The effect of transgenesis on rabbit thyroid tissue structure

This study was aimed to compare structures of the thyroid tissue of transgenic rabbits expressing the human clotting factor VIII under the murine whey acidic protein promoter (mWAP-hFVIII rabbits) with the non-transgenic controls. Thyroid tissue samples were taken from transgenic and non-transgenic New Zealand White rabbits, examined by optical microscopy and analysed morphometrically. The analysis revealed no significant differences (P > 0.05) in the relative volume of basic thyroid structures. Furthermore, no significant differences (P > 0.05) were observed when measuring the epithelial height and nuclear diameter of the follicular cells. Altogether, this study demonstrates no negative effect of the mWAP-hFVIII transgenesis on the rabbit thyroid gland structure.     

Characteristics of Rabbit Transgenic Mammary Gland Expressing Recombinant Human Factor VIII

The objective of this research was to compare (i) the content of milk protein and recombinant human factor VIII (rhFVIII) in the milk of transgenic and non-transgenic rabbit females at three lactations and (ii) histological structure, ultrastructural morphology and occurrence of apoptosis in rabbit transgenic and non-transgenic mammary gland during third lactation and involution. Significant differences (t_0.05) in milk protein content were found between transgenic and non-transgenic at all three lactations. The percentage of apoptotic cells was significantly higher (t_0.01) in non-transgenic ones compared with transgenic mammary gland tissues (6.5% versus 2.4%) taken at the involution stage. Morphometrical analysis of histological preparations at the involution stage detected a significantly higher (t_0.05) relative volume of lumen in transgenic animals compared with non-transgenic ones (60.00 versus 46.51%). Ultrastructural morphology of the transgenic mammary gland epithelium at the involution stage revealed an increased relative volume of protein globules (t_0.05); at the lactation stage, a significantly higher volume of mitochondria (13.8%) compared with the non-transgenic (9.8%) ones was observed. These results, although revealing differences in some parameters of ultrastructure and histology, indicate no harmful effect of the mouse whey acid protein-hFVIII transgene expression on the state of mammary gland of transgenic rabbit females.     

Evaluation of haematological, biochemical and histopathological parameters of transgenic rabbits

The aim of our study was to compare the hFVIII mRNA expression in different organs, pathological changes and selected haematological and biochemical blood parameters between transgenic and non-transgenic rabbits from F3 generation. Selected physiological parameters of 3- to 4-month-old transgenic rabbits from F3 generation carrying human factor VIII gene (hFVIII) were analysed and compared with those of non-transgenic ones. Before slaughtering, the blood for haematological and biochemical analysis was taken from the central ear artery. Pathological and histological examination of vital organs and RT-PCR analysis of several tissue organs of transgenic and non-transgenic animals were performed after slaughtering. Except for the mammary gland tissue, slight hfVIII mRNA expression in the spleen, lung and brain and none expression in the liver, kidney, skeletal muscle and heart of rabbits were recorded. pathological examination of vital organs showed some pathological changes in both transgenic and non-transgenic rabbits which were confirmed by histological qualitative evaluations. Statistically significant lower values of blood haemoglobin in blood of transgenic (11.86+/-0.86) animals compared with non-transgenic (12.41+/-1.02, P<0.05) ones and lower parameters of HCT (39.22+/-2.44 versus 40.89+/-2.26, P<0.01) in blood of transgenic rabbits were observed. Parameters of WBC, RBC and PLT showed no significant differences between the analysed groups. All biochemical serum parameters of transgenic rabbits were higher in comparison with non-transgenic ones. Significant differences were found in the concentration of the urea, AST and GMT between transgenic and non-transgenic animals (P<0.001) and in the total protein content, the difference was significant at P<0.05. In conclusion, our results showed that no considerable impact on the general health was found in transgenic rabbits.     

Morphology of Testes from Transgenic Rabbits: Histological and Ultrastructural Aspects

The aim of this study was to compare morphological characteristics of testes from transgenic (the WAP-hFVIII gene) and non-transgenic rabbits with emphasis on the histological and ultrastructural aspects. Samples of testes from both groups were fixed and embedded into Durcupan ACM for transmission electron microscopy. For histological analysis, semi-thin toluidine blue-stained sections were evaluated under a Jenaval light microscope. Male fertility was tested based on egg fecundity and blastocyst yield; transgene transmission was proved using PCR assay. Spermatogenesis in rabbit testes had not been destroyed both in transgenic and non-transgenic rabbits. No significant differences were found in the occurrence of individual cell organelles of the Sertoli cells in transgenic and non-transgenic rabbits. The ultrastructure of Leydig cells in testes of transgenic and non-transgenic rabbits was rather similar. No differences in the occurrence of individual organelles of Leydig cells between transgenic and non-transgenic males were found. These results were in concert with fertilizing capacity of transgenic spermatozoa. The presented status of organelles in this study indicates functional activity of the analysed cells.     

乳头管灌注脂多糖对兔血清酶活性的影响及病理组织学观察

探讨经乳头管灌注脂多糖(LPS)对兔血清酶活性的影响及乳腺病理组织学变化.12只泌乳兔(产后第7天)经乳头管灌注LPS(150 μg/kg体重)建立急性临床型乳房炎模型,在灌注前2 h,灌注后6、12、24、48、72 h以及5 d和7 d分别测定血清中乳酸脱氢酶(LDH)、髓过氧化物酶(MPO)、过氧化物酶(LP)及...     

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.     

猪乳腺细胞分离培养及EGFP基因转化

本研究旨在从猪乳腺组织中分离得到上皮细胞和成纤维细胞,并将EGFP基因导入这些细胞.利用乳腺细胞堵养体系从成年猪乳腺组织中分离培养上皮细胞和成纤维细胞,并利用脂质体介导转染技术将EGFP基因导入这些细胞.结果,从成年猪乳腺组织中成功分离培养出上皮细胞和成纤维细胞,获得转EGFP基因上皮细胞和成纤维细胞.上皮细胞呈短梭形或多角形,细胞之间紧密相靠,互相衔接,连接成片;细胞核呈圆形或椭圆形,核仁2～4枚,比较明显.成纤维细胞呈长梭形.结果表明,可以从猪乳腺组织中分离上皮细胞和成纤维细胞,EGFP可以在这些细胞中表达.     

Mensuration of the rabbit pituitary gland from computed tomography

The aim of this retrospective reference interval observational study was to determine the mensuration of the pituitary gland (hypophysis cerebri) by analyzing CT studies in rabbits without clinical evidence of pituitary disease or central neurologic signs. Though diseases of the rabbit pituitary gland are uncommon, the pituitary gland is essential in regulation of the rabbit's endocrine system, as in other species. Currently, there are minimal published studies that detail the rabbit head anatomy on cross‐sectional imaging, and even less specifically examining the pituitary gland. The pituitary gland was measured by one observer at a single time point from transverse and reconstructed sagittal CT images in a soft‐tissue algorithm in 62 rabbits for a total of 66 rabbit head CT studies. The rabbits ranged from 0.84 to 14 years in age (mean ± SD: 5.46 ± 3.05 years) and 0.92 to 4.95 kg in weight (2.21 ± 0.83 kg). Linear pituitary measurements were performed using electronic calipers. The mean ± SD pituitary height was 4.22 ± 0.57 mm, width was 4.48 ± 0.71 mm, and length was 6.02 ± 0.70 mm. The pituitary gland height‐to‐brain area ratio was 1.10 ± 0.16 mm^?1, which is much higher than the values reported in normal dogs and cats. The age, weight, and sex of the rabbits were not found to have a significant impact on pituitary gland mensuration. These measurements could be useful as a reference range for future rabbit head CT studies and to rule out pituitary enlargement or disease when evaluating rabbit pituitary glands.     

Changes in gene expression of glucose transporters in lactating and nonlactating cows

Glucose delivery and uptake by the mammary gland are a rate-limiting step in milk synthesis. It is thought that insulin-independent glucose uptake decreases in tissues, except for the mammary gland, and insulin resistance in the whole body increases following the onset of lactation. To study glucose metabolism in peak-, late-, and nonlactating cows, the expression of erythrocyte-type glucose transporter (GLUT1) and the insulin-responsive glucose transporter (GLUT4) in the mammary gland, adipose tissue, and muscle were assessed by Western blotting and real-time PCR. Our results demonstrated that the mammary gland of lactating cows expressed a large amount of GLUT1, whereas the mammary gland of nonlactating cows did not (P < 0.05). On the other hand, adipose tissue of late and nonlactating cows expressed a large amount of GLUT1, whereas the adipose tissue of peak-lactating cows did not (P < 0.05). There were no significant differences in the abundance of GLUT4 mRNA in adipose tissue and muscle, whereas GLUT4 mRNA was not detected in the mammary gland. The plasma insulin concentration was greater (P < 0.05) in nonlactating cows than in peak- and late-lactating cows. The results of the present study indicate that in lactation, GLUT1 expression in the mammary gland and adipose tissue is a major factor for insulin-independent glucose metabolism, and the expression of GLUT4 in muscle and adipose tissue is not an important factor in insulin resistance in lactation; however, the plasma insulin concentration may play a role in insulin-dependent glucose metabolism. Factors other than GLUT4 may be involved in insulin resistance.     

转基因牛与非转基因牛性控精液分离及配种效果初探

本研究对转基因牛以及非转基因牛精液经流式细胞仪分离冷冻后精子活力、分离准确率进行了比较,同时对分离的性控冷冻精液进行了人工输精,对受体牛的情期受胎率进行了统计分析。结果表明,转基因牛与非转基因牛精液在冷冻解冻后活力以及分离准确率方面差异不显著(P>0.05);转基因牛与非转基因牛的性控冷冻精液的情期受胎率分别为57.4%、59.3%,二者之间差异不显著(P>0.05)。     

Immunolocalization of lingual antimicrobial peptide (LAP) in the bovine mammary gland

Lingual antimicrobial peptide (LAP), a member of the β-defensin family in cows, is involved in the innate immune system and plays a crucial role in killing a large variety of microorganisms. The aim of the present study was to demonstrate the immunolocalization of LAP in the mammary glands of cows. A LAP antibody was raised in a rabbit by immunity with a synthetic 11 amino acid sequence out of a 42-amino acid sequence of the mature form of LAP. The specificity of the LAP antibody was checked using a competitive immunoassay and Western blotting. Paraffin sections of the mammary gland were immunostained with LAP antibody. In the competitive immunoassay, an increase of synthetic LAP concentration suppressed the optical density. Western blotting analysis for LAP revealed the presence of the LAP peptide in mammary alveolar tissue. When the mammary gland was immunostained with LAP antibody, epithelial cells of both infected and non-infected alveoli were immunopositive. These results indicate that LAP is localized in the epithelium of non-infected as well as infected alveolus in the mammary gland in cows.     

Adrenergic and cholinergic innervation of the mammary gland in the pig

Adrenergic and acetylcholinesterase-positive (AChE-positive) innervation of the mammary gland in the sexually immature and mature pigs was studied using histochemical methods. Upon examining the adrenergic and cholinergic innervation, the adrenergic innervation was found to be much more developed. The majority of both sub-populations of the nerve fibres studied was localized in the subcutaneous tissue of the mammary gland. Adrenergic and AChE-positive nerve fibres also supplied structures of the nipple (subcutaneous tissue, blood vessels, smooth muscles fibres) and glandular tissue (blood vessels, lactiferous ducts). The glandular tissue contained the smallest number of adrenergic and AChE-positive nerve fibres. No distinct differences were observed in the adrenergic and AChE-positive innervation of the porcine mammary gland between the juvenile and non-pregnant adult animals.     

转基因动物乳腺生物反应器研究进展

文章综述了转基因动物乳腺生物反应器的原理、应用、优点、存在问题及国内外研究进展,并对其发展方向进行了小结及展望。     

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.     

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.     

乳腺生物反应器的研究和产业化进展

随着转基因技术的发展,利用乳腺生物反应器生产珍贵药用蛋白已成为一种极具潜力的生物技术,它为商业化前景广阔的药用蛋白提供了高产量、低成本的生产方式,开创了基因工程制药的新途径。目前,应用乳腺生物反应器已表达百余种药用蛋白,其中近30种进入临床试验不同阶段,用于治疗抗凝血酶缺乏症的抗凝血酶Ⅲ（ATryn^R）已上市,乳腺生物反应器生产重组蛋白显示出广阔的产业化前景。作者概述了乳腺生物反应器的原理、优点、国内外研究进展和产业化进程,讨论了其目前存在问题和应对策略。     

Telomere length and telomerase activity in canine mammary gland tumors

OBJECTIVE: To measure telomere length and telomerase activity in naturally occurring canine mammary gland tumors. SAMPLE POPULATION: 27 mammary gland tumor specimens obtained during resection or necropsy and 12 mammary gland tissue specimens obtained from healthy (control) dogs. PROCEDURE: Telomere length in tissue specimens was measured by use of restriction endonuclease digestion and Southern blot analysis. Telomerase activity was measured by use of a telomeric repeat amplification protocol assay. RESULTS: Telomere length in mammary gland tumors ranged from 11.0 to 21.6 kilobase pairs (kbp; mean +/- SEM, 14.5+/-0.5 kbp) but did not differ among tumor types. Telomeres in mammary gland tumors were slightly shorter than in normal tissue specimens, but telomere length could not be directly compared between groups, because mean age of dogs was significantly different between groups. Age was negatively correlated with telomere length in control dogs but was not significantly correlated with length in affected dogs. Telomerase activity was detected in 26 of 27 mammary gland tumors and in 4 of 12 normal tissue specimens. However, telomerase activity and telomere length were not correlated in tumor specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Telomere length is maintained in canine mammary gland tumors regardless of the age of the affected dog. Measurement of telomere length may be a useful tool for monitoring the in vivo effects of telomerase inhibitors in dogs with tumors.     

转基因动物乳腺生物反应器研究进展

转基因动物乳腺生物反应器是利用动物乳腺特异性启动子调控元件指导外源基因在乳腺中特异性表达 ,并从转基因动物乳汁中获取重组蛋白。近年来 ,生物学和分子生物学研究领域的成就促进了该技术的蓬勃发展 ,并在生产药用蛋白领域展现出广阔前景。本文简要概述了转基因动物乳腺生物反应器的基本概念、原理、特点及应用 ,并重点阐述其研究现状及前景 ,从而探讨其存在的问题     

家兔金黄色葡萄球菌性乳房炎模型的建立及中药组方治疗试验

选择泌乳期（7～10日）家兔,用10^4CFU、10^6CFU、10^8CFU三个不同浓度的金黄色葡萄球菌悬液人工诱导家兔乳房炎模型,观察临床表现,耳缘静脉采血进行血常规检测,确定10^6CFU为建立乳房炎模型的最佳剂量。造模后,分别采用不同剂量的中药组方制剂,通过乳池灌注和肌肉注射两种方式治疗患病家兔,观察临床治疗效果并检测血常规,确定乳池灌注和肌肉注射的最佳治疗剂量分别为35mg／乳区、100μg／kg,治愈率均为100％（8／8）。