中药白芍总苷预防非肥胖型糖尿病小鼠自发性涎腺炎的研究

目的 探讨中药白芍总苷对非肥胖型糖尿病(NOD)小鼠自发性涎腺炎的预防作用及机制.方法 4周龄雌性NOD小鼠27只,随机分成3组:生理盐水组、羟氯喹组及白芍总苷组.自5周龄开始,将等效剂量药物溶于04 mL生理盐水中每天灌胃给药,直到20周龄处死.10、15及20周龄时收集每组小鼠的唾液流量以及血清、颌下腺组织.采用苏...     

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。     

TLR9依赖的p38MAPK信号通路对鼠原发性舍格伦综合征的影响

目的:在动物模型NOD(非肥胖型糖尿病)鼠中,观察研究Toll样受体9(Toll Like Receptor 9,TLR9)依赖的p38MAPK信号通路在原发性舍格伦综合征发病机制中的作用。方法:实验选取5周龄的雌性NOD小鼠,分别给予3种抑制剂:ODN2088、VX-792、羟氯喹。利用流式细胞学技术检测小鼠外周血淋巴细胞的情况。利用免疫组化检测小鼠下颌下腺TLR9及p-p38 MAPK的表达情况。利用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测小鼠外周血中血浆抗体的表达。利用Tunel方法检测小鼠下颌下腺腺上皮细胞的凋亡。同时,观察小鼠刺激性唾液流率的改变以及下颌下腺病理学改变。结果:只有ODN2088组的NOD小鼠的唾液流率显著增加。在所有被给予羟氯喹的NOD鼠和未接受治疗的NOD鼠中,下颌下腺均有淋巴细胞浸润灶的出现。但在ODN2088组中,只有1只NOD小鼠出现下颌下腺的淋巴细胞浸润灶。在VX-702组中,所有NOD小鼠均未发现淋巴细胞浸润灶。所有实验组的外周血淋巴细胞的数目显著减少。ODN2088组NOD小鼠的抗SSA/Ro抗体和抗SSB/La抗体的浓度是所有实验组中最低的。结论:TLR9依赖的p38MAPK信号通路的抑制,能一定程度上减轻原发性舍格伦综合征动物模型NOD鼠的临床表现。     

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.     

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。     

Toll样受体9依赖的p38MAPK信号通路在原发性舍格伦综合征中的作用机制

目的 :在动物模型NOD鼠中,研究Toll样受体9(Toll like receptor 9,TLR9)依赖的p38MAPK信号通路在原发性舍格伦综合征发病机制中的作用,从而寻找疾病药物治疗的新靶点。方法:选取4、5、8、10、15周龄的NOD雌性小鼠,利用流式细胞学技术检测小鼠外周血单个核细胞中TLR9、p-p38 MAPK双阳性细胞的比率。利用免疫组化检测小鼠下颌下腺TLR9及p-p38 MAPK的表达情况。同时,观察小鼠刺激性唾液流率的改变以及下颌下腺的病理学改变。结果:TLR9、p-p38MAPK双阳性细胞在4、15周龄NOD鼠外周血单个核细胞中的表达,相对于正常对照组Balb/c小鼠无显著性差异。而自第5周开始,NOD鼠中双阳性细胞的比率逐渐升高,到第8周达到最高,第10周后逐渐下降。TLR9在NOD鼠下颌下腺的浸润淋巴细胞和部分腺上皮细胞中呈阳性表达,p-p38在NOD鼠下颌下腺的浸润淋巴细胞和周围少量腺上皮细胞中呈阳性表达。NOD鼠刺激性唾液流率自第5周起逐渐减少,相较于正常小鼠降低50%~60%。结论 :从第5周到第10周,TLR9、p-p38MAPK双阳性细胞在NOD鼠中显著升高,同时伴随着刺激唾液流率的降低以及下颌下腺TLR9、p-p38MAPK阳性的淋巴细胞浸润。结果提示,外周血单个核细胞中TLR9依赖的p38MAPK信号通路的激活,可能在原发性舍格伦综合征发病早期起到重要作用,NOD鼠可用于p38 MAPK或TLR9抑制实验的动物模型。     

涎腺疾病

非肥胖型糖尿病小鼠自发性涎腺炎的发生与发展;家兔失神经支配腮腺导管改道术后唾液成分的分析;涎腺导管癌9例报道及文献复习;颌下腺导管内涎石取出术的疗效分析;副腮腺涎石病;     

阳离子脂质体乳剂增强防龋疫苗黏膜免疫效果的研究

目的：观察阳离子脂质体增强防龋疫苗诱导的小鼠特异性IgG和唾液IgA抗体水平。方法：以阳离子脂质体乳剂分别包被防龋DNA疫苗（pGJA-P/VAX）和基因重组rPAc蛋白疫苗,用DNA初免,2周后蛋白加强免疫的策略滴鼻免疫6～8周龄的BALB/c小鼠,分别收集初免后0、2、4、6、8、10、12、16周的血清和唾液,用酶联免疫吸附实验（ELISA）检测血清和唾液中特异性抗体的水平。结果：制备的乳剂疫苗CLE/DNA和CLE/rPAc的粒径分别为134.6nm和230nm,ZETA电位为36mV和18.5mV,乳剂疫苗免疫小鼠后可以诱导血清特异IgG和唾液特异IgA产生,并且实验组（LE/DNA初免＋LE/rPAc加强免疫组）能产生较对照组（DNA初免＋rPAc加强免疫组）更持久的黏膜免疫反应,两组比较差异有显著性（P〈0.05）。结论：阳离子脂质体乳剂是有效的基因疫苗载体,与防龋疫苗（DNA和rPAc蛋白）合用免疫小鼠,可增强血清特异性IgG和唾液特异性IgA抗体水平,延长抗体维持时间,具有较好黏膜免疫反应增强作用。     

防龋基因疫苗pcDNA3-PAc不同途径免疫BALB/c小鼠的实验研究

目的:观察防龋基因疫苗pcDNA3- PAc经不同途径免疫BALB/c小鼠后的免疫效果.方法: 重组质粒pcDNA3- PAc经颌下腺区皮下注射、股四头肌注射和鼻腔黏膜滴注免疫BALB/c小鼠,共免疫2 次,每次间隔2 周.分别于首次免疫前1 d和免疫后第1、2、4 周采集血液和唾液样品,采用酶联免疫吸附方法检测血清特异性IgG抗体和唾液特异性IgA抗体.结果: 各实验组免疫后1 周血清和唾液中特异性抗体水平开始升高,第4 周时达最高水平.颌下腺区皮下注射免疫产生的唾液IgA和股四头肌注射免疫诱导的血清IgG抗体水平明显高于其它实验组(P<0.05).颌下腺区皮下注射免疫和鼻腔黏膜滴注免疫可诱导较高水平的唾液IgA和一定水平的血清IgG;股四头肌注射免疫诱导的血清特异性IgG抗体水平较高(P<0.05),而唾液IgA抗体水平不如颌下腺区皮下注射免疫和鼻腔黏膜滴注免疫(P<0.05).结论: 防龋基因疫苗pcDNA3- PAc能够诱导动物机体产生有效的系统免疫应答和黏膜免疫应答.     

羟氯喹治疗口腔扁平苔藓前后的唾液双向电泳研究

目的：比较口腔扁平苔藓（OLP）经羟氯喹治疗后,唾液中差异表达的蛋白质,探讨羟氯喹治疗OLP的可能作用机制。方法：采用固相pH梯度（immobilized pH gradient,IPG）双向凝胶电泳（two-dimensional gel electrophoresis,2-DE）分离OLP经羟氯喹治疗三个月后唾液中的差异蛋白质,银染显色,Imagemaster图像分析软件分析差异蛋白质点。结果：①羟氯喹治疗OLP前和治疗后的唾液2-DE图谱的平均蛋白质点数分别为1894±183个和1920±341个,凝胶图谱分辨率、重复性较好;②通过Imagemaster图像分析比较治疗前后的唾液2-DE图谱,差异表达的蛋白质点数为21个,其中6个点在治疗3个月后高表达,15个点在治疗后低表达。结论：本研究建立了分辨率高且重复性较好的OLP治疗前后的唾液双向凝胶电泳图谱,发现存在差异表达的蛋白质,为进一步研究羟氯喹治疗OLP的作用机制奠定了基础。     

Effects of total glucosides of paeony for delaying onset of Sjogren's syndrome: An animal study

ObjectiveTo investigate the effectiveness of total glucosides of paeony (TGP) on Sjogren's syndrome (SS) using non-obese diabetic (NOD) mice model.Study designTwenty-seven 8-week-old female NOD mice were assigned into TGP group, hydroxychloroquine (HCQ) group and normal saline (NS) group, receiving corresponding drugs respectively and sacrificed at 24-week-old. Saliva flow rate (SFR), ration of regulatory T cells, level of anti-SSA/SSB, histological changes in submandibular glands (SMG) and microarray analysis were assessed. The data were analyzed using SPSS.ResultsCompared to NS group, in TGP group, SFR, SMG index and the ration of regulatory T cells were significantly higher, while anti-SSA/SSB and lymphocytic foci were significantly lower. HCQ group demonstrated similar results except SMG index. Altered gene expression was found in 10.71% of TGP and 13.09% of HCQ of the profile.ConclusionTGP demonstrated a similar effectiveness as HCQ in delaying the onset of SS-like disease in NOD mice.     

舌癌酸性环境下雷公藤多甙对单核/巨噬细胞杀伤活性的影响

目的　通过模拟舌癌局部的酸性环境,加入雷公藤多甙（TWP）,检测其对单核/巨噬细胞杀伤肿瘤细胞功能的影响。方法在pH值6.6,6.8酸性环境中,人单核/巨噬细胞THP-1与人舌鳞癌细胞Tca8113共同培养,经不同浓度的TWP干预,MTT法检测Tca8113细胞数量变化,观察此舌癌酸性环境下TWP对单核/巨噬细胞杀伤舌癌细胞作用的影响。结果酸性环境下,经不同浓度TWP干预,THP-1细胞与Tca8113细胞共同培养4小时,THP-1细胞杀伤活性随TWP浓度增加而增大,与浓度成正相关(P<0.05);pH值为6.6,TWP浓度为5×10-1μg/ml时,THP-1细胞杀伤活性随时间延长而增加,与时间成正相关(P<0.05)。结论体外模拟的舌癌酸性环境中,经一定浓度的雷公藤多甙干预,可使单核/巨噬细胞杀伤活性较未干预组增强,与药物浓度及时间成正相关。     

Periapical lesion progression with controlled microbial inoculation in a type I diabetic mouse model.

Uncontrolled or poorly controlled diabetes mellitus may be a risk factor for the development of large and/or debilitating periapical infections. The objectives of this investigation were to: (i) determine the effect of diabetes mellitus on the pathogenesis of periapical lesions with or without specific bacterial inoculations at the exposure sites, and (ii) test the sensitivities of two microbiological techniques in detecting the persistence of the bacterial inoculum in exposed pulps of nonobese diabetic (NOD) mice. Periapical lesions were induced in first molars of 29 female NOD mice and 31 BALB/c controls. Acute (1-2 wk) or chronic (5 wk) exposures were either inoculated with a mixture of facultative and anaerobic bacteria or exposed to oral flora without inoculations. After death the teeth in the chronic groups were analyzed for the presence of the inoculated bacteria by culturing and by polymerase chain reaction amplification of 16S rDNA. Periapical lesion size was measured histomorphometrically and the interleukin-6 content was measured immunohistochemically. The mortality among NOD mice with inoculated and sealed exposures was 83%, compared with 29% for BALB/c mice. In the inoculated and uninoculated chronic NOD mice groups, 38% of the animals versus none of the BALB/c mice died. The chronic uninoculated NOD mice lost significantly more weight at the time of death than controls. Polymerase chain reaction was more sensitive than culturing in detecting the inoculated anaerobic bacteria. In the animals that survived to the predetermined time periods, lesion size and interleukin-6 content in NOD and BALB/c mice were not statistically different.     

Review of genes potentially related to hyposecretion in male non-obese diabetic (NOD) mice,a Sjögren's syndrome model

BackgroundSjögren's syndrome (SS) is known to cause dry eyes and mouth due to inflammation of the lacrimal and salivary glands. However, some reports imply that other factors trigger dry eyes and mouth. We previously investigated various factors using RNA-sequencing analysis of lacrimal glands from male non-obese diabetic (NOD) mice, an SS model. In this review, we described (1) the exocrine features of male and female NOD mice, (2) the up- and down-regulated genes in the lacrimal glands of male NOD mice as revealed by our RNA-sequencing data, and (3) comparisons between these genes and data in the Salivary Gland Gene Expression Atlas.HighlightsMale NOD mice exhibit a steady worsening of lacrimal hyposecretion and dacryoadenitis, whereas females exhibit a complex pathophysiological condition that includes diabetic disease, salivary hyposecretion, and sialadenitis. Ctss, an up-regulated gene, is a potential inducer of lacrimal hyposecretion and is also expressed in salivary glands. Two other up-regulated genes, Ccl5 and Cxcl13, may worsen the inflammation of SS in both the lacrimal and salivary glands. The genes Esp23, Obp1a, and Spc25 were detected as down-regulated, but judging the relationship between these genes and hyposecretion is difficult as only limited information is available. Another down-regulated gene, Arg1, is involved in lacrimal hyposecretion, and it also has the potential to cause salivary hyposecretion in NOD mice.ConclusionIn NOD mice, males may be better than females at evaluating the pathophysiology of SS. Some regulated genes revealed by our RNA-sequencing data might be potential therapeutic targets for SS.     

Temporal changes in salivary glands of non-obese diabetic mice as a model for Sjögren's syndrome

Oral Diseases (2011) 18 , 96–106 Objective: Non‐obese diabetic (NOD) mice develop an autoimmune exocrinopathy that shows similarities with Sjögren’s syndrome. They provide an experimental model to study the pathoetiogenesis of this disease. Materials and Methods: Salivary gland (SG) function and salivary sodium content were measured in 8‐, 12‐, 16‐ and 20‐week‐old NOD and age‐matched CB6 mice. In NOD mice, SG expression of phenotypic cell markers, B cell‐stimulating and costimulatory molecules were evaluated. Cytokine levels were measured in serum and SG homogenates. Results: Microscopically evident SG inflammation in NOD mice was preceded by expression of intercellular adhesion molecule 1 on epithelial cells in the presence of macrophages and relatively high levels of cytokines. Next, an influx consisting of mainly T, B, natural killer, plasma and dendritic cells was seen. Most cytokines, except for interleukin (IL)12/IL23p40 and B cell‐activating factor, decreased or remained stable over time, while glandular function deteriorated from 16 weeks of age onward compared with CB6 mice. Conclusion: Sjögren’s syndrome‐like disease in NOD mice occurs in multiple stages; immunological and physiological abnormalities can be detected before focal inflammation appears and salivary output declines. Extrapolating this knowledge to human subjects could help in understanding the pathogenesis and aid the identification of potential therapeutic targets.     

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.