王不留行对奶牛乳腺上皮细胞磷酸化雌激素受体的影响

通过研究王不留行对奶牛乳腺上皮细胞中磷酸化ERα的影响揭示王不留行促进乳腺泌乳的分子机制。应用Western blot和实时荧光RT-PCR检测P-ERα的变化;免疫荧光观察P-ERα定位表达变化。结果表明,中药王不留行增乳活性成分可通过调控奶牛乳腺上皮细胞雌激素信号转导途径中P-ERα的表达来增强其泌乳功能,但详细机制尚需进一步研究。     

奶牛乳腺中瘦素及其受体的表达与定位

为探讨瘦素在奶牛乳腺发育、泌乳及退化各时期的表达变化规律及在乳腺组织中的具体位置,采用免疫印迹(western blot-ting)技术及激光共聚焦技术检测奶牛乳腺组织中瘦素及瘦素受体(OB-Rb)的表达变化及其定位。结果表明,瘦素在青春期表达量较高,在泌乳期表达量最低。瘦素受体在青春期和妊娠期表达量较高,泌乳期表达量相对较低,退化期逐渐恢复到妊娠期水平。     

催乳素对奶牛RANKL基因启动子活性的研究

文章以奶牛乳腺上皮细胞为实验模型,探讨催乳素对RANKL基因启动子活性的调节。采用Western Blot方法检测催乳素对RANKL蛋白表达的影响,构建RANKL启动子荧光素酶载体,采用双荧光素酶报告基因检测系统检测催乳素对RANKL基因启动子活性影响,采用定点合成突变的方法,构建突变型RANKL启动子荧光素酶载体,运用双荧光素酶报告基因检测系统检测催乳素对突变型RANKL启动子活性影响。结果表明,催乳素能够显著提高乳腺上皮细胞RANKL启动子活性及RANKL蛋白表达,催乳素响应元件在奶牛RANKL基因启动子-826～-814 bp区域。     

DNA甲基化对奶牛乳腺上皮细胞乳脂和乳糖合成的影响

以健康的泌乳期中国荷斯坦奶牛的乳腺上皮细胞(DCMECs)为实验模型,构建DNMT3A过表达载体pcDNA3.1-DNMT3A,用脂质体转染奶牛乳腺上皮细胞,采用荧光定量PCR技术检测奶牛乳腺上皮细胞中DNMT3A的mRNA表达,采用Bovine milk fat ELISA KIT检测细胞中的乳脂含量,采用Bovin...     

小G蛋白RagD对奶牛乳腺上皮细胞乳蛋白合成的影响

以体外培养的奶牛乳腺上皮细胞为模型,研究了RagD对细胞中β-酪蛋白(CSN2)的合成及蛋氨酸调节CSN2合成信号通路的影响。采用实时荧光定量PCR(q RT-PCR)和蛋白质免疫印迹(WB)检测添加蛋氨酸后细胞中CSN2与Rag D的表达;对Rag D进行过表达和抑制,用q RT-PCR和WB检测细胞中CSN2、mTOR和p-mTOR的表达。结果表明:添加蛋氨酸使细胞中CSN2和RagD的表达显著增加;RagD过表达时,细胞中CSN2与p-mTOR的表达显著增加,mTOR表达变化不显著,此时添加蛋氨酸,细胞中CSN2与p-m TOR的表达继续显著增加;RagD抑制时,细胞中CSN2与p-m TOR的表达显著下降,mTOR表达变化不显著(P0.05),此时添加蛋氨酸,不能使细胞中CSN2与p-m TOR的表达恢复。RagD作为信号分子,能接收并传递蛋氨酸信号,使细胞中mTOR磷酸化,正向调节奶牛乳腺上皮细胞中CSN2的表达。     

microRNA-148b对奶牛乳腺上皮细胞乳蛋白合成的影响

以奶牛乳腺上皮细胞(Dairy cow mammary epithelial cells,DCMECs)为研究模型,探讨microRNA-148b对DCMECs增殖和β-酪蛋白合成的影响.应用脂质体转染技术将miR-148b转染DCMECs,qRT-PCR观测其表达量,采用双荧光素酶实验验证miR-148b与转化生长因...     

人α-乳白蛋白在奶牛乳腺上皮细胞中的表达

将人α-乳白蛋白(α-LA)0.76 kb的cDNA序列连接到PSV载体SV40启动子后,构建真核表达载体hα-LA-psv.将构建的真核表达载体转染奶牛乳腺上皮细胞,Aα-乳白蛋白的表达量约为0.85 g/L.结果表明,构建的真核表达载体能够在体外培养的牛乳腺上皮细胞中高效表达Aα-乳白蛋白.     

Thrsp在不同乳品质奶牛乳腺中的表达及其对乳脂合成的影响

利用数字基因表达谱及荧光定量PCR对不同乳品质奶牛及干奶期奶牛乳腺甲状腺激素应答蛋白(Thyroid Hormone Responsive Spot 14 protein,THRSP)基因Thrsp mRNA差异表达进行分析,并克隆奶牛乳腺上皮细胞THRSP基因,构建THRSP过表达载体后转染奶牛乳腺上皮细胞,检测Thrsp过表达对奶牛乳腺上皮细胞乳脂合成的影响。结果表明:泌乳期高乳品质奶牛Thrsp mRNA表达高于低乳品质奶牛(P0.05),过表达Thrsp后奶牛乳腺上皮细胞甘油三酯浓度升高(P0.05)。结论:Thrsp参与调控奶牛乳脂合成。     

奶山羊乳腺中胰岛素样生长因子及其受体的表达与作用

为研究胰岛素样生长因子及胰岛素样生长因子受体在奶山羊乳腺组织青春期、妊娠期、泌乳期及退化期表达的差异及变化,探索其在奶山羊乳腺发育过程中的作用,根据奶山羊乳腺发育特点及生长阶段不同分为青春期、妊娠期、泌乳期及退化期,采用激光共聚焦显微镜检测胰岛素样生长因子在奶山羊乳腺各生长阶段的表达及定位,通过定量分析来比较胰岛素样生长因子在奶山羊乳腺各生长阶段表达的差异,描述其在奶山羊乳腺发育过程中的作用.结果显示:胰岛素样生长因子及胰岛素样生长因子受体在奶山羊乳腺法如各时期均有表达,表达水平与乳腺发育密切相关.     

王不留行促进奶牛乳腺泌乳的信号转导途径

应用Western blot检测王不留行水浸提液作用后,乳腺上皮细胞泌乳信号转导基因P-AKT1、cyclinD1、PRLR、P-STAT5a和AMPK、GULT1的变化,结果表明:中药王不留行对P-AKT1、cyclinD1、PRLR、P-STAT5a和AMPK、GULT1基因表达具有一定作用,可能通过mTOR信号转导途径促进乳腺上皮细胞的增殖;通过激活PRLR从而激活Stat5a泌乳信号转导途径激活其下游β-酪蛋白基因的表达;且可能通过激活AMPK而促进GLUT1表达的增强,从而增加细胞对葡萄糖的摄取能力促进乳糖的合成。     

Role of prolactin, growth hormone and insulin-like growth factor 1 in mammary gland involution in the dairy cow

Bovine mammary involution, an important process for subsequent lactations, is characterized by loss of epithelial cells by apoptosis, but its hormonal regulation is still not well defined. Prolactin (PRL) and growth hormone (GH) play a specific role on rat mammary gland apoptosis, through insulin-like growth factor 1 (IGF-1) and the IGF binding protein (IGFBP) system. The purpose of our investigation was to determine the possible role of PRL, GH, and IGF-1 on cell survival and on IGFBP-5 expression in the bovine mammary gland. Mammary gland explants were cultured in the presence of cortisol, 17beta-estradiol, progesterone, insulin, PRL, GH, and IGF-1 and with the same treatment but without PRL, GH or IGF-1, respectively. After 24 h of culture, we determined the level of apoptosis through evaluation of DNA laddering in the oligonucleosomal fraction and examined IGFBP-5 messenger RNA (mRNA) expression. The results show a high level of DNA laddering and an increase in IGFBP-5 mRNA content in mammary explants cultured in the absence of PRL, GH, or IGF-I with respect to explants treated with all hormones. Moreover, explants cultured in presence of PRL, GH, or IGF-I show a low level of DNA laddering and IGFBP-5 expression with respect to explants cultured without any hormones. These data demonstrate a relationship between levels of apoptosis and IGFBP-5 mRNA expression in the bovine mammary gland and confirm the involvement of this binding protein programmed cell death and its relationship with the main lactogenic hormones.     

Secretion and mammary gland uptake of prolactin in dairy cows during lactogenesis

Mammary arteriovenous differences of prolactin concentration and net mammary uptake of prolactin from blood were quantified near parturition in 9 dairy cows. Six cows were milked once daily for at least 6 d before parturition, and prepartum lactogenesis occurred in 3 of 6 cows. Prepartum milking 2 or more d before parturition abruptly increased secretion of prolactin into blood, but milkings within 1 d before or after parturition did not increase prolactin secretion. Concentrations of prolactin in whole milk sampled over 8 d before parturition (64.5 ng/ml) were substantially greater than those occurring several days after parturition (19 ng/ml). Milk prolactin concentrations were unaffected by the successful induction of prepartum lactogenesis, which greatly increased prepartum yields of milk (2 to 8 kg/milking). Therefore, the alveolar lumenal content of prolactin was greatest in pregnant cows with prepartum lactogenesis. This enhanced content of intraalveolar prolactin before parturition was associated with an absence of mammary uptake of prolactin immediately prior to ejection of the prolactin-containing milk from the alveoli. However, prolactin uptake was quickly restored to about 2 micrograms/min per half udder shortly after milk ejection. During the prepartum period, an enhanced intraalveolar reservoir of 200 to 400 micrograms prolactin, due to induction of prepartum lactogenesis, appears to saturate temporarily all putative sites for uptake of prolactin from blood.     

Relationship between glucocorticoids and prolactin during mammary gland stimulation in dairy cows

The objectives of this study were to determine the role of glucocorticoids in the regulation of prolactin (PRL) release induced by mammary gland stimulation and to investigate whether the milk depression induced by glucocorticoids in dairy cows is due to a decrease in PRL release. In experiment 1, 8 dairy cows were used in a 4 × 4 Latin square design. Four hours after the morning milking, the cows received 1 of the following treatments: (1) a 5-min manual stimulation of the mammary gland; (2) an i.v. injection of 1 mg of dexamethasone; (3) 2 infusions of 2.5 g of metyrapone (an inhibitor of cortisol biosynthesis) in the omasum 4 and 2 h before a 5-min stimulation of the mammary gland; or (4) no treatment. Sixty minutes later, the mammary gland of each cow was stimulated for 5 min. Blood samples were collected from 20 min before to 120 min after the start of the treatment. When the mammary gland was stimulated twice in 60 min, less PRL and cortisol were released during the second stimulation. Metyrapone did not affect PRL or cortisol release. Dexamethasone decreased serum cortisol concentration but did not affect PRL concentration. In experiment 2, 16 cows were used in a crossover experimental design consisting of 2 experimental weeks separated by 1 resting week. During the first week, cows were treated as follows: (1) 4 cows were injected with 0.5 g of domperidone (a PRL secretagogue) in canola oil on d 1 and 2 and 20 mg of dexamethasone on d 1; (2) 4 cows were injected with 0.5 g of domperidone on d 1 and 2; (3) 4 cows were injected with canola oil on d 1 and 2 and with 20 mg of dexamethasone on d 1; and (4) 4 cows were injected with canola oil on d 1 and 2. During the second experimental week, the same 4 treatments were repeated, except the cows that did not receive dexamethasone in the first week received it on d 1 of the second week, and cows that did receive it in the first week did not receive it in the second week. On d 1 and 2 of each week, blood samples were collected during morning milking for PRL determination. Dexamethasone reduced milk production and decreased both basal and milking-induced PRL release. It also increased milk fat and protein percentages and decreased milk lactose content. Domperidone increased basal PRL levels in serum and milk but did not affect milk yield. Although we cannot rule out the possibility that inhibition of PRL secretion or reduction of mammary gland PRL responsiveness play a role in the inhibition of milk production by glucocorticoids, the fact that enhancement of PRL secretion by domperidone could not prevent the depression of milk yield suggests that other mechanisms are involved.     

Influence of estrus of dairy goats on somatic cell count, milk traits, and sex steroid receptors in the mammary gland

Two experiments were conducted to study the effect of the stage of a spontaneous estrus cycle on milk yield and constituents [somatic cell count (SCC), fat, protein, caseins, lactose, and urea content] and on estrogen receptor-α (ERα) and progesterone receptor (PR) immunostaining in the mammary gland. In experiment I, the major components of milk and SCC were monitored weekly in 80 lactating Saanen goats for 6 wk, whereas detection of estrus was daily. In experiment II, milk samples were collected daily for SCC determination during 1 spontaneous estrus (d 0) until the second spontaneous estrus in 14 Saanen goats. The day of the estrous cycle was confirmed by plasma progesterone and 17β-estradiol levels. Immunoreactivity of ERα and PR was analyzed in mammary gland samples of 8 Saanen goats (d 0, n = 4; d 10, n = 4) and the number of positive nuclei and intensity of the staining were evaluated in 1,000 cells. In experiment I, milk casein and protein percentages were significantly affected by the stage of estrous cycle; during proestrus and estrus, these variables were higher (3.32 ± 0.06 and 4.44 ± 0.08) than during metestrus (3.03 ± 0.07 and 4.07 ± 0.10), but not higher than during diestrus (3.23 ± 0.06 and 4.35 ± 0.09, respectively). In experiment II, daily measurement of SCC revealed higher levels at estrus (7,195 ± 672 × 10³ cells/mL) and a decline toward the luteal phase (1,694 ± 672 ± 10³ cells/mL). Estrogen receptor-α and PR immunostaining were exclusively detected on epithelial cells. The percentage of positive nuclei to ERα was higher on d 0 than on d 10 (75.4 ± 8.8 vs. 68.3 ± 8.8%), but no change was observed for PR (4.0 ± 0.3 vs. 3.5 ± 0.4%). The average immunostaining intensity for both receptors was greater on d 0 than on d 10 (ERα : 1.44 ± 0.02 vs. 1.35 ± 0.02; PR: 0.079 ± 0.008 vs. 0.057 ± 0.008). The high SCC at estrus in experiment II was associated with high plasma estradiol and low progesterone, suggesting that the increased SCC could be brought about by the estrogen-induced proliferation and exfoliation of epithelial cells. In addition, this action may be supported by the higher sensitivity to estrogens (ERα content) found at d 0.     

Effect of estrogen on leptin and expression of leptin receptor transcripts in prepubertal dairy heifers

Plasma leptin concentrations increase as growing dairy heifers approach puberty and have greater plasma estrogen. In intact and ovariectomized rodents, estrogen has been shown to modulate expression of leptin and its receptor (Ob-R). To determine if estrogen regulates the bovine leptin system, prepubertal dairy heifers were ovariectomized at 140 d of age or left intact. A month later, both groups received a subcutaneous injection of excipient or 17β-estradiol for 3 consecutive days. Neither ovarian status nor 17β-estradiol injection altered plasma leptin or leptin mRNA abundance in adipose tissue depots. To assess whether these factors affected Ob-R expression, we tested 20 bovine tissues for leptin receptor (Ob-R) by using quantitative real-time PCR assays for the short receptor isoform (Ob-Ra), the long receptor isoform (Ob-Rb), and all receptor isoforms (Ob-R_TOTAL). Ob-R_TOTAL was detected in all tissues, with copy numbers covering 3 orders of magnitude between the lowest and highest expressing tissues (kidney cortex vs. liver). The Ob-Rb isoform accounted for 40% of Ob-R_TOTAL in the hypothalamus, but averaged less than 3% of Ob-R_TOTAL in peripheral tissues. Reciprocally, Ob-Ra accounted for only 19% of Ob-R_TOTAL in the hypothalamus and for nearly all of Ob-R_TOTAL in most peripheral tissues. Finally, we evaluated the effects of ovarian status and 17β-estradiol on Ob-R expression in selected tissues. Treatment with 17β-estradiol reduced Ob-R_TOTAL, Ob-Rb, and Ob-Ra expression by 70% in the uterine endometrium and tended to do the same in mammary adipose tissue. There was no effect of 17β-estradiol on Ob-R in the hypothalamus, liver, soleus muscle, or subcutaneous adipose tissue. We conclude that greater estrogen secretion does not cause increased plasma leptin in prepubertal dairy heifers but estradiol can modulate Ob-R expression in some estrogen-responsive tissues.     

奶牛不同发育时期乳腺组织LAT1和4F2hc的表达

为研究氨基酸转运载体LAT1的表达与奶牛乳腺发育的关系,应用免疫荧光技术和免疫印迹方法,检测奶牛乳腺组织中LAT1及其辅助蛋白4F2hc表达的情况.结果表明,LAT1和4F2hc共表达于乳腺导管或腺泡的基底侧和顶膜侧;且泌乳期LAT1蛋白表达显著高于其他发育时期,4F2hc蛋白表达量在围产前期达到峰值,泌乳期4F2hc蛋白的表达量恢复至妊娠中期水平.结论:LAT1及4F2hc均特异性表达于不同发育时期奶牛乳腺组织中,提示LAT1参与了奶牛乳腺的发育和泌乳的调控.     

Inhibiting prolactin by cabergoline accelerates mammary gland remodeling during the early dry period in dairy cows

The inhibition of prolactin release using cabergoline, a dopamine agonist, is an effective strategy to accelerate the changes in mammary secretion composition after drying-off. The objective of this study was to determine how cabergoline may affect mammary tissue remodeling during early involution. Holstein dairy cows were treated with either a single i.m. administration of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n = 7) or placebo (n = 7) at the time of drying-off. Mammary biopsy samples were collected 1 wk before drying-off (d ?6), after 30 h of milk accumulation (d 1), and again 8 d following drying-off (d 8) to determine changes in gene expression, lactoferrin content, and cell turnover. Blood and mammary secretion samples were collected at d ?6 and again at d 1, 2, 3, 4, 8, and 14 following the abrupt cessation of lactation to evaluate indicators of blood-milk barrier integrity and other markers of mammary tissue remodeling. Cabergoline induced less SLC2A1, BAX, CAPN2, and IGFBP5 mRNA expression. In contrast, cabergoline did not modify changes in cell proliferation and apoptosis. Following the cessation of lactation, changes in mammary secretion composition (Na⁺ and K⁺) and blood lactose concentrations were indicative of a loss in the blood-milk barrier function in both treatment groups. Cabergoline treatment affected only Na⁺ and K⁺ concentrations at d 1, suggesting a moderate increase in tight junction permeability. The increase in the activity of MMP9 and in mammary epithelial cell concentration in mammary secretions was greater in cabergoline-treated cows than in control cows, suggesting more mammary tissue remodeling. The increase in lactoferrin immunostaining in the mammary tissue occurred earlier for cabergoline-treated cows than for control cows, and was essentially localized in the stroma. Changes in some key markers of mammary involution suggest that cabergoline accelerates mammary gland remodeling. Thus, a single injection of cabergoline after the last milking would facilitate drying-off by enhancing mammary gland involution.