IGF-I retards proper development of acinar structures formed by bovine mammary epithelial cells via sustained activation of Akt kinase

Insulin-like growth factor-I is involved in mammary gland development, promoting proliferation and inhibiting apoptosis of mammary epithelial cells (MECs). Mitogenic actions of IGF-I are mainly mediated by the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. We have found that in the presence of IGF-I bovine BME-UV1 MECs cultured on reconstituted basement membrane form large spheroids with disrupted polarity and no cavity in the center. These cells showed enhanced phosphorylation of Akt, decreased level of cleaved caspase-3, and sustained proliferative activity throughout the 16-d period of 3-dimensional culture. Inhibition of the PI3K/Akt pathway by a specific inhibitor of PI3K, LY294002, resulted in the restoration of the normal acinar phenotype. However, this effect was noted only when LY294002 was added in the second week of 3-dimensional culture, which corresponded with the time of cell cycle arrest and polarity formation under control conditions. Normal development of acini was also obtained when BME-UV1 cells were treated simultaneously with IGF-I and 17β-estradiol. The addition of 17β-estradiol regulated Akt activation, enabling the subsequent initiation of polarization processes. 17β-Estradiol also increased the level of IGFBP-3 protein in MECs cultured on Matrigel in the presence of IGF-I. The presented results indicate important interactions between signaling pathways activated by estrogen and IGF-I, which regulate alveologenesis in bovine mammary gland.     

牛乳腺上皮细胞的分离培养

为了对牛乳腺上皮细胞(MECs)进行分离、培养和鉴定,并研究细胞分泌功能,试验通过胶原酶消化法分离得到了牛乳腺上皮细胞,采用传代法对细胞进行纯化,对细胞标志蛋白进行免疫荧光染色鉴定,通过体外诱导和RT-PCR分析鉴定细胞的分泌功能。结果表明:分离到的牛乳腺上皮细胞具有典型乳腺上皮细胞的形态特征,表达广谱角蛋白,经诱导后可分泌β-酪蛋白。     

奶牛乳腺上皮细胞和成纤维细胞的体外培养与鉴定

为体外培养纯化出稳定的奶牛乳腺上皮细胞和成纤维细胞,试验通过外科手术的方法取妊娠后期或泌乳期的荷斯坦奶牛乳腺组织,分离乳腺腺泡,用组织块法体外培养奶牛乳腺细胞,应用差时胰酶消化法和差速贴壁法将奶牛乳腺上皮细胞和成纤维细胞分别纯化出来,并用免疫组化的方法对细胞的纯度进行鉴定。结果表明:纯化的奶牛乳腺上皮细胞多为多角形,细胞核呈圆形或椭圆形,核仁清晰可见,多呈鹅卵石样或铺路石样生长,并可分泌乳滴,角蛋白-18反应阳性,波形蛋白反应阴性;纯化的奶牛乳腺成纤维细胞多为长梭形,呈旋涡状或放射状生长,角蛋白-18反应阴性,波形蛋白反应阳性;经纯化后2种细胞的纯度均可达95%以上,可满足后续试验的要求。     

细胞因子对牛乳腺上皮细胞体外增殖作用评价

分离纯化了乳腺上皮细胞,对细胞进行角蛋白免疫组化鉴定后,比较不同细胞因子对乳腺上皮细胞生长的影响.结果显示,生长因子EGF或HGF对牛乳腺上皮细胞的增殖具有重要作用,17β-E2不能促进牛乳腺上皮细胞的增殖,但可与EGF协同促进牛乳腺上皮细胞的增殖,表明17β3-E2对EGF诱导的乳腺上皮细胞增殖具有重要作用.     

山羊乳腺上皮细胞的生长特性

用组织块培养法获得山羊乳腺细胞原代培养物。根据山羊乳腺成纤维细胞与上皮细胞对胰蛋白酶的敏感性不同将二者分离纯化，对细胞生长特性进行了光镜观察。结果如下：细胞可形成闭合型细胞群和开放型细胞群。乳腺上皮细胞与成纤维细胞混生时，细胞之间形成许多腔状结构。纯化的乳腺上皮细胞通过单细胞悬浮后传代，部分细胞形成岛屿状聚集，部分细胞以贴壁的自由单个细胞散在形式存在。上皮细胞增殖可形成圆顶型结构，呈乳头状，称之为乳球体；可产生乳腺上皮细胞并分泌乳汁。山羊乳腺上皮细胞含不同的细胞类型．大多数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，呈蜂窝状；细胞核呈圆形或椭圆形．核仁2～4枚；部分细胞呈圆饼状，体积较大；出现部分长形细胞；接触抑制的上皮细胞形态不均一。纯化的山羊乳腺上皮细胞传代至第15代时其生长仍正常，经透射电镜观察发现细胞表面微绒毛极为发达，细胞质中线粒体和粗面内质网丰富，细胞质内有大量脂滴及小泡，表明第15代山羊乳腺上皮细胞增殖活力旺盛。染色体数目分析表明．该细胞系稳定，在离体培养条件下细胞不发生转化。山羊乳腺上皮细胞系的细胞染色体数目为60．染色体组型为2n=60。     

IGF-Ⅰ对奶牛乳腺上皮细胞合成乳脂、乳蛋白的影响

分离培养奶牛乳腺上皮细胞,添加不同质量浓度的IGF-Ⅰ（insulin-like growth factor-Ⅰ）（0,10,100,200μg/L）刺激24h后,提取细胞总RNA,用荧光定量RT-PCR方法检测β-酪蛋白（CSN2）和乙酰辅酶A羧化酶α（ACA-CA）基因的转录水平变化。结果显示,IGF-Ⅰ能够显著促进CSN2和ACACA基因的转录,并且具有浓度依赖性。结果表明,IGF-Ⅰ可作为信号分子调节乳腺的泌乳功能。     

In vitro development of bovine nuclear transfer embryos reconstructed with mammary gland epithelial cells at different passages

In the present study, the effect of passage of nuclear donor cells on the in vitro development of nuclear transfer (NT) embryos was investigated using colostrum‐derived mammary gland epithelial (MGE) cells at different passages (3–30 passages) to find reliable passages for the efficiency of cloning. Development of NT embryos to the blastocyst stage was affected by the number of passages of MGE cells (P < 0.05). Nuclear transfer embryos reconstructed with MGE cells at 3–7 passages showed a significantly higher blastocyst development (31.3–48.5%) than those with the cells at 10–30 passages (2.5–12.5%, P < 0.05). No difference in the proportion of the MGE cells with normal diploid was observed among passage of 3, 15 and 30 (P > 0.05). The use of MGE cells at early passages for nuclear donor cells may be advantageous for the production of NT embryos.     

Retinoids,retinoid analogs,and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (P < 0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P < 0.05). The MeBo cells exhibited some inhibition with these natural ligands (P < 0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects (P < 0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability (P < 0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability (P < 0.05) and inhibited BME-UV1 cell viability (P < 0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type (P < 0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.     

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

无乳链球菌体外感染奶牛乳腺上皮细胞

用分离到的无乳链球菌(Streptococcus agalactiae,GBS)及标准菌株侵袭原代奶牛乳腺上皮细胞建立体外感染细胞模型,并进行细胞凋亡检测。同时,采用qRT-PCR方法检测细胞受到侵袭后白细胞介素6(IL-6)、白细胞介素8(IL-8)、肿瘤坏死因子(TNF-α)等细胞炎性因子mRNA的转录水平。结果显示,标准菌株组(WL组)与空白组相比凋亡率差异极显著(P<0.01),分离菌株组(FL组)与空白组相比凋亡率差异极显著(P<0.01),分离菌株组与其标准菌株组的凋亡率差异也极显著(P<0.01)。qRT-PCR结果分析显示,标准株组TNF-α、IL-6及IL-8 mRNA转录水平与对照组相比,差异极显著(P<0.01);而分离菌株组TNF-αmRNA转录水平与对照组相比差异显著(P<0.05),IL-6及IL-8 mRNA转录水平差异不显著(P>0.05)。结果表明,与标准菌株相比,无乳链球菌分离株对奶牛乳腺上皮细胞侵袭和损伤能力较低,乳腺细胞的凋亡率也较低,当奶牛乳腺上皮细胞受到细菌侵袭的时候,细胞中IL-6、IL-8及TNF-α等炎性因子mRNA的转录水平都显著性提高,但在相同浓度下GBS标准株诱导细胞炎性因子的表达量显著高于GBS分离株。本试验结果为以后预防和控制GBS引起的隐性乳房炎提供试验依据。     

Roles of IGF-I and the estrogen, androgen and IGF-I receptors in estradiol-17beta- and trenbolone acetate-stimulated proliferation of cultured bovine satellite cells

Although numerous studies have shown that both androgenic and estrogenic steroids increase rate and efficiency of muscle growth in steers, there is little consensus as to their mechanism of action. A combined estradiol 17beta (E2)/trenbolone acetate (TBA) implant causes a significant increase in muscle IGF-I mRNA and both E2 and TBA stimulate a significant increase in IGF-I mRNA level in bovine satellite cell (BSC) cultures in media containing 10% fetal bovine serum (FBS). Consequently, increased IGF-I expression may play a role in anabolic-steroid-enhanced muscle growth. However, even though treatment of cultured BSC with E2 or TBA in media containing 1% IGFBP-3-free swine serum (SS) results in increased proliferation there is no effect on IGF-I mRNA expression, suggesting that increased IGF-I expression may not be responsible for anabolic-steroid-enhanced BSC proliferation. To further examine the role of estrogen, androgen and IGF-I receptors and their respective ligands in E2- and TBA-stimulated BSC proliferation, we assessed the effects of specific inhibitors on E2- or TBA-stimulated proliferation of BSC. Both ICI 182 780 (an estrogen receptor blocker) and flutamide (an inhibitor of androgen receptor) suppressed (p<0.05) E2- and TBA-stimulated BSC proliferation, respectively. JB1 (a competitive inhibitor of IGF-I binding to type I IGF receptor) reduced (p<0.05) both E2- and TBA-stimulated proliferation in BSC cultures. Both the Raf-1/MAPK kinase (MEK)1/2/ERK1/2, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathways play significant roles in the actions of IGF-I on proliferation and differentiation of myogenic cells. PD98059, an inhibitor of the MAPK pathway, and wortmannin, an inhibitor of the PI3K pathway, both suppressed (p<0.05) E2- and TBA-stimulated proliferation of cultured BSC. Our data suggest that IGF-I plays a role in E2- and TBA-stimulated proliferation of cultured BSC even in the absence of increased IGF-I expression.     

Serum and intratumoural GH and IGF-I concentrations: Prognostic factors in the outcome of canine mammary cancer

The biological implication of the growth hormone/insulin like growth factor-I (GH/IGF-I) axis in canine mammary tumours (CMT) has been recently demonstrated, however its clinical and prognostic implications are unknown. Our aim was to investigate its prognostic significance.Hormonal determinations were done by enzyme immunoassays techniques validated for canine species in serum and tumour tissue from 32 bitches with CMT and in serum and normal mammary tissue from 10 controls. Serum and tissular GH and IGF-I concentrations were significantly higher in the case of malignant tumour compared with benign and controls. GH and IGF-I elevated concentrations were significantly associated with tumour relapse and/or metastases during follow-up and in dogs with reduced survival times; however these parameters were not independent prognostic factors in multivariate analysis. This association demonstrates a link between high serum and intratumoural GH and IGF-I concentrations and a worse prognosis and opens the possibility to new anticancer endocrine therapies in dogs.     

组织块再移法分离培养奶山羊乳腺上皮细胞

通过比较组织块贴壁培养法、组织块再移法、胰蛋白酶消化法、胶原酶Ⅱ消化法和胶原酶消化配合组织块贴壁法分离、培养、纯化山羊乳腺上皮细胞,绘制细胞生长曲线并计算细胞群体倍增时间,研究乳腺上皮细胞培养效果。结果显示,利用胶原酶消化配合组织块贴壁法不能获得正常生长的乳腺上皮细胞;利用组织块贴壁培养法、组织块再移法和胶原酶Ⅱ消化法获得大量的乳腺上皮细胞,所得到的上皮细胞生长曲线呈典型的＂S＂型,符合细胞生长的一般规律。组织块再移法不仅可快速获得大量纯化的乳腺上皮细胞,而且所得到的细胞经传代后,细胞群体倍增时间最短、增殖能力最强,表明,该法是获得山羊乳腺上皮细胞最适宜的方法。     

IGF-I与IGFBP-1基因对京海黄鸡生长性状的遗传效应分析

旨在对IGF-I和IGFBP-1基因部分SNPs与鸡生长性状进行关联分析。试验以京海黄鸡为材料,采用PCR-SSCP方法检测2个候选基因的单核苷酸多态性(SNPs),采用一般线性模型(GLM)分析基因型与生长性状的遗传效应。结果表明,IGF-I基因外显子3序列的60bp处有A→G的点突变,在京海黄鸡中检测到AA、AB、BB 3种基因型,A等位基因频率为0.613,B等位基因的频率为0.387;IGFBP-1基因外显子2序列的21bp处有A→T的点突变,104bp处有T→C的突变,在京海黄鸡中检测到CC、CD和DD 3种基因型,C等位基因频率为0.558,D等位基因频率为0.442。IGF-I基因BB基因型个体4周龄体质量显著高于AA和AB型个体(P<0.05);IGFBP-1基因DD基因型个体8周龄体质量显著高于CC基因型个体(P<0.05),4、12和16周龄体质量差异极显著(P<0.01)。因此,推测这些SNPs对京海黄鸡生长性状具有一定的影响,应用于鸡育种过程中的标记辅助选择可以加快育种进程。     

Effects of induced energy deficiency on lactoferrin concentration in milk and the lactoferrin reaction of primary bovine mammary epithelial cells in vitro

A dietary energy restriction to 49% of total energy requirements was conducted with Red Holstein cows for three weeks in mid‐lactation. At the last day of the restriction phase, primary bovine mammary epithelial cells (pbMEC) of eight restriction (RF) and seven control‐fed (CF) cows were extracted out of one litre of milk and cultured. In their third passage, an immune challenge with the most prevalent, heat‐inactivated mastitis pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was conducted. Lactoferrin (LF) was determined on gene expression and protein level. An enzyme‐linked immunosorbent assay (ELISA) was developed to determine LF in milk samples taken twice weekly throughout the animal trial, beginning on day 20 pp (post‐partum) until day 150 pp, in cell culture total protein and in cell culture supernatant. Milk LF increased throughout the lactation and decreased significantly during the induced energy deficiency in the RF group. At the beginning of realimentation, LF concentration increased immediately in the RF group and reached higher levels than before the induced deficit following the upward trend seen in the CF group. Cell culture data revealed higher levels (up to sevenfold up‐regulation in gene expression) and significant higher LF protein concentration in the RF compared to the CF group cells. A further emphasized effect was found in E. coli compared to S. aureus exposed cells. The general elevated LF levels in the RF pbMEC group and the further increase owing to the immune challenge indicate an unexpected memory ability of milk‐extracted mammary cells that were transposed into in vitro conditions and even displayed in the third passage of cultivation. The study confirms the suitability of the non‐invasive milk‐extracted pbMEC culture model to monitor the influence of feeding experiments on immunological situations in vivo.     

Potential role of G-protein-coupled receptor 30 (GPR30) in estradiol-17beta-stimulated IGF-I mRNA expression in bovine satellite cell cultures

Androgenic and estrogenic steroids enhance muscle growth in animals and humans. Estradiol-17beta (E2) and trenbolone acetate (TBA) (a synthetic testosterone analog) increased IGF-I mRNA expression in bovine muscle satellite cell (BSC) cultures. The goal of this study was to evaluate the mechanisms responsible for this increase by evaluating the effects of ICI 182 780 (an E2 receptor antagonist), flutamide (an androgen receptor inhibitor), G1 (a GPR30 agonist), and BSA-conjugated E2 on E2 and/or TBA-stimulated IGF-I mRNA expression in BSC cultures. Flutamide completely suppressed TBA-stimulated IGF-I mRNA expression in BSC cultures. ICI 182 780 did not suppress E2-stimulated IGF-I mRNA expression and 100nM ICI 182 780 enhanced (93%, p<0.05) IGF-I mRNA levels in BSC cultures. G1 (100nM) stimulated IGF-I mRNA expression (100%, p<0.05) but had no effect on proliferation in BSC cultures. E2-BSA, which cannot cross the cell membrane, stimulated IGF-I mRNA expression (approximately 100%, p<0.05) in BSC but even at extremely high concentrations had no effect on proliferation. In summary, our data indicate the E2-stimulation of proliferation and E2-stimulation of IGF-I mRNA expression in BSC cultures occur via different mechanisms. Our previous results showing that ICI 182 780 inhibited BSC proliferation and results of the current study showing lack of response to E2-BSA or G1 suggest that E2-stimulated proliferation in BSC cultures is mediated through classical estrogen receptors. Stimulation by ICI 182 780, G1 and E2-BSA suggests the E2-stimulated IGF-I mRNA expression in BSC cultures is mediated through the GPR30 receptor.