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Culture Models to Investigate Mechanisms of Milk Production and Blood-Milk Barrier in Mammary Epithelial Cells: a Review and a Protocol. 研究乳腺上皮细胞产乳和血-乳屏障机制的培养模型:综述和方案。
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2023-05-01 DOI: 10.1007/s10911-023-09536-y
Ken Kobayashi

Mammary epithelial cells (MECs) are the only cell type that produces milk during lactation. MECs also form less-permeable tight junctions (TJs) to prevent the leakage of milk and blood components through the paracellular pathway (blood-milk barrier). Multiple factors that include hormones, cytokines, nutrition, and temperature regulate milk production and TJ formation in MECs. Multiple intracellular signaling pathways that positively and negatively regulate milk production and TJ formation have been reported. However, their regulatory mechanisms have not been fully elucidated. In addition, unidentified components that regulate milk production in MECs likely exist in foods, for example plants. Culture models of functional MECs that recapitulate milk production and TJs are useful tools for their study. Such models enable the elimination of indirect effects via cells other than MECs and allows for more detailed experimental conditions. However, culture models of MECs with inappropriate functionality may result in unphysiological reactions that never occur in lactating mammary glands in vivo. Here, I briefly review the physiological functions of alveolar MECs during lactation in vivo and culture models of MECs that feature milk production and less-permeable TJs, together with a protocol for establishment of MEC culture with functional TJ barrier and milk production capability using cell culture inserts.

乳腺上皮细胞(MECs)是哺乳期唯一产生乳汁的细胞类型。mec还形成不易渗透的紧密连接(TJs),以防止乳和血液成分通过细胞旁途径(血-乳屏障)渗漏。包括激素、细胞因子、营养和温度在内的多种因素调节着mec的产奶量和TJ形成。多种细胞内信号通路正向和负向调节产奶量和TJ的形成。然而,它们的调控机制尚未完全阐明。此外,mec中调节牛奶产量的未知成分可能存在于食物中,例如植物。功能mec的培养模型概括了牛奶产量和tj是他们研究的有用工具。这种模型能够消除通过mec以外的细胞产生的间接影响,并允许更详细的实验条件。然而,功能不合适的mec培养模型可能导致体内泌乳乳腺中从未发生过的非生理性反应。在这里,我简要回顾了哺乳期间肺泡MEC的生理功能,以及具有产奶和低渗透性TJ的MEC培养模型,以及使用细胞培养插入物建立具有功能性TJ屏障和产奶能力的MEC培养方案。
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引用次数: 2
The Serotonergic System and Bone Metabolism During Pregnancy and Lactation and the Implications of SSRI Use on the Maternal-Offspring Dyad. 妊娠期和哺乳期的羟色胺能系统和骨代谢以及使用 SSRI 对母子二人的影响。
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2023-04-22 DOI: 10.1007/s10911-023-09535-z
Hannah P Fricke, Laura L Hernandez

Lactation is a physiological adaptation of the class Mammalia and is a product of over 200 million years of evolution. During lactation, the mammary gland orchestrates bone metabolism via serotonin signaling in order to provide sufficient calcium for the offspring in milk. The role of serotonin in bone remodeling was first discovered over two decades ago, and the interplay between serotonin, lactation, and bone metabolism has been explored in the years following. It is estimated that postpartum depression affects 10-15% of the population, and selective serotonin reuptake inhibitors (SSRI) are often used as the first-line treatment. Studies conducted in humans, nonhuman primates, sheep, and rodents have provided evidence that there are consequences on both parent and offspring when serotonin signaling is disrupted during the peripartal period; however, the long-term consequences of disruption of serotonin signaling via SSRIs during the peripartal period on the maternal and offspring skeleton are not fully known. This review will focus on the relationship between the mammary gland, serotonin, and bone remodeling during the peripartal period and the skeletal consequences of the dysregulation of the serotonergic system in both human and animal studies.

哺乳是哺乳纲动物的一种生理适应,是 2 亿多年进化的产物。哺乳期间,乳腺通过血清素信号协调骨代谢,以便通过乳汁为后代提供充足的钙。血清素在骨重塑中的作用在二十多年前首次被发现,随后几年人们一直在探索血清素、泌乳和骨代谢之间的相互作用。据估计,产后抑郁症影响着 10-15% 的人口,选择性血清素再摄取抑制剂(SSRI)通常被用作一线治疗药物。在人类、非人灵长类动物、绵羊和啮齿类动物中进行的研究已经提供了证据,证明围产期血清素信号转导中断会对父母和后代造成影响;但是,围产期通过 SSRIs 干扰血清素信号转导对母体和后代骨骼造成的长期影响尚不完全清楚。本综述将重点探讨围产期乳腺、血清素和骨骼重塑之间的关系,以及血清素能系统失调对人类和动物骨骼造成的后果。
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引用次数: 1
Associations Between Dog Breed and Clinical Features of Mammary Epithelial Neoplasia in Bitches: an Epidemiological Study of Submissions to a Single Diagnostic Pathology Centre Between 2008-2021. 犬种与母狗乳腺上皮瘤临床特征之间的关系:2008-2021年间提交给单一诊断病理中心的流行病学研究
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2023-03-24 DOI: 10.1007/s10911-023-09531-3
Grace Edmunds, Sam Beck, Kedar Umakant Kale, Irena Spasic, Dan O'Neill, David Brodbelt, Matthew J Smalley

Mammary cancer is one of the most common neoplasms of dogs, primarily bitches. While studies have been carried out identifying differing risk of mammary neoplasia in different dog breeds, few studies have reported associations between dog breeds and clinical features such as number of neoplastic lesions found in an individual case or the likelihood of lesions being benign or malignant. Such epidemiological studies are essential as a foundation for exploring potential genetic drivers of mammary tumour behaviour. Here, we have examined associations between breed, age and neuter status and the odds of a diagnosis of a mammary epithelial-origin neoplastic lesion (as opposed to any other histopathological diagnosis from a biopsied lesion) as well as the odds of a bitch presenting with either a single mammary lesion or multiple lesions, and the odds that those lesions are benign or malignant. The study population consisted of 129,258 samples from bitches, including 13,401 mammary epithelial neoplasms, submitted for histological assessment to a single histopathology laboratory between 2008 and 2021.In multivariable analysis, breed, age and neuter status were all significantly associated with the odds of a diagnosis of a mammary epithelial-origin neoplastic lesion. Smaller breeds were more likely to receive such a diagnosis. In cases diagnosed with a mammary epithelial neoplasm, these three factors were also significantly associated with the odds of diagnosis with a malignant lesion and of diagnosis with multiple lesions. Notably, while neutered animals were less likely to have a mammary epithelial neoplasm diagnosed, and were less likely to have multiple neoplasms, they were more likely to have malignant disease. Exploration of the patterns of risk of developing malignant disease, or multiple lesions, across individual breeds showed no breed with increased odds of both outcomes. Breeds with altered odds compared to the Crossbreed baseline were either at increased risk of malignant disease and decreased risk of multiple lesions, or vice versa, or they were at significantly altered odds of one outcome with no change in the other outcome. Our analysis supports the hypothesis that age, neuter status and intrinsic biological and genetic factors all combine to influence the biological heterogeneity of canine mammary neoplasia.

乳腺癌是狗最常见的肿瘤之一,主要是母狗。虽然已经进行了研究,确定了不同犬种患乳腺肿瘤的不同风险,但很少有研究报道犬种与临床特征之间的关系,例如单个病例中发现的肿瘤病变数量或病变是良性还是恶性的可能性。这种流行病学研究对于探索乳腺肿瘤行为的潜在遗传驱动因素至关重要。在这里,我们研究了犬种、年龄和中性状态与乳腺上皮源性肿瘤病变的诊断几率(相对于任何其他活检病变的组织病理学诊断)之间的关系,以及母狗单发或多发乳腺病变的几率,以及这些病变是良性或恶性的几率。研究人群包括来自母狗的129258份样本,其中包括13401份乳腺上皮肿瘤,在2008年至2021年期间提交给单一组织病理学实验室进行组织学评估。在多变量分析中,品种、年龄和中性状态都与乳腺上皮源性肿瘤病变的诊断几率显著相关。较小的品种更有可能得到这样的诊断。在诊断为乳腺上皮肿瘤的病例中,这三个因素也与诊断为恶性病变和诊断为多发性病变的几率显著相关。值得注意的是,虽然绝育的动物不太可能被诊断出患有乳腺上皮肿瘤,也不太可能患有多发性肿瘤,但它们更有可能患有恶性疾病。对个体品种发生恶性疾病或多重病变的风险模式的探索表明,没有品种增加了这两种结果的几率。与杂交品种基线相比,几率发生改变的品种要么恶性疾病的风险增加,多发性病变的风险降低,反之亦然,要么一种结果的几率显著改变,而另一种结果没有变化。我们的分析支持这样的假设,即年龄、中性状态和内在的生物学和遗传因素共同影响犬乳腺肿瘤的生物学异质性。
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引用次数: 1
Correction to: Valine Treatment Enhances Antimicrobial Component Production in Mammary Epithelial Cells and the Milk of Lactating Goats Without Influencing the Tight Junction Barrier. 修正:缬氨酸处理提高乳腺上皮细胞和泌乳山羊乳中抗菌成分的产生,而不影响紧密连接屏障。
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2023-03-14 DOI: 10.1007/s10911-023-09533-1
Yusaku Tsugami, Takahiro Nii, Naoki Isobe
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引用次数: 1
Correction to: Rank ectopic expression in the presence of Neu and PyMT oncogenes alters mammary epithelial cell populations and their tumorigenic potential. 更正:Neu和PyMT癌基因存在时,Rank异位表达改变了乳腺上皮细胞群及其致瘤潜能。
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2023-03-07 DOI: 10.1007/s10911-023-09532-2
Alex Cordero, Patricia G Santamaría, Eva González-Suárez
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引用次数: 0
Valine Treatment Enhances Antimicrobial Component Production in Mammary Epithelial Cells and the Milk of Lactating Goats Without Influencing the Tight Junction Barrier. 缬氨酸处理在不影响紧密连接屏障的情况下提高了哺乳山羊乳腺上皮细胞和乳汁中抗菌成分的产生。
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2023-02-18 DOI: 10.1007/s10911-023-09529-x
Yusaku Tsugami, Takahiro Nii, Naoki Isobe

The production of antimicrobial components and the formation of less-permeable tight junctions (TJs) are important in the defense system of lactating mammary glands and for safe dairy production. Valine is a branched-chain amino acid that is actively consumed in the mammary glands and promotes the production of major milk components like β-casein; additionally, branched-chain amino acids stimulate antimicrobial component production in the intestines. Therefore, we hypothesized that valine strengthens the mammary gland defense system without influencing milk production. We investigated the effects of valine in vitro using cultured mammary epithelial cells (MECs) and in vivo using the mammary glands of lactating Tokara goats. Valine treatment at 4 mM increased the secretion of S100A7 and lactoferrin as well as the intracellular concentration of β-defensin 1 and cathelicidin 7 in cultured MECs. In addition, an intravenous injection of valine increased S100A7 levels in the milk of Tokara goats without influencing milk yield and milk components (i.e., fat, protein, lactose, and solids). In contrast, valine treatment did not affect TJ barrier function either in vitro or in vivo. These findings indicate that valine enhances antimicrobial component production without influencing milk production and TJ barrier function in lactating mammary glands; thus, valine contributes to safe dairy production.

抗菌成分的产生和不易渗透的紧密连接(TJs)的形成对泌乳乳腺的防御系统和安全乳制品生产至关重要。缬氨酸是一种支链氨基酸,在乳腺中被积极消耗,并促进乳中主要成分如β-酪蛋白的产生;此外,支链氨基酸刺激肠道中抗菌成分的产生。因此,我们假设缬氨酸在不影响产奶量的情况下增强了乳腺防御系统。研究了缬氨酸在体外培养的乳腺上皮细胞(MECs)和在体内哺乳的托卡拉山羊乳腺中的作用。缬氨酸在4 mM处理时增加了培养mec细胞中S100A7和乳铁蛋白的分泌,以及细胞内β-防御素1和抗菌素7的浓度。此外,静脉注射缬氨酸增加了托卡拉山羊乳中的S100A7水平,而不影响产奶量和乳成分(即脂肪、蛋白质、乳糖和固体)。相比之下,缬氨酸治疗在体内和体外均未影响TJ屏障功能。上述结果表明,缬氨酸在不影响泌乳乳腺泌乳量和TJ屏障功能的情况下提高了抗菌成分的产生;因此,缬氨酸有助于安全的乳制品生产。
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引用次数: 3
Rank ectopic expression in the presence of Neu and MMTV oncogenes alters mammary epithelial cell populations and their tumorigenic potential. Neu和MMTV癌基因的Rank异位表达改变了乳腺上皮细胞群及其致瘤潜能。
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2023-02-18 DOI: 10.1007/s10911-023-09530-4
Alex Cordero, Patricia G Santamaría, Eva González-Suárez

Determination of the mammary epithelial cell that serves as the cell of origin for breast cancer is key to understand tumor heterogeneity and clinical management. In this study, we aimed to decipher whether Rank expression in the presence of PyMT and Neu oncogenes might affect the cell of origin of mammary gland tumors. We observed that Rank expression in PyMT+/- and Neu+/- mammary glands alters the basal and luminal mammary cell populations already in preneoplasic tissue, which may interfere with the tumor cell of origin restricting their tumorigenesis ability upon transplantation assays. In spite of this, Rank expression eventually promotes tumor aggressiveness once tumorigenesis is established.

确定作为乳腺癌起源细胞的乳腺上皮细胞是了解肿瘤异质性和临床管理的关键。在这项研究中,我们旨在揭示在PyMT和Neu癌基因存在的情况下,Rank表达是否会影响乳腺肿瘤的起源细胞。我们观察到,PyMT+/-和Neu+/-乳腺中Rank的表达改变了肿瘤前组织中的基底和腔内乳腺细胞群,这可能会干扰肿瘤起源细胞,限制其在移植试验中的肿瘤发生能力。尽管如此,一旦肿瘤发生,Rank表达最终会促进肿瘤的侵袭性。
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引用次数: 1
Characterization of Hyaluronan Localization in the Developing Mammary Gland and Mammary Tumors. 透明质酸在发展中的乳腺和乳腺肿瘤中的定位特征。
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2023-01-27 DOI: 10.1007/s10911-023-09528-y
Patrice M Witschen, Alexis K Elfstrum, Andrew C Nelson, Kathryn L Schwertfeger

The extracellular matrix (ECM) is biochemically and biomechanically important for the structure and function of the mammary gland, which undergoes vast structural changes throughout pubertal and reproductive development. Although hyaluronan (HA) is a ubiquitous glycosaminoglycan (GAG) of the mammary gland ECM, extensive characterization of HA deposition in the mammary gland is lacking. Understanding physiologic HA metabolism is critical as this tightly controlled system is often hijacked in cancer. In the current studies, we characterize HA regulation throughout mammary gland development to better understand subsequent dysregulation of HA in mammary tumors. Using immunofluorescence (IF) imaging, we demonstrate that organized HA-rich septa exist in the mammary gland stroma throughout puberty, pregnancy, and involution. Furthermore, we find heterogeneous HA deposition within two murine models of breast cancer. Using cell specific isolation techniques, we characterize expression of genes associated with HA binding, synthesis, and degradation within EpCAM + epithelial cells, CD90.2 + fibroblasts, and F4/80 + macrophages isolated from mammary glands and tumors. Most notably, we identify elevated levels of the hyaluronidases Hyal1 and Hyal2 in tumor-association macrophages (TAMs), suggesting a role for TAM-mediated turnover of HA in the tumor microenvironment (TME). Gene expression is supported functionally by in vitro experiments in which macrophages treated with tumor-cell conditioned media exhibit increased hyaluronidase activity. These findings link TAMs to the direct degradation of HA within the TME of mammary tumors, which has negative implications for patient survival.

细胞外基质(ECM)在生物化学和生物力学上对乳腺的结构和功能具有重要意义,在整个青春期和生殖发育过程中,乳腺经历了巨大的结构变化。虽然透明质酸(HA)是乳腺ECM中普遍存在的糖胺聚糖(GAG),但缺乏对HA在乳腺中沉积的广泛表征。了解生理上的透明质酸代谢是至关重要的,因为这个严格控制的系统经常在癌症中被劫持。在目前的研究中,我们描述了HA在乳腺发育过程中的调节,以更好地了解乳腺肿瘤中HA的后续失调。利用免疫荧光(IF)成像,我们证实在青春期、妊娠期和复老期乳腺间质中存在有组织的富含ha的间隔。此外,我们在两种乳腺癌小鼠模型中发现了异质HA沉积。利用细胞特异性分离技术,我们在乳腺和肿瘤分离的EpCAM +上皮细胞、CD90.2 +成纤维细胞和F4/80 +巨噬细胞中表征了与HA结合、合成和降解相关的基因表达。最值得注意的是,我们发现肿瘤相关巨噬细胞(tam)中透明质酸酶Hyal1和Hyal2水平升高,这表明tam在肿瘤微环境(TME)中介导HA的转换中起作用。基因表达在功能上得到了体外实验的支持,在实验中,巨噬细胞用肿瘤细胞条件培养基处理后,显示出更高的透明质酸酶活性。这些发现将tam与乳腺肿瘤TME内HA的直接降解联系起来,这对患者的生存有负面影响。
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引用次数: 1
Comparative Analysis of microRNAs that Stratify in vitro Mammary stem and Progenitor Activity Reveals Functionality of Human miR-92b-3p. 对体外乳腺干细胞和祖细胞活性分层的microrna的比较分析揭示了人类miR-92b-3p的功能。
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2022-12-01 Epub Date: 2022-10-03 DOI: 10.1007/s10911-022-09525-7
James L Miller, Matt Kanke, Gat Rauner, Kimaya M Bakhle, Praveen Sethupathy, Gerlinde R Van de Walle

Mammary stem/progenitor cells are fundamental for mammary gland development and function. However, much remains to be elucidated regarding their function in mammals beyond the traditionally studied rodents, human, and to a lesser extent, ruminants. Due to the growing appreciation for microRNAs (miRNAs) as regulators of stem cells and their progenitors, we compared miRNA expression in mammary stem/progenitor cells from mammals with varying mammary stem/progenitor activity in vitro, in order to identify miRNA candidates that regulate stem/progenitor self-renewal and function. Mammosphere-derived epithelial cells (MDECs), which are primary cell lines enriched in mammary stem and progenitor cells, were generated from six mammalian species (i.e., cow, human, pig, horse, dog, and rat) and small RNA sequencing was performed. We identified 9 miRNAs that were significantly differentially expressed in MDEC cultures with a low versus high mammary stem/progenitor activity. miR-92b-3p was selected for functional follow-up studies, as this miRNA is understudied in primary mammary cells but has well-described gene targets that are known to regulate mammary stem/progenitor activity. Altering the expression of miR-92b-3p in MDECs from species with low stem/progenitor activity (human and cow) and those with high stem/progenitor activity (dog and rat) via inhibition and overexpression, respectively, resulted in significantly decreased mammosphere formation of human MDECs, but showed no significant effects in cow, dog, or rat MDECs. This study is the first to perform small RNA sequencing in MDECs from various mammals and highlights that conserved miRNAs can have different functions in mammary stem/progenitor cells across species.

乳腺干细胞/祖细胞是乳腺发育和功能的基础。然而,除了传统上研究的啮齿动物、人类和反刍动物之外,关于它们在哺乳动物中的功能,还有很多有待阐明。由于越来越多的人认识到microrna (miRNA)是干细胞及其祖细胞的调节剂,我们在体外比较了具有不同乳腺干/祖细胞活性的哺乳动物乳腺干/祖细胞中miRNA的表达,以确定调节干/祖细胞自我更新和功能的候选miRNA。乳腺球源性上皮细胞(MDECs)是一种富含乳腺干细胞和祖细胞的原代细胞系,从6种哺乳动物物种(即牛、人、猪、马、狗和大鼠)中获得,并进行小RNA测序。我们鉴定出9个mirna在乳腺干/祖细胞活性低与高的MDEC培养物中显著表达差异。miR-92b-3p被选择用于功能随访研究,因为该miRNA在原代乳腺细胞中的研究尚不充分,但已知其具有调节乳腺干细胞/祖细胞活性的良好描述的基因靶点。通过抑制和过表达分别改变低干/祖细胞活性物种(人和牛)和高干/祖细胞活性物种(狗和大鼠)MDECs中miR-92b-3p的表达,可显著降低人MDECs的乳腺球形成,但对牛、狗或大鼠MDECs无显著影响。这项研究首次对来自各种哺乳动物的MDECs进行了小RNA测序,并强调了保守的mirna在不同物种的乳腺干细胞/祖细胞中具有不同的功能。
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引用次数: 2
Protein Expression of PI3K/AKT/mTOR Pathway Targets Validated by Gene Expression and its Correlation with Prognosis in Canine Mammary Cancer. 犬乳腺癌中PI3K/AKT/mTOR通路的蛋白表达及其与预后的关系
IF 2.5 4区 医学 Q2 ENDOCRINOLOGY & METABOLISM Pub Date : 2022-12-01 DOI: 10.1007/s10911-022-09527-5
Isabela F S Perossi, Mylena M Saito, Giovanna Rossi Varallo, Bianca Lara Venâncio de Godoy, Jucimara Colombo, Debora A P C Zuccari

Mammary cancer is the main type of neoplasia in female dogs and is considered an adequate model for the biological and therapeutic study of cancer in women. The PIK3CA/AKT/mTOR pathway plays a central role in cellular homeostasis and is often dysregulated in cancer. The increased expression of PI3K protein in the literature is associated with a poor prognosis, and alterations in the PIK3CA gene can lead to changes in downstream pathways. Thus, the objective of this study was to validate the protein expression to confirm the gene expression of proteins belonging to the main pathway PI3K and PTEN, and their downstream pathways through ZEB1, ZEB2, HIF1A, VHL, CASP3 and PARP1 relating to prognosis in canine mammary cancer. For protein studies, the samples came from 58 female dogs with mammary neoplasia, immunohistochemistry was performed and its analysis by the histoscore method. For the genetic evaluation, the samples came from 13 patients, the DNA was extracted and the analysis for quantitative expression. Through immunohistochemistry, PI3K positivity was significantly associated with affected regional lymph node, distant metastasis, patients with HER2+, Triple Negative and Luminal B phenotypes, and the lowest survival rates. Through gene expression, we observed higher gene expression of ZEB2 and PARP1 both among patients who were alive and who died, which was not true for the expressions of PIK3CA and HIF1A. In conclusion, the data observed in this work are promising in the study of new molecular prognostic markers such as PI3K, ZEB2 and PARP1 for canine mammary cancer.

乳腺癌是雌性犬的主要肿瘤类型,被认为是女性癌症生物学和治疗研究的适当模型。PIK3CA/AKT/mTOR通路在细胞稳态中起核心作用,在癌症中经常失调。文献中PI3K蛋白表达升高与预后不良相关,PIK3CA基因的改变可导致下游通路的改变。因此,本研究的目的是验证蛋白表达,以确认犬乳腺癌中PI3K和PTEN主通路及其下游通路ZEB1、ZEB2、HIF1A、VHL、CASP3和PARP1蛋白的基因表达与预后的关系。蛋白研究取材于58只雌性乳腺肿瘤犬,进行免疫组化,并采用histoscore法进行分析。遗传学评估,样本来自13例患者,提取DNA并进行定量表达分析。免疫组化结果显示,PI3K阳性与受影响的区域淋巴结、远处转移、HER2+、三阴性和Luminal B表型患者显著相关,生存率最低。通过基因表达,我们观察到存活和死亡患者中ZEB2和PARP1的基因表达均较高,而PIK3CA和HIF1A的表达则不如此。综上所述,本工作所观察到的数据为研究新的犬乳腺癌分子预后标志物如PI3K、ZEB2和PARP1提供了前景。
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引用次数: 1
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Journal of Mammary Gland Biology and Neoplasia
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