SOEing法在构建人瘦蛋白乳腺表达载体中的应用

目的 :将奶牛CSN2 5′端启动子区与人瘦蛋白cDNA序列进行拼接 ,进而构建人瘦蛋白乳腺特异性表达载体。方法 :设计特殊的“巨型引物” ,运用PCR方法分别从质粒pBBC和pHL上扩增了牛CSN2 5′端启动子 ( 2 8kb)和有完整读码框的人瘦蛋白基因。利用改进的重叠区扩增基因拼接法 (SOEing法 ) ,将两个独立的片断进行了拼接。结果 :得到了两者线形融合基因 ,为构建人瘦蛋白乳腺特异性表达载体打下了基础。     

人尿激酶原乳腺定位转基因小鼠的建立

应用大鼠β乳酪蛋白基因的上游调控序列和人尿激酶原ｃＤＮＡ构建成功了乳腺定位表达载体．用显微注射的手段导入到受精卵的雄前核,从注射的３００枚受精卵中,１４０枚被移植到９只假孕的受体小鼠．结果从获得的子一代小鼠中,经ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ证实,有３只转基因阳性的小鼠．     

乳腺特异高表达人促红细胞生成素转基因奶山羊的制备

目的制备乳腺特异性高表达人促红细胞生成素（hEPO）转基因奶山羊。方法采用牛β-乳球蛋白基因（BLG）调控元件和hEPO全长编码序列基因组DNA构建真核表达载体,应用受精卵原核注射的方法制备hEPO转基因山羊。结果在原核注射获得的188头羔羊中,经Southern blot法检测有4头羊含有hEPO基因,其中3头为母羊,1头公羊于出生后20d死亡;3头转基因母羊hEPO基因的拷贝数分别为1、10、2;Western blot检测结果显示转基因羊乳中的hEPO分子质量为32kDa;MTT法检测结果表明,在泌乳10d的3只转基因羊乳汁中,每毫升乳汁中hEPO活性分别达到1.17×10^2IU、1.90×10^4IU、1.91×10^4IU。结论牛BLG能够调控hEPO基因在山羊乳腺中高表达,为实现其他药用蛋白在山羊乳腺中表达奠定了基础。     

Hormone Action in the Mammary Gland

A woman’s breast cancer risk is affected by her reproductive history. The hormonal milieu also influences the course of the disease. The female reproductive hormones, estrogens, progesterone, and prolactin, have a major impact on breast cancer and control postnatal mammary gland development. Analysis of hormone receptor mutant mouse strains combined with tissue recombination techniques and proteomics revealed that sequential activation of hormone signaling in the mammary epithelium is required for progression of morphogenesis. Hormones impinge on a subset of luminal mammary epithelial cells (MECs) that express hormone receptors and act as sensor cells translating and amplifying systemic signals into local stimuli. Proliferation is induced by paracrine mechanisms mediated by distinct factors at different stages. Tissue and stage specificity of hormonal signaling is achieved at the molecular level by different chromatin contexts and differential recruitment of coactivators and corepressors.Breast cancer is the most frequent cancer in women and the second leading cause of cancer deaths among women. To better understand the genetic alterations responsible for breast cancer, it is critical to first understand the mechanisms regulating normal mammary gland development. Increased interest in the field has led to the identification of a large number of genes important for mammary gland development (reviewed in Tanos and Brisken 2008).A woman’s risk for breast cancer is linked to her reproductive history and with her lifetime hormonal exposure; hormones also influence the course of the disease. The same hormones that affect breast carcinogenesis control postnatal mammary gland development. The mouse mammary gland has been instrumental in providing new insights into the mechanisms by which hormones act in the mammary gland.A number of features make the mouse mammary gland a particularly attractive experimental system. Being the only organ that undergoes most of its development postnatally, it is particularly suited for studying developmental processes; it is readily amenable to experimental manipulation and can be easily accessed as it localizes to the underside of the ventral skin. Furthermore, mammary gland tissue is abundant; there are 5 pairs of mammary glands in mice, and cells can be isolated in large numbers. The versatile tools of mouse genetics can be combined with powerful tissue recombination techniques to generate chimeric glands, as we will illustrate in this article.     

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.     

Immunocytochemical Localization of Sex Steroid Hormone Receptors in Normal Human Mammary Gland

The sex steroids, estrogens, progesterone, and androgens, all play a role in mammary development and function. To precisely identify the sites of action of these steroids, we studied the localization of the estrogen receptor α (ERα) and ERβ, the progesterone receptor A (PRA) and PRB, and androgen receptors (AR) in the normal human mammary gland. Immunocytochemical localization of ERα, ERβ, PRA, PRB, and AR was performed with reduction mammoplasty specimens from premenopausal women. ERα, PRA, PRB, and AR were localized mostly to the inner layer of epithelial cells lining acini and intralobular ducts, as well as to myoepithelial cells scattered in the external layer of interlobular ducts. AR was also found in some stromal cells. ERβ staining was more widespread, resulting in epithelial and myoepithelial cells being labeled in acini and ducts as well as stromal cells. These results suggest that all sex steroids can directly act on epithelial cells to modulate development and function of the human mammary gland. Estrogens and androgens can also indirectly influence epithelial cell activity by an action on stromal cells. (J Histochem Cytochem 58:509–515, 2010)     

Dynamic Control of Oligosaccharide Modification in the Mammary Gland: Linking Recombinant Human Erythropoietin

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin α and β are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and α1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin α derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin α. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use. These authors are equal contributors to this work.     

On Hormone Action in the Mammary Gland

The importance of systemic reproductive hormones in mammary gland development and breast cancer has been known for more than a century. In fact, the first targeted therapy for cancer was the development of tamoxifen, as an estrogen receptor (ER) antagonist. Based on studies performed primarily in a few breast cancer cell lines, the textbook concept of steroid hormone action at present is that on ligand binding, steroid receptors translocate into the nucleus and stimulate proliferation, and that this effect is mediated by specific coregulators. More recently, as nicely discussed by Brisken and O’Malley (2011), the concepts of specific receptor-positive sensor cells for systemic hormones, and paracrine mediators regulating the development and proliferation of proximal cells has been elegantly shown by the use of genetically engineered mice and chimeric transplantation experiments. One key question raised by these studies is, “How is the patterning of hormone receptor-positive sensor cells established during normal development?” As described by Visvader and Smith (2011), mammary stem cells lack the estrogen and progesterone receptors, and these receptors are first expressed at a still-undefined stage of the mammary cell hierarchy following the appearance of ductal and alveolar progenitors. So how is this process regulated appropriately to provide the correct temporal and spatial expression of the receptor-positive ductal and alveolar cells needed for normal ductal morphogenesis and alveolargenesis? Furthermore, what happens when this process is inappropriately regulated during early breast cancer progression when there may be a switch from paracrine to autocrine signaling mechanisms?Until recently, it was not possible to study these processes in primary mammary epithelial cells, because when these cells are grown under conventional cell culture conditions they rapidly lose the expression of steroid receptors. However, some recent success in culturing both primary mouse and human mammary cells in embedded 3D Matrigel cultures have provided at least a surrogate system to help dissect some of these paracrine mechanisms (Novaro et al. 2003; Graham et al. 2009). Still, it has not been possible to precisely mimic the patterning of receptor-positive cells observed in vivo in these surrogate in vitro models. So how can we specifically target steroid receptor-positive sensor cells to perform gain- and loss-of-function experiments in vivo? Recent advances using genetically engineered mouse models (Jeong et al. 2010; Mukherjee et al. 2010) may provide the key. In these models, Lydon, Demayo, and colleagues (Jeong et al. 2010) have inserted the Cre recombinase into the progesterone receptor gene allowing specific gene deletion only in that subset of mammary epithelial cells. Because the majority of ER positive cells are also progesterone receptor positive, this should facilitate loss-of-function studies of paracrine mediators for both steroid hormone receptors. Conversely, using a clever bigenic system for doxycycline-inducible expression, these same investigators have expressed one of the identified paracrine mediators, RANKL, in the mammary epithelium of progesterone receptor knockout mice exclusively in ER positive cells. Thus, this gain-of-function approach should help define the critical paracrine mediators of progesterone action and perhaps even the role of specific coregulators in this subset of cells.Downstream from the nuclear receptors, hormonal signaling is regulated by different chromatin contexts and differential recruitment of coactivators as well as corepressors (Brisken and O’Malley 2011). Numerous posttranslational modifications also play key roles in modulating the effects of coregulators, but these have been studied primarily in the HeLa, and to a lesser extent in MCF7, cell lines. Thus, we still know very little about these coregulators and their modifications in normal mammary epithelial cells. Because cell context and architecture are critical, studies, therefore, should be performed in primary mammary epithelial cells to provide a better understanding of how these coregulators and their posttranslational modifications affect normal mammary gland development. No doubt, coregulators may differentially influence hormone receptor-positive cells as compared to the receptor-negative adjacent cells, because most coregulators can also affect cells lacking steroid hormone receptors. Clearly, we are only at the tip of the iceberg when it comes to understanding the precise molecular mechanisms of hormone action in the normal mammary gland, and this will be critical for identifying alterations which occur during breast cancer progression.     

Human Breast Cancer Cells Are Redirected to Mammary Epithelial Cells upon Interaction with the Regenerating Mammary Gland Microenvironment In-Vivo

Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display ‘normal’ behavior when placed into ‘normal’ ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for ‘normal’ gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts) confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini) were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic) respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo.     

人G—CSF基因组基因的克隆及其在转基因小鼠乳腺表达的研究

采用ＰＣＲ方法以正常中国人脐带血提取总ＤＮＡ为模板,扩增出１．５Ｋｂ的粒细胞集落刺激因子（Ｇ－ＣＳＦ）基因组基因,序列分析证实其正确性,将其插入小鼠乳清酸蛋白（ＷＡＰ）基因的起支密码子ＡＴＧ臆的ＫｐｎＩ位点,使其受控于２．６ｋｂ的ＷＡＰ调控序列,从而构建乳腺表达载体ｐＷＧＧ。回收经ＥｃｏＲＩ酶切后的８．７ｋｂ片段用于显微注射,共注射１２００枚受精卵,移植至受体３４母鼠,产生仔鼠８５只,经ＰＣＲ检测     

Knocking Off SOCS Genes in the Mammary Gland

Suppressors of cytokine signaling (SOCS) proteins are critical regulators of cytokinemediatedresponses in diverse tissues. In the mammary gland, signal transductionpathways elicited by cytokines and hormones have been shown to control distinct stagesof development. In vivo evidence points to essential roles for Socs1 and Socs2 as keyphysiological attenuators of prolactin receptor (PRLR) signaling during pregnancy andlactogenesis. Recently, Socs3 has been shown to be a critical regulator of involution, thecoordinated process of programmed cell death and tissue remodelling that is initiatedafter the cessation of lactation. This review will predominantly focus on the antiapoptoticfunction of Socs3 during mammary gland involution in which it acts as a keyattenuator of Stat3-mediated signal transduction. Perturbation of this pathway leads to anincrease in the levels of c-myc and its likely target genes, p53, bax and E2F-1, providingevidence that c-myc is a central effector of apoptosis during involution.     

表达人溶菌酶基因牛乳腺特异表达载体的构建

利用高保真PCR法,分别扩增了牛乳腺酪蛋白基因的1.8kb和1.1kb的5'和3'调控 序列,克隆入TA载体.经测序鉴定后,利用DNA重组技术依次亚克隆入改造过的真核表达载体 pcDNA3(切除CMV启动子),并插入人溶菌酶基因(hLYZ)的cDNA, 构建成牛乳腺特异表达 载体.获得的重组载体经限制性内切酶酶切鉴定、PCR验证等表明,成功克隆了酪蛋白基因5 '和3'的调控区,并成功构建了表达人溶菌酶基因的牛乳腺特异表达载体.     

乳腺增生病的治疗现状

乳腺增生病是最常见的乳房疾病,发病率高,占全部乳房病的75%。目前,其治疗方面,单用中药治疗虽疗效肯定,但起效较慢,如单用西药治疗虽起效快,但作用难于持续,长期服用毒副反应多,复发率较高。目前在临床上对乳腺增生病尚无疗效肯定、起效快、毒副作用小的治疗方法,本文就当前乳腺增生病的治疗及临床应用进行概括。