乳腺脂肪组织调节乳腺发育的研究进展

乳腺是哺乳动物哺育子代的重要器官,其通过分泌乳汁给子代提供充足的营养物质,乳腺的健康发育对泌乳以及提高子代的存活率具有重要意义.脂肪组织是乳腺重要的组成部分,在乳腺发育和循环重构过程中,乳腺脂肪组织随之呈现规律性的形态和功能变化,乳腺脂肪组织的动态变化是乳腺循环性发育重构的重要特征.脂肪组织能够分泌特殊的"脂肪因子"调节上皮细胞的功能和乳腺的发育,并且存在与上皮细胞相互转换的潜能.本综述综合近年来乳腺脂肪组织的相关研究进展,为后续研究脂肪组织调节乳腺发育的机制提供基础数据.     

Maternal Obesity Reduces Milk Lipid Production in Lactating Mice by Inhibiting Acetyl-CoA Carboxylase and Impairing Fatty Acid Synthesis

Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.     

Pten对奶牛乳腺上皮细胞活力及乳蛋白合成的影响

Pten作为抑癌基因,参与调控细胞生长、粘附、凋亡以及其它细胞活动.目前,国内外关于Pten在奶牛乳腺发育过程中表达及调节的研究鲜有报道.为了揭示Pten的表达与奶牛乳腺发育与泌乳之间的关系,本研究应用qRT-PCR技术检测Pten在不同泌乳时期和不同乳品质的奶牛乳腺组织中的表达差异,进而应用脂质体转染方法,通过siRNA介导的RNA干扰技术改变Pten基因在奶牛乳腺上皮细胞中的表达量,CASY法检测细胞活力,用ELISA试剂盒检测细胞分泌β-酪蛋白的含量,采用qRT-PCR、Western 印迹等技术检测Pten对奶牛乳腺上皮细胞中乳蛋白相关信号通路基因表达的影响.结果显示,泌乳期高乳品质奶牛乳腺组织中Pten表达水平显著低于泌乳期低乳品质及干乳期奶牛;Pten基因沉寂后,细胞活力提高,β-酪蛋白质量浓度增加,CSN2、AKT、MTOR、STAT5表达量增加.研究表明,Pten可通过抑制细胞活力和乳蛋白分泌而影响泌乳.     

The epithelial glucocorticoid receptor is required for the normal timing of cell proliferation during mammary lobuloalveolar development but is dispensable for milk production

Glucocorticoids have been shown to influence mammary gland function in vivo and to stimulate milk protein gene expression in vitro. Here, we describe the generation and analysis of a mouse model to study glucocorticoid receptor (GR, NR3C1) function in mammary epithelial cells. Using the Cre-loxP system, mutant mice were obtained in which the GR gene is specifically deleted in epithelial cells during lobuloalveolar development, leading to a complete loss of epithelial GR at the onset of lactation. Mice harboring the mammary-epithelial-specific GR mutation are able to nurse their litters until weaning. During pregnancy, however, GR deficiency delays lobuloalveolar development, leading to an incomplete epithelial penetration of the mammary fat pad that persists throughout lactation. We identified a reduced cell proliferation during lobuloalveolar development as reason for this delay. This reduction is compensated for by increased epithelial proliferation after parturition in the mutant glands. During lactation, GR-deficient mammary epithelium is capable of milk production and secretion. The expression of two milk proteins, namely whey acidic protein and beta-casein, during lactation was not critically affected in the absence of GR. We conclude that GR function is not essential for alveolar differentiation and milk production, but influences cell proliferation during lobuloalveolar development.     

Bovine FcRn-Mediated Human Immunoglobulin G Transfer across the Milk-Blood Barrier in Transgenic Mice

Maternal-fetal IgGs transport occurs either prenatally or postnatally, which confers the newborns with passive immunity before their own immune system has matured. However, little is known about the mechanisms of postnatal IgGs passage in the mammary gland. To investigate how FcRn mediates the IgGs transport in the mammary gland, we first generated bFcRn and anti-HAV mAb transgenic mice, and then obtained HF transgenic mice expressing both transgenes by mating the above two strains. Transgene expression of bFcRn in the four lines was determined by qRT-PCR and western blot. We then localized the expression of bFcRn to the acinar epithelial cells in the mammary gland, and anti-HAV mAb was mainly detected in the acini with weak staining in the acinar epithelial cells. Human IgGs could be detected in both milk and serum of HF transgenic mice by western blot and ELISA. A significantly lower milk to serum ratio of human IgGs in HF mice compared with that of anti-HAV mAb mice, indicating that bFcRn could transport human IgGs across the milk-blood barrier from milk to serum during lactation in HF mice. While, there were no transport of murine IgGs, IgAs, or IgMs. These results provide understandings about the mechanisms of maternal-fetal immunity transfer in the mammary gland.     

Distribution and source of lipoprotein lipase in mouse mammary gland

During lactation lipoprotein lipase (LPL) is elevated in mammary tissue and depressed in adipose tissue to redirect lipids for incorporation into milk fat. The cellular origin of lipoprotein lipase in mammary tissue is thought to be the mammary epithelial cell which is the predominant cell type noticeable in the lactating gland; however, mammary adipocytes are also present. If lipoprotein lipase is produced by adipocytes in other sites of the body, then the question remains as to why mammary adipocytes have not been shown to produce lipoprotein lipase. In this study we present several lines of evidence that indicate that the mammary adipocyte is a source of LPL in the lactating mammary gland of mice. This evidence includes the absence of extracellular and intracellular lipoprotein lipase activity in two types of primary mammary epithelial cell cultures and a similarity in the changes of lipoprotein lipase activity in genital adipose tissue from nonpregnant mice and lactating mammary tissue to the nutritional state of the animal. Other evidence presented here includes strong localization of lipoprotein lipase protein and messenger RNA by fluorescence immunohistochemistry and in situ hybridization, respectively, to interstitial cells located between epithelial structures. We postulate that these interstitial cells are regressed, lipid-deleted mammary adipocytes.     

Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland

During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.     

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation

The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.     

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lactation-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.     

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.     

Leptin mRNA expression in the rat mammary gland at different activation stages

Leptin is expressed in various tissues, suggesting that this protein is effective not only at the central nervous system level, but also peripherically. Recent studies have shown leptin production by other tissues, including the placenta, stomach, and mammary tissues, but there is no information available concerning expression levels of leptin in the rat mammary gland at different activation stages. We used semi-quantitative RT-PCR to investigate leptin mRNA expression levels in the rat mammary gland at different activity stages. Rat mammary gland samples were collected from virgin females and on days 6, 12, 18 of pregnancy and of lactation (six rats per group). The expression levels of leptin mRNA were measured by semi-quantitative RT-PCR, with β-actin as an internal control. Leptin mRNA was highly expressed in virgin rat mammary glands (leptin(IOD)/β-actin(IOD) = 1.60). It decreased gradually during pregnancy, being lowest at 18 days of pregnancy, when the levels were significantly lower than in virgin mammary tissue. Leptin mRNA increased slightly during lactation, but the difference was not significant. By day 18 of lactation, expression levels of leptin mRNA reached the same values as in virgin mammary tissue (leptin(IOD)/β-actin(IOD) = 1.65). Based on these results, we suggest that leptin has an important regulation role in rat mammary gland activation.     

Expression of Human NSAID Activated Gene 1 in Mice Leads to Altered Mammary Gland Differentiation and Impaired Lactation

Transgenic mice expressing human non-steroidal anti-inflammatory drug activated gene 1 (NAG-1) have less adipose tissue, improved insulin sensitivity, lower insulin levels and are resistant to dietary induced obesity. The hNAG-1 expressing mice are more metabolically active with a higher energy expenditure. This study investigates female reproduction in the hNAG-1 transgenic mice and finds the female mice are fertile but have reduced pup survival after birth. Examination of the mammary glands in these mice suggests that hNAG-1 expressing mice have altered mammary epithelial development during pregnancy, including reduced occupancy of the fat pad and increased apoptosis via TUNEL positive cells on lactation day 2. Pups nursing from hNAG-1 expressing dams have reduced milk spots compared to pups nursing from WT dams. When CD-1 pups were cross-fostered with hNAG-1 or WT dams; reduced milk volume was observed in pups nursing from hNAG-1 dams compared to pups nursing from WT dams in a lactation challenge study. Milk was isolated from WT and hNAG-1 dams, and the milk was found to have secreted NAG-1 protein (approximately 25 ng/mL) from hNAG-1 dams. The WT dams had no detectable hNAG-1 in the milk. A decrease in non-esterified free fatty acids in the milk of hNAG-1 dams was observed. Altered milk composition suggests that the pups were receiving inadequate nutrients during perinatal development. To examine this hypothesis serum was isolated from pups and clinical chemistry points were measured. Male and female pups nursing from hNAG-1 dams had reduced serum triglyceride concentrations. Microarray analysis revealed that genes involved in lipid metabolism are differentially expressed in hNAG-1 mammary glands. Furthermore, the expression of Cidea/CIDEA that has been shown to regulate milk lipid secretion in the mammary gland was reduced in hNAG-1 mammary glands. This study suggests that expression of hNAG-1 in mice leads to impaired lactation and reduces pup survival due to altered milk quality and quantity.     

The regulation of Na(+)-dependent anionic amino acid transport by the rat mammary gland.

The regulation of anionic amino acid transport, using radiolabelled D-aspartate as a tracer, by rat mammary tissue explants has been examined. Na(+)-dependent D-aspartate uptake by mammary tissue increased between late pregnancy and early lactation and again at peak lactation but thereafter declined during late lactation. In contrast, the Na(+)-independent component of D-aspartate uptake by mammary explants did not change significantly with the physiological state of the donor animals. Premature weaning of rats during peak lactation markedly decreased Na(+)-dependent D-aspartate uptake by mammary tissue. In addition, premature weaning also reduced the effect of reversing the trans-membrane Na(+)-gradient on the fractional loss of D-aspartate from mammary tissue explants. Unilateral weaning of rats during peak lactation revealed that milk accumulation per se reduced the Na(+)-dependent moiety of D-aspartate uptake by mammary tissue suggesting that the transport of anionic amino acids is regulated to match supply with demand. Treating lactating rats with bromocryptine reduced D-aspartate uptake by mammary tissue explants suggesting that the transport of anionic amino acids by the rat mammary gland is regulated by prolactin.     

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.     

转基因小鼠乳腺上皮细胞的体外培养及其对催乳素反应的研究

实现转基因生物乳腺反应器对外源蛋白的高效表达是目前生物制药亟待解决的难题。催乳素对泌乳期乳蛋白的合成与分泌具有重要的调控功能。通过转基因小鼠乳腺上皮细胞模型的建立,研究催乳素如何调控乳蛋白的表达,为提高乳腺反应器高效表达外源蛋白提供技术及理论支撑。应用机械破碎及胶原酶消化法,经差速贴壁纯化,成功培养含人转铁蛋白基因的小鼠乳腺上皮细胞,细胞上清液中检测到人转铁蛋白表达。细胞经牛催乳素诱导后人转铁蛋白的表达水平明显升高。利用转基因小鼠乳腺上皮细胞模型,可以进行催乳素和环境因素等对乳腺上皮细胞合成及分泌蛋白能力影响的研究。