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。     

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。     

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)。     

奶牛乳腺肥大细胞免疫形态学的增龄性变化

应用改良甲苯胺蓝染色法研究不同年龄奶牛乳腺肥大细胞分布特点的增龄性变化，并用Alcian bluc-safranin染色法对肥大细胞进行细胞化学分型研究。结果：青年奶牛乳腺中肥大细胞数量较少，中年奶牛乳腺中肥大细胞的数量增多，有趣的是，老年奶牛乳腺肥大的数量更多，AB-S染色显示，各年龄奶牛乳腺中只有黏膜型肥大细胞。结果表明：奶牛乳腺肥大细胞数量具有随年龄增长而增加的特点，但肥大细胞的类型没有发生改变。     

肥·大·细·胞·与·奶·牛·蹄·叶·炎

奶牛蹄叶炎的确切病因和发病机制没有一致性的结论。陈家璞认为蹄叶炎的主要原因是组胺、毒素和代谢紊乱，而组胺本身就是肥大细胞所释放的生物活性物质之一。关于肥大细胞及其所释放的多种生物活性物质，在生理和病理过程中发挥重要作用已有许多文献报道。尤其是分布于皮肤内的肥大细胞释放组胺、肝素、类胰蛋白酶、类糜蛋白酶、肿瘤坏死因子（TNF—α）、白三烯、前列腺素（PG）、细胞因子等参与Ⅰ型变态反应及许多皮肤性疾病如银屑病、增生性瘢痕、瘢痕性疙瘩、特应性皮炎等疾病的病理过程。但肥大细胞及其所释放的以上生物活性物质在奶牛蹄叶炎发病机制中的作用尚未见报道，故本文应用组织化学方法观察蹄叶炎奶牛蹄部的组织结构、肥大细胞的数量、分布及组化性质，探讨肥大细胞在奶牛蹄叶炎发病过程中的作用，为奶牛蹄叶炎的防治提供新的理论依据。     

六种动物含类胰蛋白酶肥大细胞的酶组化分析

使用鉴定人类肥大细胞类胰蛋白酶的酶组织化学技术，证实猪，牛，绵羊，狗，猫及小鼠的肥大细胞中均信在胰蛋白酶。与采用甲七胺兰的常规组织化学色比较，类胰蛋白酶阳性肥大细胞与甲苯胺兰阳性肥大细胞在分布，形态和密度方面是相亿主的。     

泌乳周期中外源物质对大鼠乳腺内肥大细胞数量的影响及乳腺组织内组胺含量的研究

选择泌乳期和静止期大鼠，经乳腺局部（乳导管）或外周（跖部皮下）注射辣根过氧化物酶（HRP）后，应用改良甲苯胺蓝染色法（MTB）对乳腺及其引流（鼠蹊部）和非引流（腘）淋巴结内的肥大细胞数量变化进行了研究，用荧光测定改良法检测了乳腺组织中组胺含量。结果显示，对照组乳腺及引流和非引流淋巴结内的肥大细胞数量均为静止期显著多于泌乳期（P〈0．01），乳腺中组胺含量也呈相同的变化趋势。在泌乳期从不同部位引人HRP后，乳腺及乳腺引流淋巴结内肥大细胞数量明显增多，与对照组相比差异极显著（P〈0．01）；而乳腺非引流淋巴结内肥大细胞数量则略有减少，与对照组相比差异不显著（P〉0．05）。在静止期从不同部位引人HRP后，乳腺及乳腺非引流淋巴结内肥大细胞数均减少，而乳腺引流淋巴结内的肥大细胞数则随引人部位不同而有差别：乳腺引人时增加，而外周引入时减少。本研究结果表明肥大细胞数量随生理周期的改变而增减的趋势与乳腺的发育、乳腺局部免疫状态及内分泌调节都有关系，而且不同泌乳阶段乳腺内肥大细胞对异物的易感性也存在差异。     

奶牛蹄叶炎的防治措施

奶牛蹄叶炎是危害奶牛生产的（除乳腺炎、繁殖障碍疾病）第3大疾病。蹄叶炎的确切病因和发病机制没有一致性的结论。一般认为，蹄叶炎主要是组胺、毒素和代谢紊乱导致，而组胺本身就是肥大细胞所释放的生物活性物质之一。尤其是分布于皮肤内的肥大细胞释放组胺、肝素、类胰蛋白酶、类糜蛋白酶、肿瘤坏死因子（TNF—α）、白三烯、前列腺素（PG）、细胞因子等。由于蹄叶炎可导致蹄变形、蹄底溃疡病及白线病等多种蹄病，给奶牛养殖造成巨大的损失。     

绒山羊肥大细胞类胰蛋白酶的免疫组化及图像分析研究

[目的]探讨绒山羊肥大细胞中是否存在类胰蛋白酶及不同固定液对其常规染色和免疫组化染色结果的影响。[方法]采用兔抗羊肥大细胞类胰蛋白酶多克隆抗体间接免疫过氧化物酶技术检测由Carnoy液和中性福尔马林液(NBF)固定的绒山羊空肠、瓣胃和肺等组织肥大细胞中是否存在类胰蛋白酶。同时采用Image-ProPlus图像分析软件和人工计数法对结果进行分析,比较经常规染色和免疫组化染色后不同固定液固定组织中肥大细胞的形态及数量,进而判断Carnoy液和中性福尔马林液(NBF)对该检测技术的影响。[结果]兔抗羊多克隆抗体与绒山羊肥大细胞中的类胰蛋白酶具有良好的交叉反应,证实绒山羊肥大细胞胞浆颗粒中存在类胰蛋白酶。同时,与Carnoy液相比较,NBF液固定组织能较好地反映肥大细胞在组织中的数量和形态变化。[结论]绒山羊肥大细胞中存在类胰蛋白酶,且与Carnoy液相比较,NBF液是一种更为适合绒山羊肥大细胞常规染色和免疫组化染色的固定剂。     

敲低SQLE基因对奶牛乳腺上皮细胞增殖及凋亡的影响

为了探讨角鲨烯环氧酶（squalene epoxidase,SQLE）基因对体外培养奶牛乳腺细胞凋亡及增殖的影响,本研究用RNAi技术敲低奶牛乳腺上皮细胞中SQLE基因;用实时荧光定量PCR方法检测奶牛乳腺上皮细胞中SQLE基因及凋亡和周期相关基因的表达;用CCK-8法检测其对细胞增殖的影响;用周期和凋亡检测试剂盒筛选细胞,用流式细胞术准确计数处于不同周期的细胞数和凋亡细胞数。结果显示,奶牛乳腺上皮细胞转染siRNA后,SQLE基因相对表达量显著下降（P<0.05）,细胞增殖受到极显著抑制（P<0.01）;G1期细胞数量显著下降（P<0.05）,G2期细胞数量没有显著性改变,S期细胞数量显著上升（P<0.05）;肿瘤坏死因子超家族成员6（又称Fas或CD5）的相对表达量显著上升（P<0.05）,细胞周期蛋白依赖性激酶抑制剂1B（cyclin dependent kinase inhibitor 1B,P27）相对表达量显著上升（P<0.05）,而细胞周期素D1（Cyclin D1）、B淋巴细胞瘤因子2（B-cell lymphoma-2,Bcl-2）、细胞周期蛋白依赖性激酶抑制剂1A（cyclin dependent kinase inhibitor 1A,P21）、Bcl-2相关X蛋白（Bcl-2 associated X,apoptosis regulator,Bax）显著下降（P<0.05）。综上所述,敲低SQLE基因通过调节相关基因的表达抑制乳腺上皮细胞的增殖。     

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.     

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。     

Immunohistolocalization of carbonic anhydrase isozyme (CA-VI) in bovine mammary glands

The localization of bovine carbonic anhydrase isozyme VI (CA-VI) was examined immunohistochemically in bovine mammary glands during early lactation period (after 2-3 days of postpartum) and dry period (at about 2 months preparturition in adults), and young calves (at 30 and 150 days after birth) using specific CA-VI antiserum. The immunoreaction for anti-CA-VI antiserum was very weak in the mammary glands in young (prepubescent) calves. In dry period, CA-VI was also weakly expressed in secretory epithelial (acinar) and ductal cells. In contrast, the reaction was intense in mammary gland cells in early lactation period. Dot blotting analysis indicated that anti-CA-VI reacted positively to beastings and mature saliva, but weakly or not at all to milk during the dry period or calf saliva, respectively. The intense expression of CA-VI in the mammary glands in early lactation period might compensate for low levels of secretion from functionally and structurally immature salivary glands in young calves.     

Aberrant development of mammary glands, but precocious expression of beta-casein in transgenic females ubiquitously expressing whey acidic protein transgene

It has been suggested that whey acidic protein (WAP) may function as a protease inhibitor. However, the actual function of WAP remains obscure. We investigated the histological development of the mammary glands of transgenic mice ubiquitously expressing WAP and CAG/WAP transgene. Ubiquitous expression of WAP induced aberrant development of the lobular alveoli of the mammary glands: mammary alveoli that were either aberrantly large or small in size increased in number in the developing mammary glands of these transgenic females during pregnancy and lactation. The expression of beta-casein was precociously induced in the mammary glands of the transgenic females during early pregnancy and accompanying this was a histological observation that abnormally developed lobular alveoli filled with milk proteins appeared in the mammary glands of transgenic females during early pregnancy. However, during lactation, the development of mammary glands was impaired in transgenic females. To investigate the possible paracrine action of WAP associated with mammary gland aberration, we transplanted the mammary tissue of CAG/EGFP transgenic females into the fat pad of virgin CAG/WAP transgenic females and initiated pregnancy by mating. The development of mammary tissue transplanted to the recipient was histologically examined on day 3 of lactation. The results revealed that the development of grafted mammary tissues was impaired in a manner similar to that of the mammary glands of CAG/WAP transgenic females, indicating that the inhibitory effect of WAP acts via a paracrine mechanism. In vitro experiments using HC11 cells with forced expression of exogenous WAP demonstrated the inhibitory function of WAP on proliferation of mammary epithelial cells.     

S‐100 Protein‐Positive Mast Cells in Canine Paranal Sinus (Sinus paranalis)

The aim of this study was to establish the immunocytochemical expression of S‐100 protein in mast cells, localized in the wall of dog's paranal sinus. Control serial sections were used for immunocytochemical detection of tryptase‐positive mast cells. It was observed that S‐100‐positive cells have the same morphology and localization as the tryptase‐positive mast cells, which indicated that S‐100‐positive cells are most probably mast cells with abilities as dendritic cells. In conclusion, for the first time, the current study gave evidence that mast cells in this organ possess one more function, such as dendritic cells.     

Reduced mucosal injury of SPF chickens by mast cell stabilization after infection with very virulent infectious bursal disease virus

Recent studies here have demonstrated that increased mast cell populations and tryptase activity contribute to lesion formation in regions of immune organs in special-pathogen-free chickens after infection with very virulent infectious bursal disease virus (vvIBDV). Mast cells and their mediators have been implicated in acute inflammatory injury after vvIBDV infection, but their precise role in this process remains elusive. In this study, the role of mast cells in the vvIBDV infection process was examined using ketotifen, a mast cell membrane stabilizer. On days 1, 2, and 3 postinfection, the bursa of Fabricius (BFs) were collected to quantify mast cells, tryptase and histamine contents by cytochemistry, immunohistochemistry and fluorospectrophotometry analyses, respectively. The results showed that the mast cell populations, tryptase expression, and histamine released increased significantly in the BFs (p < 0.01) of infected birds compared to controls, and acute inflammatory responses were observed in the former. In contrast, in infected chickens pretreated with ketotifen, mast cells, tryptase, and histamine were markedly decreased (p < 0.01) and probably as a result, the BFs remitted significantly. The overall results suggest that mast cells are positively involved in BF injury induced by vvIBDV infection. Inhibition of mast cell degranulation and concurrent mediator release may represent a novel strategy to modulate this process. This study, thus, advances the understanding of the acute inflammatory injury mechanisms triggered by vvIBDV infection and the contribution of mast cell activity in this process.