Distinct stem cells contribute to mammary gland development and maintenance

The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.     

奶牛金黄色葡萄球菌乳腺炎小鼠模型的建立

目的利用从临床乳腺炎奶牛乳样中分离纯化的金黄色葡萄球菌（S.aureus）感染小鼠乳腺,建立奶牛金黄色葡萄球菌乳腺炎小鼠模型。方法将产后8～10 d的ICR远交系母鼠随机分为3组,包括无菌生理盐水对照组,低剂量攻菌组（含金黄色葡萄球菌2×102CFU）和高剂量攻菌组（含金黄色葡萄球菌5×106CFU）。以小鼠第4对乳腺为攻菌部位,用非损伤方法经乳头管注入生理盐水或金黄色葡萄球菌。攻菌24h后采集乳汁进行白细胞计数;从眼球采血,进行血常规检测和流式细胞计数;之后处死母鼠,取乳腺组织进行匀浆细菌计数,并做乳腺组织切片。结果高剂量攻菌组不论是体温、组织学观察,还是乳汁白细胞计数、乳腺匀浆细菌计数、血常规指标和流式细胞计数,都和对照组有显著或极显著差异（P〈0.05）;而低剂量攻菌组与对照组相比,除血液CD4＋/CD8＋比值差异显著外（P〈0.05）,其余指标差异均不显著。结论金黄色葡萄球菌浓度为5×106CFU、攻菌时间24 h时,成功建立了远交系小鼠金黄色葡萄球菌乳腺炎模型,而2×102CFU细菌浓度则不适于远交系小鼠乳腺炎模型的建立。     

粉红丝带引发的思考——Wnt信号通路在乳腺发育与乳腺癌变中的角色

 Wnt信号转导通路是参与乳腺发育和肿瘤形成的重要机制。本文综述了Wnt信号转导通路调控乳腺发育和干细胞稳态研究的进展,讨论了Wnt信号转导中成员分子在乳腺癌形成中的角色。     

Pygo2转基因小鼠模型的建立及表型的初步分析

主要通过建立Pygo2转基因小鼠的模型对其表型进行初步分析.首先构建K14-2×flag-Pygo的转基因构件,经酶切、纯化后构建Pygo2转基因小鼠.出生后的仔鼠用PCR和Western方法检测基因型,并通过进一步的免疫组化验证Pygo2基因的表达.PCR检测获得7只转基因阳性鼠,6只Western检测为阳性.对转基因小鼠子代的胚胎和成体进行免疫组化证明,Pygo2基因在皮肤和乳腺组织中有过量表达.转基因小鼠的皮肤、乳腺以及鼠尾椎骨等组织出现了异常的表型.乳腺中有肿瘤组织的形成,且Pygo2在肿瘤中有大量表达.该模型的成功建立为进一步研究Pygo2的功能奠定了基础.     

Purification and unique properties of mammary epithelial stem cells

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.     

Generation of functional multipotent adult stem cells from GPR125+ germline progenitors

Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.     

iRhom2Uncv 小鼠乳腺发育障碍的转录组分析

摘要:目的 研究 iRhom2Uncv 小鼠乳腺表型相关的差异表达基因,为深入研究 iRhom2Uncv 小鼠乳腺发育障碍的相关机制提供靶点信息。 方法 对 12 周龄的野生型和 iRhom2Uncv 小鼠的乳腺组织进行 whole-mounts 染色,分析乳腺发育形态,收集乳腺组织进行转录组测序,筛选差异表达基因,ELISA 检测血清激素水平。 结果 与野生型小鼠相比,iRhom2Uncv 小鼠乳腺侧枝导管较稀疏,差异最显著的 18 个基因中有 6 个在 iRhom2Uncv 小鼠中表达上调,12 个表达下调。 其中 3 个催乳素家族基因在 iRhom2Uncv 小鼠中不表达,且在血清中几乎检测不到催乳素表达,而血清中的雌激素水平相比野生型小鼠较高。 结论 iRhom2Uncv 突变的小鼠存在乳腺发育障碍,可能与 iRhom2Uncv 小鼠中催乳素和 Zfp281 的缺失表达有关,生物信息学分析发现,iRhom2Uncv 小鼠神经发育可能存在不同于野生型小鼠的表型。     

Correction of RNA aberrant splice increases foreign gene expression in transgenic mice

Transgenic mice with mammary gland secreting human granulocyte colony stimulating factor (G-CSF) were produced using mice whey acid protein gene promoter. It was found that there was very low expression level in mammary gland. Human G-CSF cDNA was obtained by RT-PCR from transgenic mice mammary gland. Sequence analysis showed that this G-CSF gene deleted the 4th exon, and compared with human G-CSF genomic DNA, there were donor and acceptor splice sites in the deletion fragment. It was considered that the 3rd and 4th introns also delete in G-CSF fragment. The transgenic construct was corrected by deleting the 3rd and 4th introns to construct the minigene, which was used to produce transgenic mice by microinjection. Northern blot showed that G-CSF expression using the new construct increased 5.4 times as that before in transgenic mice. The results suggested that it was possible that RNA aberrant splice result in low expression in transgenic mice.     

Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice

Milk contains a large amount of protein, most of which consists of a few major species synthesized in the mammary gland. The genes encoding these proteins are single-copy, and expressed during pregnancy and lactation. Although beta-lactoglobulin (BLG) is the major protein in the whey of ruminants, it is not present in rodent milk. We have generated transgenic mice carrying the sheep BLG gene, and show that in such mice, BLG is specifically and abundantly expressed in the mammary gland during lactation. This results in a remarkable alteration of milk composition. These findings suggest that the manipulation of milk composition by gene transfer has considerable potential for the improvement of dairy animals.     

Mouse neural stem cells culturedin vitro and expressing an exogenous gene

Neural stem cells are the multipotential, self-renewing cells in central nerve system, and play an essential role in the development and differentiation of nerve system. Neural stem cells can be used to treat the nerve system diseases, especially, the transplantation of neural stem cells to rescue the degenerated neural cells has become a very promising therapeutic way. We successfully cultured neural stem cells isolated from the brains of embryonic micein vitro and determined their distribution in the E17 mice brains. The neural stem cells were transfected with adenoviral vector carrying GFP (green fluorescence protein) gene and then highly expressed the exogenous gene. It paves the way for gene therapy of degenerative nerve system diseases.     

Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU

Stem cells are proposed to segregate chromosomes asymmetrically during self-renewing divisions so that older ('immortal') DNA strands are retained in daughter stem cells whereas newly synthesized strands segregate to differentiating cells. Stem cells are also proposed to retain DNA labels, such as 5-bromo-2-deoxyuridine (BrdU), either because they segregate chromosomes asymmetrically or because they divide slowly. However, the purity of stem cells among BrdU-label-retaining cells has not been documented in any tissue, and the 'immortal strand hypothesis' has not been tested in a system with definitive stem cell markers. Here we tested these hypotheses in haematopoietic stem cells (HSCs), which can be highly purified using well characterized markers. We administered BrdU to newborn mice, mice treated with cyclophosphamide and granulocyte colony-stimulating factor, and normal adult mice for 4 to 10 days, followed by 70 days without BrdU. In each case, less than 6% of HSCs retained BrdU and less than 0.5% of all BrdU-retaining haematopoietic cells were HSCs, revealing that BrdU has poor specificity and poor sensitivity as an HSC marker. Sequential administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCs segregate their chromosomes randomly. Division of individual HSCs in culture revealed no asymmetric segregation of the label. Thus, HSCs cannot be identified on the basis of BrdU-label retention and do not retain older DNA strands during division, indicating that these are not general properties of stem cells.     

基于双侧TIC定量特征的乳腺肿瘤良恶性鉴别

基于DCE-MRI提出了一种利用双侧乳腺对称区域的TIC定量特征识别乳腺肿瘤良恶性的方法.使用三维区域生长算法提取乳腺的病灶区,基于病灶区及其对侧乳腺对应的ROI的TIC曲线分别提取29个特征,并定义双侧差异特征参数,经SFFS方法筛选后得到7个有效特征.使用SVM进行特征训练,基于交叉验证方法得到分类结果.本研究随机选取回顾性病例112例(良性67例,恶性45例),得到肿瘤良恶性平均分类准确率为88.39%.实验结果表明:此方法对乳腺肿瘤的良恶性鉴别有较高的准确率,对辅助医生进行乳腺病变组织的良恶性鉴别具有重要价值.     

Building of hFⅨ transgenic mice by spermatogenic cells

Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.     

Specific protection against breast cancers by cyclin D1 ablation.

Breast cancer is the most common malignancy among women. Most of these cancers overexpress cyclin D1, a component of the core cell-cycle machinery. We previously generated mice lacking cyclin D1 using gene targeting. Here we report that these cyclin D1-deficient mice are resistant to breast cancers induced by the neu and ras oncogenes. However, animals lacking cyclin D1 remain fully sensitive to other oncogenic pathways of the mammary epithelium, such as those driven by c-myc or Wnt-1. Our analyses revealed that, in mammary epithelial cells, the Neu-Ras pathway is connected to the cell-cycle machinery by cyclin D1, explaining the absolute dependency on cyclin D1 for malignant transformation in this tissue. Our results suggest that an anti-cyclin D1 therapy might be highly specific in treating human breast cancers with activated Neu-Ras pathways.     

小鼠非清髓性单倍体相合骨髓移植模型的建立

目的建立小鼠非清髓性单倍体相合骨髓移植模型,为研究移植前诱导免疫耐受或移植后输注供者细胞促进植入提供研究平台。方法以CB6F1雌性小鼠为受鼠,移植前1 d予450 cGy全身照射（TBI）后,随机分为2组,实验组移植0 d输注C57BL/6雄性小鼠骨髓有核细胞5×107/只,对照组不予移植。然后监测受鼠造血恢复、检测供鼠性别决定基因（SRY）判断植入情况,以及外周血供者细胞尤其CD3＋细胞嵌合状态,同时观察小鼠急性移植物抗宿主病（aGVHD）的发生情况。结果对照组小鼠均存活,仅表现轻度aGVHD,血象移植后30 d内基本恢复正常水平。实验组小鼠SRY基因在移植后＋14 d、＋30 d、＋60 d时检测PCR结果均阳性,供鼠外周血淋巴细胞、单核细胞、粒细胞嵌合率在移植后14 d分别为23.8%、36.9%%、19.4%;30 d分别为49.9%、53.2%、54.4%;60 d分别为67.6%、51.6%、56.9%,其中CD3＋细胞嵌合率分别为4.4%、21.2%、54.4%。结论 450 cGyTBI的非清髓性预处理方案,可以诱导受鼠免疫耐受、供者骨髓细胞植入,嵌合率处于中低水平混合嵌合状态。     

小鼠胚胎移植技术优化及对巨细胞病毒净化研究

目的 建立小鼠巨细胞病毒净化方法,以获得无MCMV感染的小鼠。方法 利用胚胎移植技术,通过使用不同激素、不同发情周期注射激素、受体鼠品系、不同的移植方法、不同时期胚胎移植,以及移植胚胎数量等对比试验,优化了净化条件。利用优化的胚胎移植净化方法,对供体鼠进行了净化。结果 SIGMA生产的激素,在10 IU剂量下超排得到的可用胚胎数量约为17枚/只;在小鼠发情间期超排得到的可用胚胎最多,超排的可用胚胎数约为23枚/只;选取C57雄性小鼠与ICR雌性小鼠交配的子一代作为受体鼠,产仔率达44.5%;输卵管移植较子宫移植效果好,产仔率为40.64%;移植2细胞胚胎的妊娠率为80%,明显优于单细胞和8细胞;受体鼠移植24枚胚胎时,产仔率达到了43.75%。利用优化的小鼠巨细胞病毒胚胎移植净化方法,净化得到了无MCMV感染的小鼠。结论 建立了小鼠巨细胞病毒净化方法,为获得无MCMV感染的小鼠种群提供了可靠的保障。     

Application of a nuclear localization signal gene in transgene mice

Efficient gene transfer by cytoplasm co-injection will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine α-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.     

Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice

Irradiated MHC-heterozygous mice often reject bone marrow cells transplanted from one of the homozygous parental strains, a phenomenon ('hybrid resistance') that appears to violate the laws of transplantation. Rejection of parental and allogeneic marrow cells also differs from conventional T cell-mediated rejection mechanisms as it is effected by NK1.1+ cells. To account for the unusual specificity of bone marrow rejection, it has been proposed that NK1.1+ cells destroy marrow cells that fail to express the full complement of self MHC class I (MHC-I) molecules. We show here that NK1.1+ cells in normal mice reject haemopoietic transplants from mice that are deficient for normal cell-surface MHC-I expression because of a targeted mutation in the beta 2-microglobulin gene. These findings demonstrate that deficient expression of MHC-I molecules renders marrow cells susceptible to rejection.     

Clonal deletion of V beta 14-bearing T cells in mice transgenic for mammary tumour virus.

Autoreactive T lymphocytes are clonally deleted during maturation in the thymus. Deletion of T cells expressing particular receptor V beta elements is controlled by poorly defined autosomal dominant genes. A gene has now been identified by expression of transgenes in mice which causes deletion of V beta 14+ T cells. The gene lies in the open reading frame of the long terminal repeat of the mouse mammary tumour virus.