Epidermal growth factor, renin, and protease in hormonally responsive duct cells of the mouse sublingual gland

In the striated ducts of the sublingual glands of normal adult male, but not female, Swiss-Webster mice a few scattered cells have apical secretion granules. These sublingual duct cells resemble the granular convoluted tubule (GCT) cells of the submandibular glands of adult female mice, in that they are smaller than submandibular GCT cells of adult males, and contain fewer apical granules, and prominent basal striations. These cells stain immunocytochemically for epidermal growth factor (EGF), renin, and protease A. Such granular striated duct cells could be induced in the sublingual glands of adult female mice by treatment with either testosterone propionate or thyroxine; the two hormones given simultaneously acted synergistically in this induction.     

Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. Immunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.     

Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. lmmunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.     

小鼠下颌下腺发育中转化生长因子β受体的表达

背景：小鼠的下颌下腺是研究唾液腺的发育的良好模型,转化生长因子β是器官发育和疾病中重要的生长因子,但是在下颌下腺中转化生长因子β受体的表达以及作用机制至今并不明确。 目的：观察胚胎小鼠下颌下腺发育过程中转化生长因子βⅠ型受体和Ⅱ型受体以及p-ERK1/2的表达,揭示转化生长因子β在小鼠涎腺发育中的作用。 方法：取C57BL/6J小鼠胚胎期第14.5天的标本,使用转化生长因子βⅠ型受体和Ⅱ型受体以及p-ERK1/2抗体,对小鼠的下颌下腺进行免疫组化染色。取新生小鼠标本,大体观察下颌下腺,并且使用苏木精-伊红染色观察其形态。 结果与结论：①小鼠出生时,下颌下腺位于下颌骨下方;苏木精-伊红染色发现小鼠下颌下腺的腺泡、导管和闰管细胞也已经分化完成。②在胚胎期第14.5天,转化生长因子βⅠ型和Ⅱ型受体在腺泡上皮和导管上皮内高表达,而腺体上皮细胞外的间充质没有表达。③p-ERK1/2主要也是表达在下颌下腺的上皮细胞中,与转化生长因子βⅠ型受体和Ⅱ型受体在下颌下腺中的表达基本一致。说明在小鼠下颌下腺的发育过程中,转化生长因子β蛋白可能通过与上皮细胞表面的受体结合,激活ERK信号通路来调节涎腺腺泡和导管的发育。     

Altered plasticity of the parasympathetic innervation in the recovering rat submandibular gland following extensive atrophy

Adult rat submandibular glands have a rich autonomic innervation, with parasympathetic and sympathetic nerves working in synergy rather than antagonistically. Ligation of the secretory duct rapidly causes atrophy and the loss of most acini, which are the main target cell for parasympathetic nerves. Following deligation, there is a recovery of gland structure and function, as assessed by autonomimetic stimulation. This study examines whether the parasympathetic nerves reattach to new target cells to form functional neuro-effector junctions. Under recovery anaesthesia, the submandibular duct of adult male rats was ligated via an intra-oral approach to avoid damaging the chorda-lingual nerve. Four weeks later, rats were either killed or anaesthetized and the ligation clip removed. Following a further 8 weeks, both submandibular ducts were cannulated under terminal anaesthesia. Salivary flows were then stimulated electrically (chorda-lingual nerve at 2, 5 and 10 Hz) and subsequently by methacholine (whole-body infusion at two doses). Glands were excised, weighed and divided for further in vitro studies or fixed for histological examination. Ligation of ducts caused 75% loss of gland weight, with the loss of most acinar cells. Of the remaining acini, only 50% were innervated despite unchanged choline acetyltransferase activity, suggesting few parasympathetic nerves had died. Following deligation, submandibular glands recovered half their weight and had normal morphology. Salivary flows from both glands (per unit of gland tissue) were similar when evoked by methacholine but greater from the deligated glands when evoked by nerve stimulation. This suggests that parasympathetic nerves had reattached to new target cells in the recovered glands at a greater ratio than normal, confirming reinnervation of the regenerating gland.     

Intraoral duct ligation without inclusion of the parasympathetic nerve supply induces rat submandibular gland atrophy

The atrophic effect of ligating the main duct of the right submandibular gland was examined in rat using a novel intraoral approach that did not include the chorda lingual (CL) nerve. Comparison was made with the effect of duct ligation including the attached CL nerve as carried out in previous studies. In all animals, the contralateral, unligated left submandibular gland was used as a control. At different times (1, 2, 7, 14 and 21 days) after ligation, glands were removed and weighed. Tissue was fixed for morphological analysis and homogenized for biochemical assay of secretory proteins. After 21 days, ligated glands showed a significant decrease in wet weight compared with unligated glands. Weight loss was the greatest (P < 0.05) in glands ligated with the CL nerve included. Light microscopy revealed that following ligation, an initial inflammatory reaction was followed by severe atrophy of acini and granular ducts. The atrophy was less severe when the CL nerve was not ligated. Secretory proteins were decreased from day 1 onwards following duct ligation in both groups. It can be concluded that most of the atrophy induced by duct ligation is independent of damage caused to the parasympathetic nerve supply, although the latter causes a greater atrophy presumably due to denervation.     

糖尿病大鼠颌下腺肥大细胞数量及转化生长因子β1的表达

背景：研究表明,持续高血糖可导致大鼠颌下腺的细胞出现退行性改变,表现为细胞因子表达增加,糖原代谢改变,分泌功能减弱。关于肥大细胞及转化生长因子β1与糖尿病颌下腺组织病变的关系研究较少。 目的：观察糖尿病状态下颌下腺组织中肥大细胞数量与形态变化和转化生长因子β1的表达。 方法：雄性SD大鼠32只,随机分为实验组和对照组,实验组注射四氧嘧啶成模后的4,8,12周测量体质量和血糖,并与对照组大鼠一同取下颌下腺组织做苏木精-伊红染色和甲苯胺蓝染色观察并计数肥大细胞,SP 免疫组织化学染色方法检测转化生长因子β1棕黄色阳性细胞数量。 结果与结论：①肥大细胞在正常鼠及糖尿病鼠颌下腺组织中均有表达,实验组与对照组比较肥大细胞数随病程延长而增多,实验组不同时期明显多于对照组(P < 0.01)。②转化生长因子β1阳性细胞数在正常大鼠及糖尿病大鼠颌下腺的腺泡细胞、颗粒曲管及纹状管细胞中均有表达,阳性颗粒位于胞浆内,糖尿病时,随病程延长而增多,实验组不同时期明显多于对照组(P < 0.01)。结果可见糖尿病大鼠颌下腺中肥大细胞与转化生长因子β1表达增强与糖尿病病程呈正相关。     

Oxygen toxicity and restructuring of pulmonary arteries--a morphometric study. The response to 4 weeks'' exposure to hyperoxia and return to breathing air.

This study describes the pulmonary vascular lesions in rat pulmonary arteries and altered right ventricular weight after 1) prolonged exposure to hyperoxia (87% O2 for 4 weeks) at ambient pressure, 2) weaning from hyperoxia to air over 7 days, and 3) return to breathing air for 2, 4, or 8 weeks. Hyperoxia for 28 days narrows the lumen of intraacinar and preacinar arteries, increasing the percent medial thickness (%MT) by reducing the external diameter and thickening medial muscle. The ratio of patent intraacinar arteries to alveoli is significantly reduced, and pulmonary vascular obstruction and obliteration is evident by electron microscopy. A higher proportion of intraacinar and preacinar arteries have muscle in their wall than in the normal lung: in alveolar wall and duct regions, the proportion of partially muscular and muscular intraacinar arteries increases at the expense of nonmuscular ones (for both regions P chi 2 less than or equal to 0.001); and in arteries associated with terminal bronchioli and bronchioli the proportion of muscular arteries increases at the expense of partially muscular ones (for both regions P chi 2 less than or equal to 0.001). Both after weaning and after return to breathing air lumen size increases; but, even after 8 weeks, the %MT remains significantly increased, and the ratio of intraacinar arteries to alveoli is less than normal. After weaning, the proportion of muscularized intraacinar and preacinar arteries is similar to that after hyperoxia. Two weeks after return to breathing air, the proportion of muscularized alveolar wall and duct arteries is greater (for both regions P chi 2 less than or equal to 0.001). Even 8 weeks after return to breathing air more arteries are muscularized than normal (for both alveolar wall and duct regions P chi 2 less than or equal to 0.001), and within the alveolar wall still more are muscularized than after hyperoxia (P chi 2 less than or equal to 0.001). Hyperoxia causes right ventricular hypertrophy, reducing the ratio of the weight of the left ventricle and septum to that of the right ventricle (P chi 2 less than or equal to 0.001). Weaning further increases the hypertrophy, the ratio being further reduced (P chi 2 less than or equal to 0.001, compared with both hyperoxia and control values). On return to breathing air the degree of hypertrophy is less, but it persists, and even after 8 weeks the ratio is still less than normal (P chi 2 less than or equal to 0.01).     

Dynamics of parenchymal cell division, differentiation, and apoptosis in the young adult female mouse submandibular gland

The submandibular salivary gland of the young adult female mouse has two secretory cell types, acinar and granular duct, which are separated by intercalated ducts. Based on the occurrence of autologous cell division in these cells, they have been traditionally classified as expanding populations. However, differentiation from stem or progenitor cells in the intercalated ducts, usually associated with renewing populations, has also been detected. The question of renewing or expanding populations is resolved by quantitating and integrating the rates of autologous cell division, differentiation, and apoptosis for each cell type. The integrated data shows that both acinar and granular duct cell populations exhibit a substantial positive growth index, whereas the growth index for the intercalated duct cells is moderately negative. On balance, it suggests that the submandibular gland of the young adult female mouse is still growing. Comparison of young female mice with older females suggests that, although overall parenchymal growth slows with age, there is no longer a net loss of intercalated duct cells. Comparison with young adult male submandibular glands indicates that gender differences exist in the rates and mechanisms used for maintaining the different cell populations. The acinar and granular duct cell populations in young adult female mouse submandibular glands are expanding at the expense of the intercalated duct cell population, which appears to be contracting.     

Endocrine regulation of thymic biosynthetic polyamine decarboxylases in adult rat.

The activities of ornithine and S-adenosyl-L-methionine decarboxylase were assayed in the thymuses of adult rats killed at 7-day intervals up to 6 wk after either pinealectomy or sham pinealectomy. The absence of the pineal gland markedly influenced the ornithine decarboxylase activity in the thymus, in which the level of the enzyme was decreased permanently by the 4th wk after the operation (P less than 0.05). The time course of the changes in S-adenosyl-L-methionine decarboxylase activity in the thymus during the entire period investigated was also significantly (P less than 0.05) modified by pinealectomy but did not show any stable trend. Adrenalectomy significantly raised (P less than 0.001) for ornithine decarboxylase; P less than 0.01 for S-adenosyl-L-methionine decarboxylase) the basal levels of the thymic biosynthetic polyamine decarboxylases. A pharmacological dose of corticosterone or cortisol produced a rapid and significant decrease in ornithine and S-adenosyl-L-methionine decarboxylase activities (P less than 0.02) in the thymus, whereas the injection of either D-aldosterone or ACTH was ineffective. Therefore, the thymic biosynthetic polyamine decarboxylases that in this organ are known to be located only in the lymphocytes appear to be regulated in opposing ways by the pineal gland and by the adrenal cortex.     

The granulated convoluted tubules of the rat submandibular glands under experimental conditions.

Granulated convoluted tubules are the specific ductal segment of the submandibular glands of mice and rats. These tubules are functionally integrated into hormonal circuits, produce regulatory peptides as well as epidermal and nerve growth factor. The experimental studies on adult male Wistar and Fischer 344 rats demonstrate acute cytotoxic lesions of the granulated tubules by means of a single dose of 2-acetylaminofluorene. After only a few administrations of the compound the intralobular duct tree is lined by an atrophic epithelium with loss of specific structures, the EGF immunoreactivity and the susceptibility to further cytolethal effects. The early selective damage of the granulated convoluted tubules indicates that the growth factor production and certain drug-metabolizing/drug-excreting capacities are situated within the same ductal segment. It is considered that other systemically administered compounds might also influence this growth factor-producing ductal segment, though less dramatically than 2-acetylaminofluorene.     

Participation of the Fas and Fas ligand systems in apoptosis during atrophy of the rat submandibular glands

Most acinar cells and some duct cells undergo apoptosis during atrophy of the submandibular gland. The present study was designed to elucidate whether Fas and its receptor ligand (FasL) are involved during apoptotic atrophy of the gland. The excretory duct of the right submandibular gland of rats was doubly ligated with metal clips from 1 to 14 days for induction of gland atrophy. Control rats were untreated. Fas and FasL expression in the atrophied submandibular gland was detected using immunohistochemistry (IHC) and Western immunoblot. Expression of activated caspase 8 and activated caspase 3 was also detected with IHC. Fas-positive acinar and duct cells and FasL-positive duct cells increased in the atrophic glands at 3 and 5 days after duct ligation when apoptotic cells were commonly observed. Thereafter, Fas- and FasL-positive cells declined in number. Patterns of expression of Fas and FasL using Western immunoblots concurred with the IHC results. Activated caspase 8-positive cells were present at every time interval but peaked at 3 and 5 days following duct ligation. The cells showing immunoreaction for activated caspase 3 first appeared on day 3, with the peak in apoptosis, after which they decreased. The results indicate that the Fas/FasL systems likely play an important role in apoptotic pathways during atrophy of the submandibular gland.     

NGF在db/db自发性糖尿病小鼠颌下腺的表达

目的　观察转基因糖尿病小鼠颌下腺的形态学改变以及神经生长因子 (NGF)在颌下腺表达的变化。　方法　引进日本C5 7BL ksj db m表型正常隐性基因小鼠 ,近亲交配 ,其纯合子后代 ,即为db db(单基因遗传自然发病型 )糖尿病小鼠。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。HE染色及SP免疫组织化学染色后行图像分析 ,统计NGF阳性表达的细胞数。　结果　随着糖尿病发展 ,颌下腺组织萎缩 ,细胞缩小 ,形态不规则 ,排列不整齐。不同月龄糖尿病小鼠NGF阳性细胞明显低于相应对照组 (P <0 0 1) ,且逐渐减少 ,呈下降趋势。　结论　NGF阳性细胞数的减少说明颌下腺颗粒曲管细胞合成和分泌NGF功能降低 ,而NGF缺乏与糖尿病性神经病变的发生与发展密切相关。     

生后小鼠颌下腺神经生长因子基因表达的发育变化

为了探讨在发育过程中小鼠颌下腺神经生长因子基因表达的变化，应用原位杂交组织化学技术和图像分析方法对新生至２月龄ＩＣＲ雄性小鼠颌下ＮＧＦｍＥＮＡ基因表达的变化，应用原位杂交研究。结果显示：新生小儿颌下腺未见ＮＧＦｍＲＮＡ杂交信号，８－１０ｄ龄小鼠颌下腺ＮＧＦ原位杂交信号呈阴性至弱阳性。     

三叶因子2在大鼠下颌下腺的表达及性差

目的:探讨三叶因子2(TFF2)在大鼠下颌下腺的定位及其在不同性别中的表达差异.方法:对大鼠下颌下腺进行TFF2免疫组织化学显色和RT-PCR检测TFF2mRNA表达.结果:TFF2免疫反应阳性物质主要位于闰管、纹状管和小叶间导管上皮细胞胞质,特别是近管腔面更多,纹状管和小叶间导管管腔内亦有阳性物质分布;颗粒区管的少颗粒暗细胞或无颗粒细胞强阳性;多颗粒细胞弱阳性或阴性.雄性大鼠的下颌下腺表达TFF2mRNA明显多于雌性.结论:TFF2主要分布在下颌下腺导管的上皮细胞,以雄性丰富,且主要以腔分泌方式发挥作用.     

Effect of advanced age on the induction by androgen or thyroid hormone of epidermal growth factor and epidermal growth factor mRNA in the submandibular glands of C57BL/6 male mice

We have compared the responsiveness of the submandibular glands of mature (12 month old) and senescent (26-28 month old) male C57BL/6 mice to dihydrotestosterone (DHT) or triiodothyronine (T3) in terms of steady state levels of epidermal growth factor (EGF) protein and EGF mRNA. Northern blot analyses did not disclose any differences with age in the apparent sizes of EGF mRNA species. In untreated animals, submandibular glands of 26-28-month-old mice contained approximately 50% less EGF, and 75% less EGF mRNA than those of 12-month-old males. With advanced age, there was a 20% reduction in the absolute volume of the granular convoluted tubule (GCT) compartment, which is the exclusive site of EGF and EGF mRNA in the gland. In general, GCTs of old mice were composed of smaller cells with fewer secretion granules, but there was considerable cell-to-cell variation. In addition, there was greater variation in the intensity of immunocytochemical staining for EGF in senescent GCT cells, which also gave a lower and more variable in situ hybridization signal for EGF mRNA. After hormonal stimulation for 1 week with either tri-iodothyronine (T3) or dihydrotestosterone (DHT), EGF protein concentration in the glands was induced to the same level at both ages. However, EGF mRNA was 50% less abundant in old hormonally stimulated glands, compared to similarly treated young ones. Although many GCT cells in treated glands of senescent males respond to hormonal stimulation by increases in size and in content of secretion granules, there was cell-to-cell variation in responsiveness, especially after treatment with T3. These findings indicate that the decreases seen in the entire gland in EGF and EGF mRNA are caused by a wide-spread deterioration of the GCT cells themselves, which apparently can be reversed in many but not all GCT cells by stimulation with supraphysiologic doses of either T3 or DHT.     

下颌下腺主导管结扎可激活损伤组织中的干/祖细胞

背景：成体干细胞的伦理学问题较少,而且某些操作技术比较成熟,利用成体干细胞进行组织工程化涎腺的构建具有十分诱人的吸引力和极其重要的应用前景。 目的：建立下颌下腺主导管结扎的涎腺组织损伤大鼠模型,探讨涎腺组织损伤模型中成体干/祖细胞存在的可能性及可能位置。 方法：SD大鼠统一行右侧下颌下腺主导管结扎,1周后处死大鼠取出两侧腺体,通过苏木精-伊红染色、PAS糖原染色及细胞角蛋白19、Bcl-2、Ki-67等指标的免疫组织化学测定,对正常涎腺组织与建立的损伤模型组织进行比较。 结果与结论：同一只大鼠,结扎侧与对照侧体积、质量有明显的差异。对照侧下颌下腺组织呈卵圆形,色泽红润,表面光滑,有完整包膜,质地柔软;结扎后腺体萎缩,组织形态欠规整,色泽暗红,包膜充血,质地变韧,周围组织血管代偿性扩张。主导管结扎的组织损伤模型可导致PAS阳性腺细胞的消失和细胞角蛋白19阳性的小导管上皮细胞增殖,并有在未结扎的腺体中很少见到的小丛层粘连蛋白阳性细胞出现在导管周围,而抑制细胞凋亡的Bcl-2和提示增殖活跃度的Ki-67的表达均有所增强。可见下颌下腺组织中可能存在着定位于涎腺周围导管区的下颌下腺干/祖细胞;下颌下腺主导管结扎导致的组织损伤模型是一种能有效激活下颌下腺组织中干/祖细胞的方法。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：