistry and for gene expression of different chemokines by quantitative PCR. Submandibular gland–infiltrating cells were characterized by flow cytometry. Results: Poly(I:C) treatment significantly upregulated the expression of multiple chemokines within the submandibular glands. The severity and incidence of sialoadenitis was considerably higher in poly(I:C)‐treated mice. There was a preponderance of dendritic cells and NK cells in the initial inflammatory cell infiltrates, and these were followed by CD4+ T cells. Conclusions: Our data clearly demonstrate that systemic activation of innate immunity accelerates sialoadenitis in a mouse model for SS‐like disease. These findings suggest that chronic activation of innate immunity can influence certain features of SS.     

TLR9在成牙本质细胞中的表达

目的：研究成牙本质细胞中TLR9、DSPP基因表达特征及信号转导途径。方法：采用RT-PCR检测TLR9在小鼠牙髓组织中及TLR9、DSPP在成牙本质细胞系中的表达。用CpGODN-A和CpGODN-B刺激细胞,在时间梯度0、3、6、9、12、24h,检测TLR9、DSPP基因的表达特征。结果：TLR9mRNA在小鼠牙髓组织中有表达。TLR9、DSPPmRNA在成牙本质细胞中都有显著的表达,在CpGODN刺激下显著上调,在6h处于峰值。结论：TLR9在成牙本质细胞中有表达;TLR9的特异性表达对DSPP基因表达量有影响。     

非肥胖型糖尿病小鼠唾液流率、颌下腺造影及病理研究

目的:研究非肥胖型糖尿病小鼠(nonobese diabetic mouse,NOD)唾液总流率;颌下腺造影及病理表现.材料及方法:本研究将56只NOD小鼠分9周、16周、20周及24周4个不同年龄组测定其唾液总流率,颌下腺造影及颌下腺病理学研究,用32只Balb/c小鼠分同样年龄组进行对照.结果:NOD小鼠唾液总流率随年龄增大而下降,Balb/c小鼠则变化不大.20周以后NOD小鼠颌下腺造影有造影剂外溢,排空功能明显迟缓.9周NOD小鼠颌下腺见淋巴细胞浸润,随年龄增长,淋巴细胞浸润加重.结论:NOD小鼠涎腺的形态及功能均明显受累,与人类SS表现类似.     

Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function

Background:  Recent studies have demonstrated the expression of Toll-like receptor 3 (TLR3) in salivary glands and epithelial cell lines derived from Sjögren's syndrome (SS) patients. As viral infections are considered to be a trigger for SS, in this study we investigated whether in vivo engagement of TLR3 affects salivary gland function.
Methods:  Female New Zealand Black/WF1 mice were repeatedly injected with polyinosinic:polycytidylic acid [poly(I:C)]. TLR3 expression within submandibular glands was studied using immunohistochemistry. RNA levels of inflammatory cytokines in the submandibular glands were determined by real time polymerase chain reaction. Pilocarpine induced saliva volume was used as an index of glandular function.
Results:  Immunohistochemical analysis of submandibular glands showed TLR3 expression in epithelium of serous and mucous acini, granular convoluted tubules, and ducts. Poly(I:C) treatment rapidly up-regulated the mRNA levels of type I interferon (IFN) and inflammatory cytokines in the submandibular glands. One week after treatment, the saliva volumes in poly(I:C) treated mice were significantly reduced in comparison with the phosphate-buffered saline (PBS) treated mice. Hematoxylin and eosin staining showed that salivary gland histology was normal and lymphocytic foci were not detected. Glandular function recovered after poly(I:C) treatment was stopped.
Conclusions:  Our results demonstrate that engagement of TLR3 within the salivary glands results in a rapid loss of glandular function. This phenomenon is associated with the production of type I IFN and inflammatory cytokines in the salivary glands. Restoration of glandular function suggests that for viral etiology of SS, a chronic infection of salivary glands might be necessary.     

Functional TLRs and NODs in human gingival fibroblasts

Since human gingival fibroblasts are the major cells in periodontal tissues, we hypothesized that gingival fibroblasts are endowed with receptors for bacterial components, which induce innate immune responses against invading bacteria. We found clear mRNA expression of Toll-like receptors (TLR)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, MD-2, MyD88, NOD1, and NOD2 in gingival fibroblasts. Gingival fibroblasts constitutively expressed these molecules. Upon stimulation with chemically synthesized ligands mimicking microbial products for these receptors, the production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1, was markedly up-regulated. Furthermore, the production of pro-inflammatory cytokines induced by TLR and NOD ligands was significantly inhibited by an RNA interference assay targeted to NF-kappaB. These findings indicate that these innate immunity-related molecules in gingival fibroblasts are functional receptors involved in inflammatory reactions in periodontal tissues, which might be responsible for periodontal pathogenesis.     

中药雷公藤多苷治疗NOD小鼠自发性涎腺炎的研究

目的观察中药雷公藤多苷对非肥胖型糖尿病（NOD）小鼠自发性涎腺炎的治疗作用,并探讨其作用机制。方法8周龄雌性NOD小鼠27只,随机分成3组：生理盐水组、羟氯喹组及雷公藤多苷组。自9周龄开始,将等效剂量药物溶于0.4ml生理盐水中每天灌胃给药,直到20周龄处死。12、16及20周龄时收集每组小鼠的唾液流量,血清、颌下腺组织。采用苏木素-伊红（HE）染色观察颌下腺组织病理学改变;酶联免疫吸附试验（ELISA）检测血清自身抗体（SSB及α-fodrin抗体）及相关细胞因子（IL-10及IFN-r）水平。结果与生理盐水组相比,雷公藤多苷组及羟氯喹组小鼠治疗后唾液流量明显增加,颌下腺炎性浸润减轻,血清自身抗体水平明显下降,TH1/TH2型细胞因子表达失调有所改善（P〈0.05）。雷公藤多苷组和羟氯喹组间差异无显著性,P0〉.05。结论雷公藤多苷对NOD小鼠自发性涎腺炎有一定治疗作用,其作用机制可能与药物减轻颌下腺淋巴细胞灶性浸润程度及改善TH1/TH2型细胞因子表达失调等有一定关系。     

S100A9在糖尿病大鼠牙周组织中的表达及其作用研究

目的 研究S100A9蛋白在糖尿病大鼠牙周组织中的表达,探讨其在糖尿病诱发的牙周病变中可能的作用机制。方法 本实验通过对SD大鼠腹腔注射链脲佐菌素（STZ）构建糖尿病大鼠模型,通过苏木精-伊红（HE）染色观察糖尿病大鼠牙周结构的变化,免疫组织化学染色观察糖尿病大鼠牙周组织中S100A9的表达与分布,同时检测其配体Toll受体4（TLR4）和核转录因子κB（NF-κB）/p-P65蛋白的表达。通过分析上述蛋白的表达规律,探讨S100A9蛋白在糖尿病诱发的牙周病变中的作用机制。结果 糖尿病大鼠的牙槽骨骨小梁结构稀疏,硬骨板消失;免疫组织化学染色显示牙周膜、牙槽骨及牙龈上皮中S100A9的表达水平比对照组明显上调,TLR4在牙槽骨、牙周膜、牙龈中的表达水平相较于对照组也显著增强;p-P65在对照组中没表达,但在糖尿病组中牙周膜和牙槽骨中呈阳性表达。结论 糖尿病导致大鼠牙周组织结构病变,其原因可能与S100A9介导的TLR4和NF-κB信号通路的活化有关。     

Systemic treatment of anti-CD4+CD25+ T cell monoclonal antibody exacerbates sialoadenitis in submandibular glands during the early life in lupus-prone female NZB × NZWF1 mice

Background:  The maintenance mechanisms of peripheral tolerance by CD4⁺CD25⁺ T cells before the development of sialoadenitis in secondary Sjögren's syndrome (sSS) are not well understood. The aim of the present study is to examine the effect of reduction of CD4⁺CD25⁺ T cells on the development of sialoadenitis during the early life in female NZB × NZWF₁ (B/WF₁) mice, a model for human sSS.
Methods:  Female B/WF₁ mice at 3 days after birth were treated with either anti-mouse CD4⁺CD25⁺ T cells rat IgG₁ monoclonal antibody (mAb) or Rat IgG₁(control). At 25 weeks of age, autoantibodies against nucleus and cytoplasm of ductal epithelial and myoepithelial cells, and histpathology of submandibular glands were examined in the mAb-treated and control groups. Also the development of anti-Ro/SS-A antibodies was examined until 25 weeks of age in both groups.
Results:  The mAb-treated group showed severe lesions with the development of autoantibodies compared to the control group.
Conclusions:  The present results suggest that peripheral CD4⁺CD25⁺ T cells may, at least in part, contribute to down-regulate the development of sialoadenitis in submandibular glands of lupus-prone female B/WF₁ mice during their early life.     

Recovery of INS-R and ER-alpha expression in the salivary glands of diabetic mice submitted to hormone replacement therapy

The interaction between proteins and cell receptors is related to tissue homeostasis such as in salivary glands. In this respect, alterations in hormone levels caused by hyperglycaemic conditions may interfere with this interaction, intensifying the damage caused by diabetes mellitus. Hormone replacement therapy is an option to reverse this damage, but doubts still exist regarding the efficacy of this procedure. The objective of this study was to evaluate the effect of oestrogen replacement therapy combined with insulin treatment on the expression of oestrogen (ER-alpha) and insulin receptors (INS-R) in the salivary glands of spontaneously diabetic mice. Twenty-five mice were divided into five group of 5 animals each: group I (NOD diabetic), group II (NOD diabetic treated with insulin), group III (NOD diabetic treated with oestrogen), group IV (NOD diabetic treated with insulin and oestrogen), and group V (control BALB/c mice). Group II received insulin, group III received oestrogen, and group IV received insulin plus oestrogen administered daily for 20 days. Groups I and V received saline for the same period of time to simulate treatment. Glucose and oestrogen levels were monitored during the experimental period and salivary gland samples were collected at the end of the experiment for fluorescence microscopy analysis of ER-alpha and INS-R. Animals receiving oestrogen replacement therapy plus insulin showed regulation of the expression of oestrogen and insulin receptors. Oestrogen treatment alone contributed to the recovery of these cell receptors. These results indicate that oestrogen replacement therapy alone, and especially when combined with insulin, is important for the recovery of the interaction between functional proteins and their receptors, thus contributing to the reestablishment of tissues damaged by the hyperglycaemic condition.     

Histamine deficiency inhibits lymphocyte infiltration in the submandibular gland of aged mice via increased anti-aging factor Klotho

ObjectivesHistidine decarboxylase (HDC), a histamine synthase, is expressed in various tissues and is induced by proinflammatory cytokines such as TNFα. As they age, C57BL/6 mice show auto-antibody deposition and lymphocyte infiltration into various tissues, including salivary glands. However, the mechanism underlying cell infiltration and the change in HDC expression in salivary glands with aging remain unclear. Thus, we aimed to elucidate the relationship between histamine and inflammaging.MethodsWe investigated the change in histology and HDC expression in the major salivary glands (parotid, submandibular, and sublingual) of 6-week- and 9-month-old wild-type mice. We also determined the histological changes, cytokine expression, and anti-aging factor Klotho in the salivary glands of 9-month-old wild-type and HDC-deficient (HDC-KO) mice.ResultsCell infiltration was observed in the submandibular gland of 9-month-old wild-type mice. Although most cells infiltrating the submandibular glands were CD3-positive and B220-positive lymphocytes, CD11c-positive and F4/80-positive monocyte lineages were also detected. HDC, TNFα, and IL-1β mRNA expression increased in the submandibular gland of 9-month-old wild-type mice. The expression of PPARγ, an anti-inflammatory protein, declined in 9-month-old wild-type mice, and Klotho expression increased in 9-month-old HDC-KO mice. Immunohistochemistry showed that Klotho-positive cells disappeared in the submandibular gland of 9-month-old wild-type mice, while Klotho was detected in all salivary glands in HDC-KO mice of the same age.ConclusionOur findings demonstrate the multifunctionality of histamine and can aid in the development of novel therapeutic methods for inflammatory diseases such as Sjogren's syndrome and age-related dysfunctions.     

Effect of Myd88 deficiency on gene expression profiling in salivary glands of female non-obese diabetic (NOD) mice

ObjectivesSjögren's syndrome (SS) is a chronic autoimmune disease characterized by inflammatory lesions in the salivary and lacrimal glands, which are caused by distinct lymphocytic infiltrates. Female non-obese diabetic (NOD) mice spontaneously develop inflammatory lesions of the salivary glands with SS-like pathological features. Previous studies have shown that MyD88, a crucial adaptor protein that activates innate immune signaling, affects lymphocytic infiltration, but its detailed role remains unclear. In this study, we investigated the role of MyD88 through gene expression profiling in the early phase of pathogenesis in the salivary glands of female NOD mice.MethodsSubmandibular glands collected from 10-week-old female wild-type and Myd88-deficient NOD mice were used for RNA preparation, followed by microarray analysis. The microarray dataset was analyzed to identify Myd88-dependent differentially expressed genes (DEGs). Data generated were used for GO enrichment, KEGG pathway, STRING database, and INTERFEROME database analyses.ResultsMyd88 deficiency was found to affect 230 DEGs, including SS-associated genes, such as Cxcl9 and Bpifa2. Most of the DEGs were identified as being involved in immunological processes. KEGG pathway analysis indicated that the DEGs were putatively involved in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, the DEGs included 149 interferon (IFN)-regulated genes.ConclusionsMyD88 is involved in the expression of specific genes associated with IFN-associated immunopathological processes in the salivary glands of NOD mice. Our findings are important for understanding the role of MyD88-dependent innate immune signaling in SS manifestation.     

Toll样受体9与头颈部鳞状细胞癌关系的研究进展

Toll样受体(TLR)9与特异性配体结合后,不仅在包括头颈鳞状细胞癌(HNSCC)在内的多种恶性肿瘤的发生发展中高表达,而且具有促进肿瘤细胞增殖、侵袭及转移的作用;但TLR9在一些肿瘤中高表达预示较高的术后复发率及较低的术后生存率,在另一些肿瘤中高表达却是肿瘤预后较好的分子指标.TLR9参与HNSCC的相关分子机制,在于促进细胞增殖、抑制程序性细胞死亡、调节血管新生、促进肿瘤侵袭和促进免疫逃逸.目前,基于TLR9的抗肿瘤治疗已被广泛用于临床,其最终的临床效果尚待进一步了解TLR9的表达调控,分析TLR9参与HNSCC的双刃剑作用机制及相关信号转导环节,开发特异性高、不良反应小和可有效用于临床的TLR9佐剂或抑制剂.     

Presence of BPIFB1 in saliva from non-obese diabetic mice

We previously showed that mRNA expression of BPIFB1 (Bpifb1), an antibacterial protein in the palate, lung, and nasal epithelium clone protein family, was increased in parotid acinar cells in non-obese diabetic (NOD, NOD/ShiJcl) mice, which is an animal model for Sjögren’s syndrome. However, we did not previously assess the protein levels. In this report, we confirmed the expression of BPIFB1 protein in the parotid glands of NOD mice. Immunoblotting of subcellular fractions revealed that BPIBB1 was localised in secretory granules in parotid glands from NOD mice, and was almost not in parotid glands from the control mice. BPIFB1 had N-linked glycan that reacted with Aleuria aurantia lectin, which caused two types of spots with a slightly different pI and molecular weight. The expression of BPIFB1 protein was also demonstrated by immunohistochemistry. BPIFB1 was detected in the saliva from NOD mice but not in the saliva from the control mice, indicating individual constitution. BPIFB1 in saliva may be applied to other research as a diagnostic marker.     

Local suppression of IL‐21 in submandibular glands retards the development of Sjögren’s syndrome in non‐obese diabetic mice

J Oral Pathol Med (2012) 41 : 728–735 Background: The aim of this study was to verify the validity of IL‐21 local suppression in submandibular glands of preventing the development of Sjögren’s syndrome in non‐obese diabetic (NOD) mice and figure out the mechanism. Methods: IL‐21 levels in submandibular glands were suppressed by ductal cannulation of IL‐21 shRNA lentivirus. Then, saliva flow rates (SFR) and histopathologic changes of submandibular glands were measured to assess the severity of disease development. Real‐time PCR, flow cytometry, and immunohistochemistry were used to detect the changes of T helper cells and related cytokines. Results: The reduction in SFRs in NOD mice was significantly alleviated from 9 to 17 weeks of age along with the suppression of IL‐21 in submandibular glands. Lymphocytic infiltration was also milder than control NOD mice. Moreover, the lower level of IL‐21 led to the down‐regulation of follicular helper T (Tfh) cells. Conclusions: Local suppression of IL‐21 in submandibular glands could retard the development of Sjögren’s syndrome in NOD mice. IL‐21 might contribute to the development of B‐cell disorder in Sjögren’s syndrome via Tfh cells pathway.     

电离辐射对大鼠腮腺旁细胞分泌途径损伤及紧密连接蛋白claudin-4的影响

目的: 探讨电离辐射对大鼠腮腺旁细胞分泌功能损伤及紧密连接 (tight junction,TJ) 蛋白claudin-4的影响与潜在作用机制。方法: 24只8周龄雄性Wistar大鼠,随机分为对照组（6只）和照射组（照射后1周组、4周组、12周组,每组6只）。照射组一次性20 Gy射线局部照射实验侧腮腺区。采用Schirmer实验,检测各组腺体唾液静息分泌量;H-E染色,光镜下观察腺体组织病理变化;透射电镜观察TJ超微结构改变;免疫荧光染色和蛋白质印迹检测毒蕈碱型乙酰胆碱受体（M受体）亚型M3、水通道蛋白5（AQP5）及claudin-4的蛋白表达。采用SPSS 23.0软件包对数据进行统计学分析。结果: 照射后1、4、12周,腺体静息分泌量较对照组显著减少（P<0.05）;12周较1周、4周时减少更显著（P<0.05）。组织学观察可见,照射后腺体早期间质血管扩张、充血,后期腺泡细胞计数显著减少（P<0.05）;TJ结构模糊、电子密度降低、宽度减少（P<0.05）。免疫荧光染色和蛋白质印迹检测结果显示,M3受体及AQP5蛋白在照射后1、4、12周表达显著下调,而claudin-4蛋白表达显著升高。结论: 电离辐射后腮腺旁细胞分泌功能降低,TJ结构改变,claudin-4表达上调,可能参与腺体分泌功能损伤。