大鼠乳腺局部免疫的调节机制--泌乳期与静止期乳腺肥大细胞分布的比较

应用改良甲苯胺蓝染色法(MTB)观察了大鼠泌乳期和静止期乳腺肥大细胞的分布、形态、数量变化规律．并用阿尔新蓝一番红鉴别染色法(AB-S)进行了细胞化学分型研究。结果：AB-S染色显示，大鼠乳腺只存在黏膜型肥大细胞；无论是泌乳期还是静止期。乳腺肥大细胞大多分布于腺泡间和小叶问结缔组织中。细胞的形态各异，但细胞数量在静止期和泌乳期有显著差异。泌乳期的乳腺肥大细胞数量明显减少(P＜0．01)。乳腺肥大细胞的动态变化，可能与乳腺泌乳期腺泡上皮的生长和静止期腺泡问结缔组织细胞增生等结构变化有关。     

奶牛发育各期乳腺肥大细胞的组织化学特性研究

本研究旨在对奶牛乳腺肥大细胞的组织化学特点进行研究。对奶牛发育各期乳腺进行取样,应用改良甲苯胺蓝染色法(MTB)和阿尔新蓝—番红鉴别染色法（AB-S）进行染色,观察肥大细胞的组织化学特点和数量变化特点。同时比较了甲苯胺蓝染色时两种固定液固定对乳腺肥大细胞着染的效果。奶牛不同发育时期乳腺肥大细胞只被阿尔新蓝染色,Carnoy氏液固定效果优于中性甲醛（NBF）,MTB的染色效果优于AB-S。奶牛乳腺肥大细胞的数量在泌乳各期和静止期有显著的变化。与静止期相比,泌乳期的乳腺肥大细胞数量明显减少(P＜0.05),分娩后60 d奶牛乳腺肥大细胞的数量最少。奶牛乳腺只存在黏膜型肥大细胞;用Carnoy氏液固定,用MTB法染色可以很好地显示肥大细胞;泌乳期乳腺肥大细胞数量与静止期相比,明显减少。     

发育各期奶牛乳腺肥大细胞类胰蛋白酶的表达

为了研究奶牛乳腺肥大细胞的组织化学特点和分布特征,对21头发育各期奶牛乳腺进行取样,应用免疫组织化学方法（SP）,观察奶牛乳腺肥大细胞类胰蛋白酶的表达。结果显示,奶牛乳腺肥大细胞表达类胰蛋白酶,肥大细胞数量在各期有显著差异,泌乳期的乳腺肥大细胞数量明显减少（P〈0.01）,分娩后60d奶牛乳腺肥大细胞的数量最少。结果表明,肥大细胞数量随生理周期的改变而改变,静止期奶牛的局部免疫能力增强。     

奶牛乳腺肥大细胞数量与胶原纤维面积的相关性研究

为了研究奶牛乳腺肥大细胞的数量与奶牛乳腺胶原纤维面积的相关性,试验对奶牛泌乳期和静止期乳腺进行采样,应用甲苯胺蓝组织化学染色法观察奶牛泌乳期和静止期乳腺肥大细胞数量的变化特点,应用苦味酸天狼猩红染色法观察奶牛泌乳期和静止期乳腺胶原纤维面积的变化特点。结果表明:静止期奶牛乳腺肥大细胞的数量明显比泌乳期多(P0.01),静止期奶牛乳腺胶原纤维的面积明显比泌乳期大(P0.01)。静止期奶牛乳腺肥大细胞的数量多,胶原纤维的面积也大;泌乳期的奶牛乳腺肥大细胞的数量少,胶原纤维的面积也小。说明奶牛乳腺肥大细胞的数量与胶原纤维的面积存在明显正相关性(r=0.677,P0.05)。     

低甲状腺素状态下小鼠甲状腺内肥大细胞数量的变化

为探讨低甲状腺素（简称低甲）状态下小鼠甲状腺内肥大细胞数量的变化，用组织学方法制做小鼠甲状腺的石蜡切片，并进行HE、TB和AB－S染色，观察甲状腺组织的一般结构及肥大细胞的数量变化。结果表明，低甲状态下，小鼠甲状腺滤泡细胞的高度（P＜0．05）、滤泡直径（P＜0．05）、肥大细胞的数量（P＜0．01）均明显大于对照组。提示肥大细胞数量增多是导致低甲状态下小鼠甲状腺肿大增生的一个因素。     

5-羟色胺和类胰蛋白酶在奶牛乳腺肥大细胞中表达的比较研究

为了对奶牛乳腺肥大细胞的组织化学特点和分布特点进行研究,试验对取自21头发育各期奶牛的乳腺样品,应用免疫组织化学方法(SP)观察奶牛乳腺肥大细胞5-羟色胺(5-HT)和类胰蛋白酶的表达。结果表明:5-羟色胺和类胰蛋白酶在奶牛乳腺肥大细胞中表达,肥大细胞数量在各期有显著差异,泌乳期的乳腺肥大细胞数量明显减少(P0.05),分娩后60天奶牛乳腺肥大细胞的数量最少;5-羟色胺在不同发育时期奶牛乳腺组织中的表达量少于类胰蛋白酶在不同发育时期奶牛乳腺组织中的表达量。说明肥大细胞的数量随生理周期的改变而增减的趋势可能与乳腺的发育、乳腺局部免疫状态及内分泌调节有密切关系。     

硒缺乏对鸡血清组胺和5-羟色含量的影响

为探讨硒缺乏对鸡血清中组胺和5-羟色胺浓度变化的影响，将40只鸡随机分为低硒试验组（饲喂低硒日粮0．032mg／kg）和正常对照组（硒含量为0．229mg／kg）。分别在30、45、60和75日龄断头处死进行取样，用ELISA法对雏鸡血清中组胺和5-羟色胺浓度进行检测和对比分析。结果表明：缺硒组血清中组织胺和5-羟色胺浓度与对照组差异极显著（P〈0．01），缺硒组不同日龄间差异极显著（P〈0．01）。在整个试验期内，缺硒组血清中硒含量和组胺浓度呈时间-效应关系；随日龄增加，缺硒组5-羟色胺浓度逐渐升高，在60日龄时达高峰而后降低，而对照组在60日龄后则趋于平稳。缺硒刺激肥大细胞脱颗粒，释放炎性介质组胺，使血清中浓度升高。但5-羟色胺属中枢神经递质，严重缺硒引起神经损伤，使其分泌受抑，含量降低。     

金黄色葡萄球菌感染对母鼠乳腺肥大细胞的影响

应用细菌计数研究不同攻菌时间金黄色葡萄球菌在乳腺内的增殖情况,并通过甲苯胺兰染色法观察乳腺内肥大细胞的数量和分布。结果显示,实验组母鼠乳腺内金黄色葡萄球菌数在攻菌后24 h达到峰值,48 h时有所下降,72 h时又有所增多;实验组脱颗粒肥大细胞数在攻菌后6h极显著多于对照组(P0.01);实验组肥大细胞数在攻菌后12 h显著多于对照组(P0.05)。脱颗粒肥大细胞数和肥大细胞总数随感染时间延长呈增多趋势。证实肥大细胞参与了金黄色葡萄球菌所致乳腺炎乳腺组织损伤的病理过程。     

鼻腔免疫禽流感灭活疫苗对鸡呼吸道肥大细胞分布的影响

用添加CpG的禽流感病毒（H5亚型）灭活疫苗对7日龄罗曼公鸡进行鼻腔免疫，14日龄加强免疫一次；分别于首免后第3、5、7周剖杀，取鼻腔、喉部、气管、气管叉及肺组织，通过甲苯胺蓝染色法显示肥大细胞，观察呼吸道各部位肥大细胞的分布及数量变化。结果显示，首免后第3周气管和气管叉黏膜中的肥大细胞数量显著增加（P〈0．05），第5周时喉部和气管叉黏膜中的肥大细胞数量显著增加（P〈0．05），气管黏膜中肥大细胞数量也极显著增加（P〈0．01），第7周肺中的肥大细胞数量显著增加（P〈0．05）；肥大细胞的数量在喉部最多，气管叉次之，气管和肺较少。试验结果表明，局部黏膜免疫可增加呼吸道肥大细胞的数量，肥大细胞可能参与黏膜免疫反应。     

let-7g对小鼠乳腺发育和泌乳相关功能基因Tgfbr1的作用及其机理

运用生物信息学方法对let-7g进行靶基因预测，构建含有与其结合位点互补序列的荧光素酶报告质粒将其与phRL-TK及let-7gmimics或negative control共转染小鼠乳腺上皮细胞，双荧光素酶报告系统检测试剂盒测定荧光素酶的表达；用let-7ginhibitor和let-7g mimics或negative control分别转染小鼠乳腺上皮细胞，并应用细胞活力分析技术、qRT—PCR技术、Western blotting、HPLC分析let-7g的表达变化及其对小鼠乳腺上皮细胞的影响。结果显示：经酶切及测序证实荧光素酶报告质粒构建成功；将荧光素酶报告基因、phRL—TK与let-7g mimics共转染小鼠乳腺上皮细胞，荧光素酶活性与对照组（即共转染荧光素酶报告基因、phRL-TK与Negative Control的小鼠乳腺上皮细胞组）相比显著降低（P〈0．05）；与阴性对照组和空白对照组相比较，let-7g inhibitor转染后，TGFβ3RI蛋白表达量显著增加（P〈0．01），细胞活性显著增强（P〈0．05），β-酪蛋白表达量增加（P〉0．05），而let-7g mimics转染后，TGFβRI蛋白表达量显著减少（P〈0．01），细胞活性显著降低（P〈0．05），β-酪蛋白表达量显著减少（P〈0．05）。结果表明，在小鼠乳腺上皮细胞中，let-7g能够靶向结合Tgfbr1，且负调控其表达；let-7g可通过抑制靶蛋白TGFβRI的表达，进而调控小鼠乳腺上皮细胞的增殖及β-酪蛋白的分泌。     

Lymphocytes from one side of the bovine mammary gland migrate to the contra lateral gland and lymph node tissue

The four quarters of bovine mammary glands are completely separated and two quarters on each side (right or left) are connected to ipsi lateral supra mammary lymph nodes. It is not clear whether cells infused into the cistern of the mammary gland are capable of migrating to lymph nodes or the general circulation. To examine cell migration, a prescapular lymph node was removed from each of two lactating and three non-lactating dairy cows, and isolated lymphocytes were stained with Hoechst 33342. Autologous stained cells were infused into the mammary gland and then activated by intramammary infusion of zymosan-stimulated serum (source of C5a). After 17 h, Escherichia coli J5 bacterin was infused into the contra lateral mammary gland to mimic infection. After 43 h cows were euthanized and tissue samples (mammary quarters, right and left supra mammary, mesenteric, ileocecal and prescapular lymph nodes, liver and spleen) were collected for microscopic examination as well as flow cytometric analysis. Hoechst stained cells were detected not only in infused quarters, but also in contra lateral quarters as well as in both supra mammary lymph nodes. This indicates that cells infused into the mammary gland migrate to contra lateral tissues and supra mammary lymph nodes.     

Effect of oestradiol on mast cell number and histamine level in the mammary glands of rat

Variations of mast cell number, histamine concentration and oestrogen receptor (ER) expression in mammary glands with the fluctuation of plasma oestradiol level were identified either in the intact rats at different oestrous stages or in the ovary-ectomized rats administrated with different doses of oestradiol benzoate. The results showed that the number of mast cells and histamine concentration fluctuated concomitantly with plasma oestradiol level during the oestrous cycle. More mast cell number and higher histamine concentrations were observed in the oestrous stage than that in the prooestrous and dioestrous stages. Ovariectomy decreased the mast cell number and histamine concentration, which were reconstituted by exogenous oestradiol. ER was mainly found in the nuclear of epithelial cells and interstitial cells of mammary glands. In addition, ER was also expressed in the cytoplasm of some stromal cells. These stromal cells were verified to be mast cells. In conclusion, our results suggested that oestradiol modulated mast cell number and its degranulation in the mammary gland through the ERs pathway.     

Expression of parathyroid hormone-related protein (PTHrP) mRNA in mammary gland of periparturient cows

The expression of parathyroid hormone-related protein (PTHrP) mRNA was examined in mammary gland with or without lactation, and during periparturient period in a Holstein cow and a Jersey cow. In the lactating mammary gland, PTHrP was detected in alveolar epithelial cells and the lumen by immunohistochemical analysis. The relative expression levels of PTHrP mRNA in mammary gland from lactating cows were significantly higher than those from non-lactating cows (P<0.05). During periparturient period, relative PTHrP mRNA level was remarkably low before the parturition in a Jersey and a Holstein cow, however, both levels were gradually increased and reached a peak level at 5-6 weeks after the parturition. In addition, the peak level in a Jersey cow was approximately 3-fold higher than that in a Holstein cow. From these results, PTHrP was synthesized and secreted in alveolar epithelial cells in mammary gland and increased subsequently with the lactation, suggesting a possible mechanism for the regulation of local calcium homeostasis.     

COMPUTED TOMOGRAPHY AND RADIOGRAPHIC INDIRECT LYMPHOGRAPHY FOR VISUALIZATION OF MAMMARY LYMPHATIC VESSELS AND THE SENTINEL LYMPH NODE IN NORMAL CATS

The potential of computed tomography indirect lymphography (CT‐indirect lymphography) and radiographic indirect lymphography to demonstrate the draining lymphatic vessels and sentinel lymph node of normal mammary glands was tested in 31 healthy female cats. The lymphatic drainage of each mammary gland was studied initially by CT‐indirect lymphography after intramammary injection of 0.5 ml of iopamidol, followed by images acquired at 1, 5, 15, and 30 min after injection. One day after CT‐indirect lymphography, the lymph drainage of the mammary gland was assessed using radiographic indirect lymphography after intramammary injection of 0.5 ml of ethiodized oil followed by radiographs made at 1, 5, 15, 30, 45, and 60 min after injection. The time between intramammary injection and opacification of the draining mammary lymphatic vessels and the sentinel lymph node, the duration of adequate opacification of the draining mammary lymphatic vessels and of the sentinel lymph node and also the number and course of draining mammary lymphatic vessels and location of sentinel lymph node were compared for CT‐indirect lymphography vs. radiographic indirect lymphography in each examined gland. This results suggest that radiographic indirect lymphography is easy to perform and can be used for accurate demonstration of the draining lymphatic pathways of mammary glands in radiographs made at 5–30 min after injection. However, CT‐indirect lymphography was able to better demonstrate small lymphatic vessels and accurately define the exact topography of the sentinel lymph node in images acquired at 1 min after injection.     

Mast cell tumor in the mandibular salivary gland in a dog

A mast cell tumor in the mandibular salivary gland was diagnosed in a 9-year-old English Setter. Dense infiltrating sheets of mast cells were present within the mandibular salivary gland, the medial retropharyngeal lymph node and in two subcutaneous masses on the flank. The salivary gland, lymph node, and masses were excised. The dog was treated postoperatively with the histamine receptor antagonist, cimetidine and the alkylating agent, chlorambucil. The tumor recurred at the angle of the mandible 4 months later and the dog was killed.     

Effect of milk stasis on Brucella abortus infection of the mammary gland in goats

To compare the effects of milk stasis and milk flow on Brucella abortus infection of the mammary gland under the same systemic conditions, primiparous goats (n = 5) were inoculated IV with B abortus on the day of parturition, and suckling by their neonates was restricted to one mammary gland. Goats were euthanatized and necropsied at 3 weeks after inoculation, and milk, mammary glands, and supramammary lymph nodes were evaluated by bacteriologic, histologic, and immunoenzymatic staining techniques. Nonnursed mammary glands had high titers of brucellae in milk, moderate interstitial mastitis, and brucellar antigen in macrophages located primarily in alveolar and ductal lumina. Brucellae often filled the macrophage cytoplasm. In contrast, nursed mammary glands had fewer brucellae in milk, minimal inflammatory changes, and no detectable brucellar antigen in histologic sections. Hyperplastic changes were only seen in supramammary lymph nodes draining nonnursed mammary glands; these contained more brucellae than lymph nodes draining nursed mammary glands. These studies show that milk stasis may be the sole cause of increased susceptibility of nonnursed mammary glands to B abortus infection.     

Study on Expression of SYK in Dairy Cow Mammary Gland

To investigate the relationship between the expression of SYK and dairy cow mammary gland development and lactation, the expression of SYK in lactating dairy cow mammary gland with high or low quality milk and dry period Holstein dairy cow mammary gland was detected by Western blotting and laser confocal microscope.The results showed that SYK expression in dry period mammary gland was significant higher than that in lactating mammary gland (P<0.05).There was no SYK differential expression detected between lactating mammary gland with high quality milk and low quality milk (P>0.05).SYK was mainly located in the cytoplasm of ductal epithelial cells in dry period mammary gland.In lactating mammary gland, SYK was existed in acinar epithelial cells.All these results revealed that SYK was a regulator in mammary epithelial cell proliferation and differentiation.It participated in mammary gland reconstitution in dry period.