犬正常发情期与患子宫蓄脓的子宫和卵巢组织结构及乳铁蛋白表达的对比

本研究旨在探明犬子宫和卵巢在正常发情期与患子宫蓄脓时，其组织结构及乳铁蛋白（lactoferrin，LF）表达的变化特征。应用Masson’s、VVG、PAS组织化学染色方法观察乏情期、发情期、患子宫蓄脓时犬子宫及卵巢的组织结构特点，用免疫组织化学SP法观察LF的分布特征。结果显示，正常发情期犬：乏情期、发情期子宫内膜肌层厚度比分别为0.762 0、0.924 3；乏情期子宫固有层中胶原纤维含量大于发情期，发情期卵巢中胶原纤维含量大于乏情期；子宫血管层及卵巢血管内弹性膜清晰完整；子宫腺在乏情期时浅层管腔小，深层较大、腺管上皮为单层柱状上皮、上皮细胞及管腔内PAS阳性反应较强，发情期子宫腺管腔变大、腺管上皮为单层立方上皮、上皮细胞及管腔内有PAS阳性反应；子宫黏膜上皮在乏情期和发情期均为单层柱状上皮，但胞核位置不同，乏情期胞核位于中央，发情期胞核位于顶部。患子宫蓄脓犬：子宫内膜肌层厚度比为1.615 0；子宫固有层和卵巢中胶原纤维含量少于正常发情期；子宫腺管腔大，且形状不规则，管腔内有炎性细胞浸润，腺管上皮为单层立方上皮，有淋巴细胞位于基膜，上皮细胞及管腔内PAS阳性反应较弱；子宫血管层及卵巢血管内弹性膜较正常发情期变薄，且有断裂现象；子宫黏膜上皮为单层柱状上皮，胞核位于基底。LF在乏情期子宫腺上皮和卵巢中的表达水平高于发情期，而在子宫黏膜上皮中发情期的表达水平高于乏情期。患子宫蓄脓时犬子宫和卵巢中LF的表达水平均较低。综上表明，犬正常发情期与患子宫蓄脓时的子宫和卵巢组织结构特点显著不同，LF的表达水平也存在差异。     

羊膜对山羊角膜缘上皮细胞培养影响的研究

比较完整羊膜和去上皮羊膜、不同冻存时间的羊膜对山羊角膜缘上皮细胞体外培养的影响,筛选出羊膜最适处理方法。将山羊角膜缘组织块分别接种于完整羊膜、去上皮羊膜、新鲜去上皮羊膜、冻存30 d去上皮羊膜、冻存90 d去上皮羊膜、冻存180 d去上皮羊膜上,比较在完整羊膜和去上皮羊膜及不同冻存时间羊膜上角膜缘上皮细胞的生长特性和组织结构。试验结果显示,在去上皮羊膜上角膜缘组织块贴壁和细胞增殖能力较完整羊膜强,角膜缘上皮细胞在2组羊膜上均能形成多层结构,但完整羊膜上形成的组织表层角质化较严重;在冻存30 d羊膜上角膜缘组织块贴壁和细胞增殖能力较新鲜羊膜、冻存90 d羊膜、冻存180 d羊膜强,角膜缘上皮细胞在各组羊膜上均形成多层结构,其结构无明显差异。结果表明,冻存30 d去上皮羊膜是构建组织工程人工角膜最适的羊膜载体材料。     

诱导发情后母犬生殖器官组织学及组织化学观察

为了进一步探讨犬的生殖生理学,试验以诱导发情后母犬生殖器官为试验材料,采用组织学和组织化学技术,观察了诱导发情后母犬卵巢、输卵管和子宫的形态学特点。结果表明:诱导发情后犬卵巢外观呈葡萄串状,镜下可见大量黄体、少量原始卵泡和初级卵泡;诱导发情后母犬输卵管壁由黏膜层、肌层和外膜构成,黏膜上皮主要是由纤毛上皮细胞、分泌细胞构成的单层柱状上皮,黏膜固有层可见由纤毛上皮和分泌细胞围绕形成的管状腺,PAS反应可见分泌细胞的游离面和管状腺内有少量的阳性物质;诱导发情后母犬子宫壁由黏膜、肌层和外膜构成,黏膜上皮是单层柱状上皮,黏膜固有层内可见由单层柱状上皮构成发达的管状腺,子宫黏膜上皮细胞内含有少量PAS阳性物质,表面覆盖一层PAS阳性物质,而腺腔内含大量PAS阳性物质。     

发情母犬阴道上皮细胞变化与最佳配种时机

母犬进入发情期后，在生殖激素的作用下，出现一系列的发情征候，包括阴唇肿胀、阴户流血、阴道分泌物成分发生改变等。Christile(1972)报道，发情母犬进入发情期后，阴道上皮细胞的形状、大小逐渐发生改变，单层的立方上皮转化为多层、形状不规则、大的、有核的鳞状细胞(中间型细胞)，最后变成无核的角化鳞状细胞(表皮细胞)。到排卵时，上皮细胞全部角化，白细胞消失。阴道上皮     

胰岛素、催乳素对奶山羊乳腺上皮细胞泌乳功能的影响

具有泌乳功能的乳腺上皮细胞可作为乳腺生长发育及泌乳功能调控机制研究的细胞模型。本研究对已建立的奶山羊乳腺上皮细胞系的泌乳功能进行评价。应用SDS-PAGE、TG试剂盒及HPLC方法对体外培养奶山羊乳腺上皮细胞总蛋白、乳脂和乳糖的分泌情况进行了测定以确定胰岛素、催乳素处理后的奶山羊乳腺上皮细胞的泌乳功能。结果表明：在添加表皮生长因子、胰岛素样生长因子-1、转铁蛋白-硒钠、标准胎牛血清的DF12培养液中奶山羊乳腺上皮细胞具有乳脂、乳蛋白及乳糖的分泌能力。胰岛素处理48 h后对细胞的泌乳功能无显著影响,催乳素处理细胞48 h后可使奶山羊乳腺上皮细胞乳蛋白、乳糖分泌能力显著升高。从而建立了具有泌乳功能的奶山羊乳腺上皮细胞系,为奶山羊、奶牛等重要经济动物泌乳机制的研究提供了便利条件。     

猪肺脏气道上皮干细胞的特异抗体筛选与抗原表达研究

猪肺脏气道上皮主要由基底细胞,克拉拉细胞,纤毛细胞,杯状细胞等上皮细胞类型组成,筛选猪气道上皮干细胞的相关特异性抗体,观察其特异抗原的表达,将有助于对猪气道上皮干细胞的分离、鉴定和生物学特性的研究.试验中,制备远端气道上皮细胞的冰冻切片与细胞爬片,利用免疫荧光染色法,分析不同物种来源的抗体对猪气道上皮细胞的免疫染色反应;同时利用Oct3/4、Sox2、CD133和SSEA-1等10种上皮干细胞相关抗体分析猪气道上皮干细胞表面抗原表达情况.通过试验,筛选出在猪气道上皮干细胞相关抗体,并发现气道组织冰冻切片未检测到这10种抗体相关的阳性细胞,而在细胞爬片中观察到CD49f和CD117阳性细胞数量较多.免疫荧光共染发现这些CD49f和CD117阳性细胞同时也表达基底细胞表面标记Keratin 14.这可能是正常情况下猪气道上皮干细胞数量极为稀少,表面抗原表达量低而不易检测到,在肺脏气道上皮受到外环境刺激时(如消化),上皮干细胞开始增殖以完成损伤修复作用发生数量上的增殖.综上所述,利用Keratin 14和CD49f或CD117(c-kit)双标染色法可以鉴定猪肺脏气道上皮干细胞的候选亚群来用于其生物学特性与功能的研究.     

1株H5N1亚型高致病力禽流感病毒人工感染鸭的抗原定位观察

通过对鸭静脉接种或眼-鼻-口腔-泄殖腔接种禽流感病毒A/duck/Guangdong/220/2004(H5N1)后,采用免疫组织化学方法检查病毒核蛋白抗原,探讨了该病毒在组织器官中的定位与分布.结果表明,病毒核蛋白抗原主要存在于鸭的心肌细胞、肝细胞、枯否氏细胞、胰腺泡上皮细胞、肾小管上皮细胞、血管内皮细胞和血液吞噬性自细胞;在气管和支气管黏膜上皮,脑组织,肠黏膜上皮,胸腺、法氏囊、脾脏和肺脏的巨噬细胞或组织细胞以及坏死的细胞碎片中也可检查到少量病毒核蛋白抗原;而在食管、胃、睾丸、卵巢、甲状腺、肾上腺、哈德氏腺、骨骼肌等未检查到该抗原.     

表皮生长因子及其受体在山羊胎儿皮肤发育中的表达特征

运用组织学和免疫组化方法对山羊皮肤发育的组织学特点、山羊皮肤发育过程中表皮生长因子(EGF)及其受体(EGFR)的分布及变化规律进行了系统研究.结果显示:(1)表皮结构形成于胎儿发育的6周龄左右,开始为单层上皮,而后逐渐增殖为复层、厚度不断增加,15周龄后逐渐变薄;真皮层在10周龄时形成,11周龄后陆续有皮肤衍生结构从中发育形成.随着胚胎的继续发育,皮肤各部分结构的发育渐趋完善.(2)在胎儿发育的第6周就出现EGF与EGFR的较弱表达,以后随着胎龄的增加,EGF与EGFR表达量逐渐增加.从表达分布看,在胎儿发育的11周前,EGF阳性反应主要定位于表皮基底层细胞、毛囊上皮细胞和真皮成纤维细胞等的胞质内,EGFR则主要位于相应细胞的膜上;11-16周,EGF和EGFR表达量逐渐增高,分布范围从表皮的基底层细胞、棘细胞、毛囊上皮细胞和成纤维细胞扩展到血管内皮细胞、汗腺上皮细胞和竖毛肌,EGF主要定位于这些细胞的胞质,EG-FR则主要分布于细胞膜;17周至出生,随着表皮的明显变薄,EGF及EGFR主要分布于表皮基底层细胞和毛囊上皮细胞,阳性反应继续增强.皮肤发育期间,EGF及EGFR的表达量总体呈增长趋势,只是EGFR的表达相对滞后,两者的表达曲线一致、相关性极显著.表明EGF及EGFR在皮肤及其衍生结构的发育中发挥着重要作用.     

免疫反应性脑啡肽在猪体外合成和分泌

试验证明,脑啡肽ir—MENK不但存在于猪的子宫、卵巢中,而且也存在于由SV40 DNA转染的猪子宫上皮细胞中。用生理盐水冲洗猪子宫腔并收集子宫液,用0.1N乙酸抽提猪卵巢及子宫上皮细胞获得抽提液。细胞培养液经纯化、冻干后再溶解作为分析液。以上几种样本在RIA系统分析。显示子宫液、卵巢、传代细胞的抽提液或培养液的抑制曲线均平衡于已知的MENK标准曲线。G—15葡聚糖凝胶过滤可见它们有相同的峰位,较大部分其分子量与标准的脑啡肽吻合Va=0.38,小部分接近孔隙率(VO)。结果显示,体外培养的猪子宫传代细胞及细胞培养液中的脑啡肽与活体的猪子宫及卵巢的脑啡肽相同。说明猪子宫上皮传代细胞不但可以合成,而且可以分泌脑啡肽。     

壮阳促孕散对雌性小鼠生殖机能的影响研究

采用常规药理学实验方法,探讨了中药壮阳促孕散对雌性小鼠子宫增重、卵巢结构和对去卵巢小鼠子宫内膜、阴道上皮结构以及阴道上皮脱落细胞角化率的影响.结果表明,壮阳促孕散能使幼年小鼠子宫显著增重,促进幼年小鼠卵巢发育,使其提早出现性成熟的形态特征;也能促进去卵巢小鼠子宫内膜和阴道上皮增生、分泌,提高阴道上皮脱落细胞角化率,从而证明中药壮阳促孕散具有雌激素样作用.     

Immunohistochemical and ultrastructural characterization of the ovarian surface epithelium of Japanese quail (Coturnix japonica)

Contrary to humans, most ovarian tumors in other species do not arise from the ovarian surface epithelium but are of follicular‐, stromal‐ or germ‐cell origin. One of the few species where ovarian cancer arises from the ovarian surface epithelium (OSE) is chicken (Gallus domesticus). Little is known about the morphology of the OSE in other avian species. In our study we analyzed the OSE morphology of Japanese quail (Coturnix japonica) using ultrastructural and histochemical techniques. Carbohydrate residues have been studied by using a panel of fluorescein isothiocyanate labeled lectins. Japanese quails are commonly used animal models in biomedical research as their housing is comparatively inexpensive and they show a short generation interval. Our ultrastructural and histochemical results demonstrate that the quail ovarian surface epithelium shows characteristic features which resemble the epithelia of both chicken and human. Additionally, the ovarian surface epithelium of the Japanese quail contains cytokeratin as well as vimentin intermediate filaments in their cytoplasm. Therewith and among other parts the quail OSE shows many characteristic features also seen in those of humans, which may qualify quail's ovary as a potential animal model for human ovarian carcinomas.     

Conditional knockout of brca1/2 and p53 in mouse ovarian surface epithelium: Do they play a role in ovarian carcinogenesis?

Alterations of genes are known to be critical for the induction of tumorigenesis, but the mechanism of ovarian carcinogenesis is little understood and remains to be elucidated. In this study, we investigated the roles of brca1, brca2 and p53 genes in the development of ovarian cancer using conditional knockout mice generated by a Cre-loxP recombinant system. Following the application of recombinant adenovirus expressing Cre in vitro, the proliferation of ovarian surface epithelium (OSE) was increased. For instance, a significant increase in cell growth was observed in OSE cells in vitro by conditional knockout isolated from the mice bearing concurrent floxed copies of brca1 and brca2/p53. However, the proliferative effect of the ovarian cells was not observed in concurrent brca1/brca2 or p53 knockout mice in vivo, indicating that we could not observe the direct evidence of the involvement of brca1, brca2, and p53 in ovarian carcinogenesis. Since morphological changes including tumor formation were not observed in mice bearing floxed copies of concurrent brca1/brca2 or p53, the inactivation of brca1/2 or p53 is not sufficient for the induction of tumor formation. Taken together, these results suggest that the deficiency of these genes may not be involved directly in the mechanism of ovarian carcinogenesis.     

Ovarian mucinous cystadenoma with smooth muscle proliferation in a cynomolgus monkey (Macaca fascicularis)

An ovarian mucinous cystadenoma was found in a 5-year-old female cynomolgus monkey (Macaca fascicularis). The tumor was composed of various sizes of multilocular cystic glands lined by a single layer of mucin-filled epithelium. Each of these cystic glands was surrounded by a large amount of solid fibrous stroma resembling smooth muscle. The ovarian surface epithelium showed partial invagination into the ovarian cortex, and a transition was observed between the surface epithelium and the mucinous cyst-forming epithelium. Immunohistochemically, the stromal cells were positive for alpha-smooth muscle actin and proliferating cell nuclear antigen. Ultrastructurally, the glandular epithelium had numerous mucinous secretory granules and microvilli. The stromal cells had numerous parallel microfibrils with focal density. It is rare to encounter evidence of a transition from the surface epithelium to the mucinous tumor epithelium and to show stromal smooth muscle proliferation in a mucinous cystadenoma.     

绵羊卵巢动脉铸型标本制作方法的建立

试验旨在探讨绵羊卵巢动脉铸型标本的制作方法,研究卵巢及其周边血管的分布与供血特征,为哺乳动物生殖系统血管的精细解剖学尤其是女性卵巢的供血机制研究奠定基础。通过采集绵羊子宫及附件样本,解剖分离出卵巢的部分螺旋动脉使其呈直线型,采用双侧卵巢动脉同时插管,用8% ABS灌注螺旋动脉及其分支,待铸型剂硬化后再用30%盐酸腐蚀软组织,经冲洗、修整获得完整的卵巢动脉血管立体铸型标本。由标本可见,绵羊卵巢动脉分支包括：卵巢动脉卵巢支、卵巢动脉子宫支和卵巢动脉输卵管支;卵巢动脉卵巢支包含：卵巢微动脉、卵巢门螺旋动脉、卵巢门轻螺旋动脉及卵巢门重螺旋动脉。观察发现,卵巢动脉卵巢支呈高度螺旋状结构,卵巢内微动脉主要集中于卵巢门附近,呈高度盘曲折叠的立体网状结构,在微动脉末端发出微血管延伸至卵巢其他区域。通过该方法制成的铸型标本血管走向清晰、表面光滑、立体感强,可作为哺乳动物卵巢血管相关研究的参考。     

Making Procedure of Vascular Corrosion Casts of Ovarian Artery in Sheep

In order to explore the making procedure of vascular corrosion casts of ovarian artery in sheep and study the distribution of the blood vessels, this study was carried out and it would provide the foundation of blood vessels anatomy of reproductive system including blood supply mechanism of female ovaries in mammals. Firstly, samples of the uterus and appendages in sheep were collected, and then ovarian arteries and their branches were infused with 8% ABS perfusion agents. After the agent became solid, the samples' soft tissues were corroded away with 30% hydrochloric acid, washed them down with running water and the moderate trim process was done for obtaining the complete vascular corrosion casts of ovarian artery in sheep.The finished casts showed that the ovarian artery in sheep arises three branches including uterine, ovarian and tubal. The ovarian branch showed complicated arrangement such as the spiral artery of ovarian hilus, light spiral piece of ovarian hilus and tight spiral piece of ovarian branch. The results confirmed that the ovarian branch was characterized by the presence of a tight spiral configuration in sheep, the ovarian arterioles were fasten on ovarian hilus, and there were some dense capillaries which extend to other regions of the ovary followed them. Through this procedure, the vascular distribution of corrosion casts was clear,three-dimensional and with smooth surface. It could provide a reference or model for the study of ovarian blood vessels in mammals.     

哺乳动物的卵巢冷冻保存

经冷冻保存后的卵巢或组织可以保存大量的原始卵泡,并且结构完整,具有活力及发育能力,且已直接用于临床获得妊娠。因此,卵巢冷冻近年来成了生殖生物学、生殖医学的研究热点。本文就卵巢冷冻的历史、基本技术原理、方案选择与评价及其影响因素作一综述。     

Activation of goat primordial follicles in vitro: Influence of alginate and ovarian tissue

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.     

Immunohistochemical localization of the progesterone and oestrogen receptors in the shell gland of sexually immature ostriches (Struthio camelus) with active or inactive ovaries

The immunohistochemical localization of progesterone and oestrogen receptors was studied in the shell gland of the immature ostrich (Struthio camelus) during periods of ovarian activity and inactivity. In birds with active ovaries moderate to strong immunostaining for the progesterone receptor was observed in the surface epithelium and tubular glands. In contrast faint progesterone receptor immunostaining was observed in the surface epithelium of the shell gland in ostriches with inactive ovaries. In addition, bud-like invaginations of the surface epithelium, which signaled tubular gland development, were negative for the progesterone receptor. Oestrogen receptor immunostaining, which was seen only in birds with active ovaries, was weak and restricted to nuclei of the surface epithelium. These results suggest that steroid hormones secreted by the active ovary regulate the differentiation of the shell gland. Furthermore, the influence of these hormones on the shell gland appears to be mediated predominantly through the activation of the progesterone receptor.     

Morphological and scanning electron microscopic study of the gonadal arterioles in the Tibetan sheep

This research aimed at revealing the structural features of Tibetan sheep's testicular and ovarian vasculature and providing theoretical basis for Tibetan sheep's physiological adaptability in plateau and low-oxygen environments. Through scanning electron microscopy, corrosion casts of the feature of the gonad arterioles were observed and analysed. The results of this study found that centrifugal arteries, centrifugal and centripetal arterioles were distributed in a ‘dendritic’ fashion in the testis lobule, the ovarian arteries and arterioles, which had a spiral shape. Testicular Tibetan Sheep knot-like artery had a denser spiral structure, and there were more branches in the microarteries. On the testis lobule and arteriole of caput epididymidis, the arteriole surface shows the ‘fusiform’ indentation to be more shallower. The ovarian artery had a higher degree of helix; its surface was distributed like a ‘bark-like’ indentation; and the ring was narrower and denser. Nevertheless, the arteriole network had a high concentration and a wide number of branches, as that of the ovarian arteriole network which had more end branches, the surface of the arterioles of ovaries and testicles was scarred and denser. The studies indicate that the structural features of testicular lobule, epididymal arterioles and ovarian arterioles are more conducive to increase the oxygen supply to the gonads tissue and controlling blood flow velocity and metabolism to respond to high altitudes and low-oxygen environments.