乳腺生物反应器特异高效表达载体的构建

乳腺生物反应器具有广阔的开发前景,而提高目的基因的表达量是该领域一个重要的研究课题。因此,选用强的非特异性启动子pCAG,而不是乳腺特异性启动子来实现高效表达;通过Cre/loxP系统和山羊β-乳球蛋白启动区（pBLG）表达Cre重组酶来实现载体的自删除以达到乳腺特异表达的目的。方法：构建含PolyA终止信号的Cre乳腺特异表达元件PolyA-pBLG-cre,插至强启动子pCAG驱动的报告基因lacZ表达载体中,构建成乳腺特异表达载体pCBCZ（pCAG-loxP-PolyA-pBLG-cre-loxP-lacZ）。转染细胞实验结果：PCR鉴定确认pCBCZ载体在小鼠乳腺上皮细胞（HC11）中发生Cre-loxP同源重组。X-Gal染色表明载体能驱动lacZ在HC11细胞中高效表达β-半乳糖苷酶,而在NIH 3T3细胞中仅少量表达。结论：构建的pCBCZ载体能高效驱动外源基因在乳腺细胞中表达,且具有较好的乳腺特异性,为研发乳腺生物反应器表达载体提供新的方法。     

Mammary cancers and pregnancy.

Uncertainties persist about management and prognosis of mammary cancers that occur during and after pregnancy and during lactation. Pathological features of mammary cancers occurring during pregnancy are the same as those in non-pregnant women and survival rates are comparable. Management should be the same as in non-pregnant patients. Termination of pregnancy does not improve survival but it should be advised if the prognosis is poor. Mastectomy apparently presents little danger to the fetus, though treatment such as chemotherapy and irradiation should be avoided. Women who have received treatment for mammary cancer need not be advised against subsequent pregnancy. Routine ovarian radiation in non-pregnant premenopausal women is not generally to be recommended, since it does not prolong survival and would deprive some of the chance of further pregnancy. In lactating women who develop mammary cancers survival is apparently not adversely affected. Lactation should be suppressed initially and followed by mastectomy. Regimens of immunotherapy, chemotherapy, or radiotherapy may then be begun. Until results of current trials of combined treatments of mammary cancers associated with pregnancy are available, management should be neither aggressive nor tentative. It should be based on a well-balanced concept of applying all available treatments, as in non-pregnant patients.     

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.     

Leukocytes in Mammary Development and Cancer

Leukocytes, of both the innate and adaptive lineages, are normal cellular components of all tissues. These important cells not only are critical for regulating normal tissue homeostasis, but also are significant paracrine regulators of all physiologic and pathologic tissue repair processes. This article summarizes recent insights regarding the trophic roles of leukocytes at each stage of mammary gland development and during cancer development, with a focus on Murids and humans.Mammary gland development can be divided into discrete phases. An initial analge is laid down from the milk-line during embryonic development resulting in a minimal ductal structure emanating from the nipple. Development of this anlage into a ductal tree is reactivated postnatally by exposure to the female sex steroid hormone estradiol-17β (E2), whose synthesis begins upon entry into puberty. In mice, this occurs at about 3 wk of age and is characterized by the formation of terminal end buds (TEB) at the ends of the ducts. These TEBs are clublike multilaminate epithelial structures that are the proliferative engines that drive mammary development. These structures also contain the mammary stem cells whose progeny differentiate into luminal and myoepithelial cells. The TEB structures disappear on their encounter with the edge of the fat pad and turn into terminal end-ducts (TED) that cease proliferation and which are bilaminar. As the primary branches grow out through the fat pad, secondary branches form to generate the mature tree that in mice is completed about 8 wk of age coincident with sexual maturity. At each estrus cycle thereafter, there is further development of the secondary branches and dependent on mouse strain, a degree of lobuloalveolar development. The next major phase of growth is during pregnancy in response to progesterone and prolactin when there is significant secondary branching morphogenesis, and the generation of the milk producing lobuloalveolar structures sprouting from these branches. At the end of the process, the gland is filled with ducts and alveolar structures with a commensurate loss of adipocytes. After birth and on suckling, lactation occurs with its effect on the secretory structure of alveoli that flatten to surround a milk-filled lumen. Weaning terminates the lactational process and the gland involutes to re-form a virgin-like structure to begin the cycle again during the next pregnancy (Daniel and Silberstein 1987; Richert et al. 2000; Neville et al. 2002). Every stage of mammary epithelial development is accompanied by changes in the surrounding stroma. This stroma is populated by many immune cells particularly those of the innate system. Although these cells undoubtedly have a role in immunological responses especially during lactation (Paape et al. 2002; Atabai et al. 2007), this review will concentrate on the trophic roles of these hematopoietic cells at each stage of development and during cancer development, with a focus on Murids and humans.     

Bone Morphogenetic Proteins Stimulate Mammary Fibroblasts to Promote Mammary Carcinoma Cell Invasion

Bone Morphogenetic Proteins (BMPs) are secreted cytokines that are part of the Transforming Growth Factor β (TGFβ) superfamily. BMPs have been shown to be highly expressed in human breast cancers, and loss of BMP signaling in mammary carcinomas has been shown to accelerate metastases. Interestingly, other work has indicated that stimulation of dermal fibroblasts with BMP can enhance secretion of pro-tumorigenic factors. Furthermore, treatment of carcinoma-associated fibroblasts (CAFs) derived from a mouse prostate carcinoma with BMP4 was shown to stimulate angiogenesis. We sought to determine the effect of BMP treatment on mammary fibroblasts. A large number of secreted pro-inflammatory cytokines and matrix-metallo proteases (MMPs) were found to be upregulated in response to BMP4 treatment. Fibroblasts that were stimulated with BMP4 were found to enhance mammary carcinoma cell invasion, and these effects were inhibited by a BMP receptor kinase antagonist. Treatment with BMP in turn elevated pro-tumorigenic secreted factors such as IL-6 and MMP-3. These experiments demonstrate that BMP may stimulate tumor progression within the tumor microenvironment.     

Mammary pathogenic Escherichia coli

Pathogenic Escherichia coli can be classified into several pathotypes, and it is believed that each pathotype carries one or more specific gene repertoire (or virulence factors combination) that distinguishes them from non-pathogenic E. coli strains and from other pathotypes. In contrast to this notion, it was proposed that this is not the case for E. coli mastitis, a common disease in farm animals and that any given E. coli isolate can cause this disease, even strains that are considered non-pathogenic. In this review we will re-examine this latter concept and recent advances in the study E. coli mastitis.     

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.¹ and the sparing procedure developed by Brill B et al.², followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn''t occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal³, in the identification of mammary stem cells by transplanting cells in limited dilution^4,5, determining if hyperplastic nodules proceed to mammary tumors⁶, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium^7,8.Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.Download video file.^{(99M, mp4)}     

Murine Mammary Tumor Virus Expression During Mammary Tumorigenesis in BALB/c Mice

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.