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。     

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。     

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.     

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。     

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。     

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...     

α1-肾上腺素受体在颌下腺的表达及苯肾上腺素对唾液分泌调节的动物实验

目的研究α-肾上腺素受体亚型在家兔颌下腺的表达和分布，及其激动剂——苯肾上腺素促家兔颌下腺唾液分泌的相关机制。方法应用RT—PCR和Western blot检测家兔正常颌下腺α1-肾上腺素受体亚型mRNA及蛋白质的表达；免疫组化法检测颌下腺α1-肾上腺素受体亚型的分布及水通道蛋白5的表达；经颌下腺导管插管给予（1×10^-8）-（1×10^-6）moL／L的苯肾上腺素，观察家兔心率和血压的变化及促颌下腺唾液分泌的量效关系。结果家兔颌下腺有α1A-α1B和α1D-肾上腺素受体3种亚型的mRNA及蛋白质的表达，并广泛分布于导管和腺泡细胞的胞膜及胞质中。给予1×10^-7moL／L苯肾上腺素7d，促进颌下腺唾液分泌增加，而对心率、血压无明显影响；水通道蛋白5在腺泡及导管细胞的顶膜和侧膜的表达增加。结论家兔颌下腺存在α1A-、α1B-和α1D-肾上腺素受体3种亚型的mRNA和蛋白质的表达。经颌下腺导管给予低剂量苯肾上腺素促进唾液分泌是安全有效的；水通道蛋白5的表达增加可能与苯肾上腺素促唾液分泌的机制有关，该研究为临床治疗领下腺功能低下提供了初步依据。     

舍格伦综合征动物模型的探讨

目的：建立类似于人类舍格伦综合征（SS）的实验动物模型。方法：运用SD大鼠颌下腺组织匀浆与完全弗氏佐剂（CFA）混匀多点注射大鼠皮下，再注射百日咳疫苗加强免疫，产生类似于人类舍格伦综合征（SS）的唾液腺改变及临床表现。结果：给SD大鼠注射同种颌下腺抗原CFA6周后，其颌下腺出现程度不一的淋巴细胞浸润，唾液流率显著减少。结论：建立的SD大鼠动物模型具有类似于人类SS表现，可作为研究SS的实验动物模型。     

失副交感神经支配对大鼠颌下腺分泌功能的影响

目的观察大鼠颌下腺失副交感神经支配后近远期分泌功能的变化,探讨失副交感神经支配调控颌下腺分泌、治疗口干的可能性。方法将20只雄性成年SD大鼠分为实验组（12只）和正常对照组（8只）。实验组行右侧鼓舌神经切除术,实验组左侧及正常对照组颌下腺均未处理。术后1、12和24周采用希尔默试验（Schirmer test）检测颌下腺的分泌功能。结果 进食刺激状态下实验组手术侧腺体的唾液流率明显低于非手术侧[(27.13±3.28)、(33.00±12.98)和(27.50±5.20) mm]和正常对照组[(30.25±3.86)、(28.00±3.46)和(27.00±4.32) mm](P＜0.05),证实术后颌下腺失去副交感神经支配。但在静息状态下,术后各时间点实验组手术侧腺体的唾液流率显著高于非手术侧[(1.91±0.62)、( 1.33±0.29)和(2.38±1.79) mm]和正常对照组[(1.90±0.34)、(1.53±0.46)和(1.48±0.39) mm](P＜0.05);非手术侧与正常对照组腺体唾液流率的差异无统计学意义。结论采用颌下腺失副交感神经支配治疗口干有一定的可能性。     

AQP6和AQP5在人下颌下腺上的免疫组化定位

目的检测水通道蛋白亚型AQP6和AQP5在人下颌下腺的分布情况。方法对源自10例行颈淋巴清扫术患者的正常颌下腺组织,通过免疫组织化学染色方法,标记确定人下颌下腺组织中AQP6和AQP5的分布情况。结果 AQP6在部分颌下腺腺泡细胞质中表达。AQP5在浆液性腺泡和粘液性腺泡顶膜以及分泌小管均有表达,而闰管和纹状管未见表达。结论 AQP6在部分颌下腺腺泡细胞中表达,与AQP5相协调,参与唾液中水和阴离子的分泌调节。     

NOD mouse model for Sjögren's syndrome: lack of longitudinal stability

Objectives: The non‐obese diabetic (NOD) mouse is not only a widely used model for diabetes mellitus type I, but also for the chronic autoimmune disease Sjögren's syndrome (SS), mainly affecting salivary and lacrimal glands. We studied the efficacy of local recombinant serotype 2 adeno‐associated viral (rAAV2) vector transfer of immunomodulatory transgenes to alter the SS‐like disease in NOD mice. Data collected over a 2‐year period indicated a changing SS phenotype in these mice and this phenomenon was investigated. Methods: 10¹⁰ particles rAAV2LacZ/gland were delivered to both submandibular glands (SMGs) of NOD/LtJ mice at 8 weeks (before sialadenitis onset) of age. Salivary flow rates were determined at 8 weeks and time of killing. Blood glucose levels and body weights were measured weekly. After killing, saliva and SMGs were harvested. Analyses of salivary output, inflammatory infiltrates (focus score), SMG cytokine profile, body weight, and diabetes mellitus status were performed. Data from six different experimental studies over 2 years were analyzed and compared. Results: Salivary flow rate, focus score, and SMG cytokines interleukin (IL)‐2, IL‐4, IL‐6, IL‐10, IL‐12(p70), tumor necrosis factor‐α and IFNγ showed changes over time. There were no differences for body weight, diabetes mellitus prevalence, or blood glucose level of non‐diabetic mice. Conclusion: This retrospective report is the first to describe longitudinal variability in the NOD mouse as a model for SS. We advise other investigators to continuously monitor the SS phenotype parameters and include appropriate controls when studying this disease in NOD mice.     

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.     

Changes in amylase activity in submandibular salivary glands of puberal male mice following castration.

Left submandibular salivary glands of 33 puberal male Swiss-Webster mice, aged 69 days, were removed for determination of amylase activity and the animals castrated. At 2-week intervals, up to 12 weeks postcastration, the paired right glands were assayed for amylase. Although there was an initial decrease in the mean amylase activity in the experimental gland compared with controls up to 6 weeks postcastration, by 8–12 weeks postcastration it returned to control level. Postcastration levels of amylase activity varied considerably, indicating that its synthesis in the submandibular salivary gland was disturbed, but not permanently suppressed, by castration.     

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.     

A mouse model linking viral hepatitis and salivary gland dysfunction

Objective:  Viral hepatitis is known to cause xerostomia in humans, but this has not been reported in an animal model. We report a severe, acute, highly reproducible saliva deficiency occurring in BALB/c mice as a result of experimental viral hepatitis.
Materials and Methods:  BALB/c mice, splenectomized or carrying genetic mutations to detect immunological contributions to the saliva deficiency syndrome, were infected intraperitoneally with a non-lethal dose of murine cytomegalovirus. Pilocarpine-stimulated saliva volumes were determined between 0 and 15 days after infection. Salivary gland, liver, spleen, and sera were analyzed for the presence of virus, cytokines, inflammatory infiltrates, and tissue damage.
Results:  Saliva deficiency was detectable 2 days after cytomegalovirus infection, peaked at 88% below normal by day 7, and resolved partially in all mice by 15 days postinfection as sialoadenitis increased. Neither salivary gland viral titers, sialoadenitis, splenectomy, nor systemic inflammatory markers correlated with hyposalivation severity. Elevated liver enzymes did correlate with hyposalivation, and mice genetically resistant to murine cytomegalovirus-induced hepatitis were significantly protected.
Conclusions:  Murine cytomegalovirus-induced salivary gland dysfunction is biphasic, with an acute hepatitis-associated phase and a later sialoadenitis-associated phase. Acute murine cytomegalovirus infection of BALB/c mice may provide a model for investigation of hepatitis-associated xerostomia.