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。     

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。     

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...     

一次性18Gy放射对大鼠颌下腺损伤的实验研究

目的:观察一次性18 Gy放射对大鼠颌下腺组织学形态和唾液流率的改变。方法:40只大鼠随机分为实验组和对照组,每组20只。实验组一次性18 Gy局部照射大鼠颌下腺区域,对照组只麻醉不放射。8周后处死所有大鼠,处死前插管法提取大鼠颌下腺唾液,称取其质量,计算唾液流率,比较两组唾液流率的改变。处死后取颌下腺组织,经4%多聚甲醛固定,切片,HE染色,镜下观察颌下腺的组织学形态。结果:放射后实验组进食进水量下降、活动减少。实验组饮水频率高于对照组。对照组的唾液流率为(18.64±8.23)μL/min,实验组的唾液流率是对照组的57.42%,为(10.70±2.22)μL/min。HE染色显示,放射后实验组颌下腺细胞变性,间质血管充血,腺泡细胞内的空泡数量明显增多。结论:放射后大鼠颌下腺唾液流率明显降低,放射后8周,组织学形态出现显著变化。放疗对大鼠颌下腺组织学形态和唾液分泌功能的远期影响,尚需进一步观察和研究。     

生理性唾液研究及对人工唾液的评价

作者搜索自80年代以来的中外文献,综述了对人口腔唾液的流率、成份的增龄性变化研究.总体上,唾液流率及成分的变化趋势具有如下特点:在达成人阶段之前随年龄增加而增加,之后随年龄增加而减少;然而颌下腺/舌下腺的唾液流率及唾液成分浓度变化仍有争议或不确定.老年人口腔防御能力下降.作者对人工唾液治疗口干症进行评价,并列出部分市售人工唾液的成分,以帮助临床医师选择.     

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。     

大鼠腮腺及颌下腺唾液的收集

目的:探讨大鼠腮腺及颌下腺唾液的收集方法。方法:采用微型Lashley吸盘法收集大鼠腮腺唾液,经口内颌下腺导管口直接插管法收集颌下腺唾液。毛果芸香碱刺激唾液分泌,假设唾液的比重为1.0 g/cm3,以唾液重量代表其体积,记录唾液流率。结果:大鼠腮腺唾液流率(9.9±1.4)μL/m in,颌下腺导管插管唾液流率(19.9±10.8)μL/m in。结论:可以采用微型吸盘法收集大鼠腮腺唾液,经口内颌下腺导管口直接插管法收集大鼠颌下腺唾液。     

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.     

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.     

Relationship of the unstimulated whole saliva flow rate and salivary gland size estimated by magnetic resonance image in healthy young humans

OBJECTIVE: The aim of this study was to examine the relationships between gland sizes and the flow rate and composition of the unstimulated whole saliva in humans. DESIGN: In 28 healthy young adults, the sizes of the three major salivary glands were estimated by use of a magnetic resonance (MR) imaging technique. Unstimulated whole saliva was collected for 5 min by the spitting method, and the flow rate and the concentrations of total protein, Na(+) and K(+) and pH were measured. RESULTS: The estimated sizes of the parotid and submandibular glands showed a significant positive correlation with the flow rate and the secretion rate of total protein in the unstimulated whole saliva, but that of the sublingual glands did not. Concerning the concentrations of Na(+) and K(+) and pH, there were no correlations with the salivary gland sizes. CONCLUSIONS: The results suggest that the larger the sizes of the parotid and submandibular glands, the faster the fluid flow and protein secretion rates in unstimulated whole saliva.     

The effect of X-ray irradiation on the function and saliva composition of rat parotid and submandibular/ sublingual glands

Radiation therapy to the head and neck area frequently causes severe salivary gland dysfunction and xerostomia. Morphological studies of irradiated salivary glands have suggested that the submandibular/sublingual gland may be less radiosensitive than the parotid gland. The purpose of this study was to evaluate the effect of radiation on major salivary gland functions in rats with radiation-induced xerostomia. The effect of salivary gland irradiation on salivary function was examined in specific pathogen-free Sprague-Dawley rats. The animals were irradiated with a single exposure of either 22 Gy or 32 Gy. Stimulated saliva excretion time was measured for the parotid and submandibular/ sublingual glands, and the total protein in saliva was analysed. Our results showed that the saliva flow rate and protein concentration of parotid saliva were significantly reduced in the 32 Gy-irradiated rats.     

The effect of food consistency and dehydration on reflex parotid and submandibular salivary secretion in conscious rats

Changes in salivary secretion with different consistency of diet and dehydration were studied in male Wistar rats under unrestricted conditions. To measure the salivary secretion, a stop-flow method was used. There was little unstimulated salivary secretion from the parotid and submandibular glands, but eating solid, powdered, and liquid diets induced parotid and submandibular saliva. There was no significant change in the volume and flow rate of saliva in bilateral parotid glands during the eating of solid diets. The solid and powdered diets induced significantly more salivary secretion from the parotid gland than did the liquid. The salivary flow rate with solid diets was significantly greater from the parotid gland than from the submandibular. On the other hand, the salivary flow rate with the liquid diet was significantly smaller from the parotid gland than from the submandibular. Appreciable amounts of submandibular saliva, but little parotid saliva were secreted during grooming. Clearly, parotid and submandibular saliva have different roles in the rat. When injected intraperitoneally with 1.5 M NaCl solution or water-deprived for 24 h, rats took longer to eat the solid diets, and had increased salivary volume and decreased flow rate from the parotid gland. These results indicate that the moisture content of the diet and the dryness of the mouth alters the volume of parotid saliva secreted in rats and show that parotid saliva plays an important part in mastication and swallowing.     

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.     

A biochemical analysis of parotid and submandibular salivary gland function with age after simultaneous stimulation with pilocarpine and isoproterenol in female NIA Fischer 344 rats.

This analysis of physiological, biochemical and molecular changes related to aging was made in 3-, 12- and 24-month-old rats. The salivary gland weight/body weight ratio and the structural membrane proteins did not change with age for either gland, but a significant age-related decline in DNA synthesis for both glands was detected, unrelated to the hormonal responsiveness at the level of the plasma membrane. There was a marked increase in the concentration of soluble proteins in adolescent parotid gland and, for the two older age groups, in submandibular gland. The saliva flow rate was different when expressed as volume per time, as volume per time and g glandular wet weight, and/or kg body weight. The concentration of secreted proteins was not affected by age in either gland. The total amount of proteins secreted over 30 min revealed no age-related perturbation for the parotid gland, but showed a significant age-related increase in submandibular saliva. Sodium dodecyl sulphate-polyacrylamide gel analysis revealed changes in the protein bands between 39 and 50 kDa in the Coomassie blue-stained gels from 12-month-old animals. Amylase showed an initial increase (12 months), followed by a marked decline in its activity in parotid saliva. The glandular supernatant had low residual cellular amylase activity after stimulation. Therefore, secretory impairment with age after pilocarpine-isoproterenol stimulation was excluded. Analysis of total RNA showed a pronounced decrease of amylase mRNA in the parotid gland between 12 and 24 months of age. No amylase mRNA was expressed in any of the submandibular samples. For epidermal growth factor, total saliva showed a decrease with age. It seemed that the submandibular gland followed the same picture with age as the parotid gland, with a specific decline in the biosynthesis of single secretory proteins.