改良小鼠胚胎下颌下腺的获取及其体外器官培养模型的建立

分支形态的形成对于保证器官可以在有限的体积内获得最有效的功能形态具有重要意义。下颌下腺是研究器官分支形态发生过程的重要模型,在小鼠胚胎中获取下颌下腺组织进行体外器官培养是一种重要的研究方法。本文介绍了一种改良的小鼠胚胎下颌下腺获取方法,并将整个获取及建立体外器官培养流程作以简介,以对其在体内发育过程中的分支形态发生过程进行更好体外的模拟,从而能够更好地开展相关研究。     

颌下腺细胞体外培养的研究进展

颌下腺缺失或功能丧失,可导致口干、咀嚼吞咽困难、语言等功能障碍,并引起一些继发疾病,严重影响患者的消化功能及生活质量.因此有必要通过颌下腺细胞体外培养,进而深入探讨颌下腺的生理和病理机制,为组织工程化修复重建颌下腺奠定实验基础.     

大鼠颌下腺肌上皮分化的研究

目的;观察组织块原代培养法是否可用于腺肌上皮细胞发育分化的研究,并探讨肌上皮细胞的组织发生,方法：采用组织块原代培养法,通过相差显微镜,透射电镜及免疫组化染色等检测手段,对不同时期大鼠颌下腺肌上皮细胞进行研究。结果：肌上皮样细胞及含有分泌颗粒的分泌细胞见于体外培养第３ｄ。肌微丝及Ａｃｔｉｎ阳性细胞分别最早出现于培养第６ｄ和７ｄ。     

体外培养大鼠颌下腺细胞的生物学特性研究

目的 :研究体外培养颌下腺细胞生物学特性。方法 :应用胰酶消化法进行颌下腺细胞的分离纯化 ,然后进行颌下腺细胞原代和传代培养。绘制培养细胞的生长曲线 ;MTT法检测培养细胞活力大小。透射电镜 (TEM )观察结合免疫组化检测鉴定细胞来源及性质。结果 :整个颌下腺细胞生长期间为呈多样性的上皮细胞 ,细胞可传 4～ 5代 ,成活 3 0～ 40d。随培养时间延长 ,各组细胞数量都有不同程度的增加 ,比较培养 72h原代、第 2代与第 4代细胞吸光度值 ,原代、第 2代与第 4代相比较有显著性差异 (P <0 .0 5 )。超微结构观察及免疫组化染色培养细胞表现为腺上皮细胞的特征。结论 :原代和传代培养第 1代、第 2代颌下腺细胞活性较强 ,可在进一步的实验得以应用     

人颌下腺腺上皮细胞体外培养及生物学特性研究

目的　建立人颌下腺腺上皮细胞的原代培养模型。方法　取口腔癌患者未受累及的颌下腺组织块 ,采用机械法和消化法 ,在 10 %血清浓度下 ,分别加入EGF、胰岛素、转铁蛋白等附加因子 ,37℃ ,5 %CO2 开放培养。选用苏木精 -伊红、PAS染色及S -P法免疫组化染色 ,扫描及透射电镜观察等方法研究其生物学性状。结果　通过特殊染色、免疫组化、电镜观察证实了体外培养细胞的腺上皮属性 ,细胞在 4d内可保持分化状态和分泌功能 ,可存活 1周以上。结论　成功建立了人颌下腺腺上皮细胞体外原代培养的模型。     

正常大鼠颌下腺上皮细胞极性培养

目的为了探索简便、有效的涎腺上皮细胞的体外培养方法。方法采用自制鼠尾胶原胶为底物．在培养液中加入一些促进上皮细胞生长分化的刺激物，对出生一天的Wistar大鼠颌下腺上皮细胞进行原代培养。通过相差显微微镜、光镜及透射电镜对上皮细胞的生长及分化进行观察和研究。结果培养的大鼠颌下腺上皮细胞呈多层生长且为极性分化，保留了腺泡导管分泌单位的三种上皮成份。结论本研究所采用的方法是一种简便、可行、有效的涎腺上皮细胞的体外培养方法，其中自制胶原胶是本研究培养成功的关键。     

db/db自发性糖尿病小鼠颌下腺PCNA的表达及细胞凋亡关系的研究

目的观察增殖细胞核抗原（PCNA）在db／db自发性糖尿病小鼠颌下腺中的表达及其与细胞凋亡的关系。方法选取3、4、6、8、10月龄db／db糖尿病小鼠及相应月龄的db／＋m正常小鼠颌下腺，应用sP免疫组织化学方法及TUNEL标记方法染色后进行图像分析，统计PCNA的表达及凋亡细胞在颌下腺组织中分布的细胞阳性率。结果①PCNA及凋亡细胞在对照组及糖尿病组颌下腺中均有表达及分布，对照组PCNA细胞阳性率高于糖尿病组。糖尿病组PCNA细胞阳性率随病程延长呈减弱趋势，且减少明显快于对照组；②糖尿病组凋亡细胞阳性率高于对照组。糖尿病组与对照组凋亡细胞阳性率随月龄增大均呈增加趋势。结论①PCNA表达逐渐减弱,较对照组明显降低，说明糖尿病可能导致颌下腺腺体增殖活动的减弱。②凋亡细胞阳性率在糖尿病组随疾病发展而增加显著，说明糖尿病可能是加速细胞凋亡的诱因之一，这与临床逐渐加重的腺体萎缩和功能受损相一致。     

SD大鼠原代颌下腺细胞体外培养的生物学特征研究

组织工程化涎腺的体外构建首先需要获得增殖活跃、有功能的种子细胞。我们通过本实验进行颌下腺腺细胞的体外培养，并对其在体外的增殖和分化进行了研究。     

SD大鼠原代颌下腺细胞体外培养的生物学特征研究

目的：研究SD大鼠颌下腺细胞体外培养的生物学特征,为组织工程化涎腺样组织的体外构建提供有功能的种子细胞。方法：获取新生SD大鼠幼崽颌下腺组织,经酶消化后体外培养,倒置显微镜、电镜下行形态学及免疫细胞化学染色观察。绘制生长曲线,对涎腺细胞的生物特征进行评价。结果：体外培养的颌下腺细胞CK(anti-cytokeralin)及淀粉酶染色阳性,体外增殖活跃。结论：体外培养的颌下腺细胞增殖活跃,保持了腺细胞的生物特征,可作为体外构建组织工程化人工涎腺研究的种子细胞。     

正常大鼠颌下腺上皮细胞极性培养

目的 为了探索简便，有效的涎腺上皮细胞的体外培养方法。方法 采用自制鼠尾胶原胶为底物，在培养液中加入一些促进上皮细胞生长分化的刺激物，对出生一天的Ｗｉｓｔａｒ大鼠颌下腺上皮细胞进行原代培养。通过相差显微镜，光镜，扫描及透射电镜对上皮细胞的生长及生化进行观察和研究。结果 培养的大鼠颌下腺上皮细胞呈多层生长且为极性分化，保留了腺泡导管分泌单位的三种上皮成份。结论本研究所采用的方法是一种简便，可行，有效     

c-Met在小鼠颌下腺胚胎发育中的表达

目的探讨c-Met在小鼠颌下腺发育不同阶段的表达特点。方法制作ICR小鼠颌下腺不同发育阶段的冰冻组织切片,利用免疫组织化学方法对小鼠颌下腺自蕾状期至出生后两天不同发育阶段c-Met的表达情况进行了研究。结果 c-Met在小鼠颌下腺发育的蕾状期上皮中表达呈明显阳性,在假腺管期早期表达减弱,而在假腺管期晚期的上皮突起中表达增强。在微管期的分枝状上皮条索和顶端胚芽中呈不对称表达,周围细胞表达较强而中心细胞表达较弱。在终末分化期的腺泡、闰管的部分上皮细胞表达,在导管上皮中持续性强表达。结论 c-Met在小鼠颌下腺发育的细胞增殖、分支形态发生及腺泡和导管的形成中可能扮演重要角色。     

人胚颌下腺细胞原代及传代培养

目的 :研究人胚颌下腺细胞原代及传代培养的方法 ,为进一步研究颌下腺细胞的功能、起源、发生及发展奠定基础。方法 :在DMEM培养基中加入FBS、表皮生长因子 (EGF)及氢化可地松 (HC) ,观察细胞的生长特点。结果 :在含有 φ =2 ?S、10ng/mlEGF、5 μg/ml胰岛素 (INS)及 5 μg/mlHC的DMEM培养基中上皮细胞生长最好 ,其它组上皮样细胞生长状态不佳。培养的细胞为圆形、三角形、多边形 ,细胞伸展良好。细胞传代后生长良好 ,形态并未发生改变。HE染色可见细胞为圆形 ,三角形 ,多边形 ,核蓝染 ,胞浆粉染。大多数细胞cytokeratin染色阳性 ,说明为上皮细胞 ,少数细胞CK18染色阳性 ,说明培养的细胞中含有一部分导管上皮细胞。结论 :含有 φ =2 ?S、10ng/mlEGF、5 μg/mlINS及 5 μg/mlHC的DMEM培养基最适合体外原代及传代培养人胚颌下腺细胞     

Postnatal expression of sialin in the mouse submandibular gland

Objective

Sialin has been identified as a sialic acid and aspartate/glutamate transporter. Both cytoplasmic localization and the plasma membrane labelling pattern suggested that sialin may possess multiple transport functions in different cell types. In mouse embryos, sialin expression was primarily detected in the central nervous system. However, sialin shows widespread and high-level expression in adult tissues. Despite its ubiquitous expression and important functions, the postnatal expression profile and subcellular localization of sialin in the salivary gland remains elusive. The aim of the present study was to investigate the expression and subcellular distribution of sialin during postnatal development in the mouse submandibular gland (SMG).

Design

Six SMGs from both female and male C57BL/6 mice were collected at P10, P30 and P90, and the material from each littermate of either sex was pooled to extract total RNA and tissue protein. The remaining tissues were immediately fixed in 10% neutral buffered formalin for histological analysis. The mRNA and protein expression levels of sialin were examined by quantitative real-time RT-PCR and Western blot analysis. The subcellular distribution of sialin was analysed by immunohistochemistry and immunofluorescence.

Results

The postnatal expression level of sialin in the mouse SMG was comparable with that in brain at each time point tested. The temporal expression of sialin in the SMG gradually increased during postnatal maturation. Immunohistochemical and immunofluorescence analysis demonstrated that sialin was predominantly expressed on the basal cytoplasmic membrane of acini and ducts, as well as in some myoepithelial cells in the SMG.

Conclusions

The high-level expression and subcellular distribution pattern of sialin in the SMG suggest that sialin may play an important role in the transport and secretion of saliva.     

Vascular clinical anatomy of the submandibular gland

ObjectiveThe aim of this study is to describe in depth the precise anatomy of the vascular supply of the submandibular gland, trying to determine the existence of patterns of glandular vascularization. Knowledge of these patterns could facilitate surgical management of the gland and the submandibular gland flap.Material and methodsNeck dissections of formaldehyde preserved human cadavers were performed. Submandibular and transmandibular approaches were used during the dissections. All the vascular branches found were registered and classified into 2groups: main or accessory branches. The anatomical data analyzed was: The diameter and length of the main and accessory branches, as well as the most important measurements of the submandibular gland flap pedicle.Results33 glands were dissected to study the arterial supply of the submandibular gland (17 right, 16 left; 17 males, 16 females) and 29 were dissected to study the venous supply (15 left, 14 right; 15 males,14 females). A total of 123 arterial branches were found reaching the 33 submandibular glands (47 main and 76 accessories) and 116 venous branches were found draining the 29 submandibular glands (47 main branches and 69 accessory branches). A constant main venous branch that ran parallel to the Wharton duct and drained in the sublingual vein was found in all of cases (Concomitant Wharton Duct Vein or CWDV).ConclusionThe CWDV is a constant venous branch for the drainage of the gland and should be considered as venous pedicle during the dissection of submandibular gland flaps.     

Expression and localization of calpain 3 in the submandibular gland of mice

ObjectivesCalpains comprise a family of intracellular Ca²⁺-dependent cysteine proteases and are considered to play roles in various physiological phenomena with limited proteolytic activities against specific substrates. We herein revealed the expression and localization of calpain 3, the muscle-type calpain, in the submandibular gland (SMG) of mice.DesignThe expression of the mRNA for conventional, ubiquitous calpains 1 and 2 and skeletal muscle-specific calpain 3 was examined in the major salivary glands of mice using RT-PCR, and the expression and localization of calpain 3 protein was examined in the SMG of mice using immunohistochemistry and Western blotting.ResultsThe large catalytic subunits of calpains 1 and 2 and the small regulatory subunit common to calpains 1 and 2 were weakly expressed in the parotid gland, sublingual gland, and SMG at similar levels in males and females. In contrast, the single large catalytic subunit of calpain 3 was expressed predominantly in the SMG at markedly higher levels in males than in females and in a manner dependent on androgens. Immunoreactivity for calpain 3 was mainly localized in cells of the granular convoluted tubules (GCT) that developed preferentially in the male SMG. In GCT cells, calpain 3 immunoreactivity was localized predominantly in the cytosolic region and was absent in the secretory granules.ConclusionsThese results revealed that the GCT is the primary site of production of calpain 3 in the mouse SMG.     

Large sialolith in the submandibular gland of a child

Sialolithiasis is a disorder encountered by oral surgeons that is rarely seen in children, although it is rather common in adults. Most sialolith found in children are smaller than 5 mm in diameter, and the majority of reported cases have been treated by surgically. We report a 9-year-old boy with a sialolith that measured 12 × 3.5 × 3 mm, which had developed in Wharton's duct and was then spontaneously passed.