奶牛乳腺上皮细胞的原代培养及其生物学特性分析

旨在从奶牛乳腺组织中分离原代乳腺上皮细胞（bovine mammary epithelial cells,BMECs）并传代培养后探究其生物学特性。本研究从屠宰场采集健康泌乳奶牛乳腺并采用改进的酶消化法从乳腺中分离得到原代奶牛乳腺上皮细胞,通过形态学观察、免疫荧光以及染色体核型分析的方法对其进行鉴定。同时,研究第3、第6和第9代乳腺上皮细胞的生长曲线、群体倍增时间和冻存复苏活力,检测不同代次细胞分泌乳蛋白、乳脂、乳糖的功能及泌乳相关基因的表达。结果表明,所分离的奶牛乳腺上皮细胞纯度较好,细胞生长呈现S型,3个代次细胞的群体倍增时间依次为34.87、41.45和65.04 h,冻存复苏活力为88%~93%;在细胞分泌功能方面,诱导培养2 d后均能检测到酪蛋白、甘油三酯和乳糖,且各代次间无显著差异;此外,3个代次的细胞诱导后均能表达乳成分合成相关基因。本研究成功培养了原代奶牛乳腺上皮细胞,并证明直到第9代细胞仍然具有正常的生物学功能,为体外探究乳腺细胞增殖与分化机制提供了良好的试验材料和技术支撑。     

奶牛原代乳腺上皮细胞的培养及β酪蛋白mRNA的表达

本试验旨在建立原代乳腺上皮细胞系的体外培养方法,并进行β酪蛋白mRNA的表达验证。取新鲜泌乳期的乳腺组织,采用组织块法培养纯化原代乳腺上皮细胞,利用显微镜观察细胞形态并进行细胞生长计数,采用实时荧光定量PCR技术检测β酪蛋白mRNA表达。结果显示,纯化培养的原代乳腺上皮细胞集聚成岛屿状生长,具有典型的铺路石和鹅卵石形状,细胞生长曲线呈"S"形,符合一般细胞的生长规律,并成功表达β酪蛋白mRNA。综上所述,本研究采用组织块法成功培养出具有正常生理功能的奶牛原代乳腺上皮细胞,为后续的乳腺上皮细胞功能研究提供了良好的细胞试验模型。     

奶牛原代乳腺上皮细胞的培养及β酪蛋白mRNA的表达

本试验旨在建立原代乳腺上皮细胞系的体外培养方法,并进行β酪蛋白mRNA的表达验证。取新鲜泌乳期的乳腺组织,采用组织块法培养纯化原代乳腺上皮细胞,利用显微镜观察细胞形态并进行细胞生长计数,采用实时荧光定量PCR技术检测β酪蛋白mRNA表达。结果显示,纯化培养的原代乳腺上皮细胞集聚成岛屿状生长,具有典型的铺路石和鹅卵石形状,细胞生长曲线呈"S"形,符合一般细胞的生长规律,并成功表达β酪蛋白mRNA。综上所述,本研究采用组织块法成功培养出具有正常生理功能的奶牛原代乳腺上皮细胞,为后续的乳腺上皮细胞功能研究提供了良好的细胞试验模型。     

奶牛乳腺上皮细胞的体外分离培养及超微结构鉴定

通过组织贴块法取部分奶牛乳腺组织进行培养,并通过在培养基中添加适当浓度的营养因子以及控制消化时间将成纤维细胞去除,纯化出原代奶牛乳腺上皮细胞。通过免疫荧光鉴定,形态学观察,扫描电镜和透射电镜检测原代乳腺上皮细胞特性。观察结果显示,本试验分离培养的牛乳腺上皮细胞角蛋白18免疫荧光染色鉴定结果呈阳性。形态学观察及超微结构观察显示,试验所分离的上皮细胞呈鹅卵石样单层聚集,胞内有丰富的线粒体和内质网,细胞状态良好,传至15代以上细胞依然增殖旺盛,因此可以作为研究奶牛乳腺机能的重要工具。     

奶牛乳腺体外培养模型应用研究进展

奶牛乳腺上皮细胞(BMEC)具有分泌乳汁的特殊功能,体外培养的奶牛乳腺细胞是研究乳成分合成调控和乳腺生理代谢的良好模型。近年来,奶牛乳腺体外培养模型受到越来越多的关注,其应用也更广泛。本文主要从奶牛乳腺体外培养模型在乳成分合成调控和乳腺生理代谢机制领域的应用两个方面进行简要综述。     

荷斯坦奶牛乳腺上皮细胞的体外培养

用外科手术的方法采取健康的泌乳期荷斯坦奶牛乳腺组织,采用机械剪切结合酶消化的方法,在体外进行原代乳腺上皮细胞分离培养。结果成功地培养出荷斯坦奶牛原代乳腺上皮细胞。显微镜下观察,细胞形成单层呈鹅卵石样;角蛋白免疫组化染色后,呈现阳性反应。经数次传代后,细胞增殖依旧旺盛。     

奶牛乳腺上皮细胞体外培养研究进展

体外培养的奶牛乳腺上皮细胞(BMECs)常被用于研究乳成分的合成调控以及乳腺的生理代谢。近年来,随着越来越多永生化奶牛乳腺上皮细胞系的建立,其应用也更为广泛。本文综述了奶牛乳腺细胞系的种类和奶牛乳腺细胞体外培养的方法,为研究乳腺上皮细胞的功能及泌乳机制提供参考。     

牛乳腺上皮细胞培养中泌乳关键激素的调控机制

乳腺的泌乳过程复杂多变,影响因素众多,加之动物体内环境的复杂性,导致乳腺泌乳的研究受到一定的制约,如何建立可长期存活且具有泌乳功能的乳腺上皮细胞是研究的基础。内分泌系统在乳腺的生长发育、分化及泌乳过程中起着关键的作用。许多激素、生长因子、受体、胞内信号转导的中间体及核内转录因子的相互作用最终会影响乳腺的发育、成熟和泌乳。原代乳腺上皮细胞的生理状态最接近于体内乳腺组织。本文就牛原代乳腺上皮细胞的培养及培养中刺激乳腺细胞泌乳的关键激素和生长因子的作用机制进行分析,以阐明激素在乳腺上皮细胞离体培养中的泌乳调控机制。     

奶牛乳腺上皮细胞系的培养与鉴定

本试验旨在培养功能性奶牛乳腺上皮细胞系,为奶牛泌乳调控和奶牛乳房炎发病机制研究提供功能性的细胞模型。采用组织块细胞培养法来分离纯化并鉴定奶牛乳腺上皮细胞;采用有限稀释法单克隆奶牛乳腺上皮细胞;采用噻唑蓝(MTT)法来分析奶牛乳腺上皮细胞的生长曲线是否为正常的"S"形;观察细胞角蛋白18免疫荧光来证明所培养的细胞为上皮型;选择培养至10和20代的奶牛乳腺上皮细胞进行染色体核型分析。结果表明:1)利用组织块细胞分离法能够成功获得奶牛乳腺上皮细胞并传至20代。2)培养5~6 d成纤维细胞迅速增殖且周围分裂出少量的上皮细胞。培养8 d奶牛乳腺上皮细胞迅速增殖,形成岛屿状集落,呈单层"鹅卵石"和"铺路石"形态生长。3)奶牛乳腺上皮细胞角蛋白18鉴定为阳性。4)培养至10和20代的奶牛乳腺上皮细胞染色体数为60条,具有正常的细胞二倍体核型。综上所述,采用组织块培养细胞能够获得具有稳定性、功能性的奶牛乳腺上皮细胞,但不是永生化细胞。     

不同方法分离奶牛乳腺上皮细胞体外培养的研究

从荷斯坦奶牛乳房无菌采取乳腺组织,通过不同的方法分离、培养、纯化牛乳腺上皮细胞,研究乳腺上皮细胞的体外培养效果.结果表明:组织块接种、0.25%的胰酶和100 u/mL透明质酸酶的混合液37℃消化组织块3 h.可以得到大量细胞用于原代培养.在以DMEM/F12为基础培养液,在培养液中添加10%的胎牛血清、100μg/mL的双抗、表皮生长因子、胰岛素、转铁蛋白、硒酸钠等,组成的完全培养液中奶牛乳腺上皮细胞生长良好.上皮细胞显微结构显示:奶牛乳腺上皮细胞在体外培养过程中舍有不同的细胞类型,大多数上皮细胞呈多角形,细胞之间连接成片,细胞界限明显,部分细胞界限不明显.     

Research Progress on Primary Culture,Purification and Identification Technique of Bovine Mammary Epithelial Cells

The bovine mammary epithelial cells not only have the function of synthesis and secretion of milk, but also play an important role in innate immunity system of the mammary gland, and there is great significance of them on studying the mechanism of lactation, mastitis pathogenesis and drug screening.Primary cultured bovine mammary gland epithelial cells are suitable for setting up cell model which can be used as the dielectric for physiological, pathological and pharmacological researching, avoiding the difficulties of in vivo test, such as the long cycle, the high cost, the individual difference, etc.The author summarized the latest researches of cell primary culture in vitro, cultivation technology, purification and identification method in order to provide reference for the studies of bovine mammary epithelial cells culture.     

金黄色葡萄球菌小菌落突变株体外感染乳腺上皮细胞的研究

为研究金黄色葡萄球菌小菌落突变株(S.aureus small colony variants,SASCVs)与正常株对原代培养的乳腺上皮细胞的侵袭情况,并观察对比其对细胞结构的损坏程度和对乳腺上皮细胞的影响,本试验对由云南农业大学动物医学实验教学中心实验室分离的一株SASCV进行体外感染小鼠乳腺上皮细胞试验,将分离出的一株SASCV及质控菌株ATCC 25923分别注入实验室体外培养的小鼠乳腺上皮细胞内,采用普通光镜和扫描电子显微镜观察质控菌株ATCC 25923及SASCVs诱导乳腺上皮细胞凋亡的细胞形态学变化;用流式细胞仪(Annexin V-FITC/PI双染法)定量检测质控菌株ATCC 25923及SASCVs感染乳腺上皮细胞凋亡的诱导作用,观察细胞的变化。结果显示,质控菌株ATCC 25923及SASCVs感染小鼠乳腺上皮细胞3 h后,可诱导乳腺上皮细胞发生凋亡,显示为细胞核皱缩,染色质边缘化和细胞浆内空泡增多等典型的凋亡特征。Annexin V-FITC/PI双染法检测后发现,与对照组相比,感染组细胞凋亡率明显升高,并且阳性对照组的细胞凋亡率明显高于试验组。结果表明,SASCVs在侵染细胞的过程中达到一定数量不再上升;SASCVs的凋亡率明显的较正常金黄色葡萄球菌低。因此,SASCVs也可诱导乳腺上皮细胞凋亡,具有细胞凋亡的典型形态特征。本试验结果为以后预防和控制SASCVs感染奶牛慢性乳房炎提供试验依据。     

Study on Infection of Staphylococcus aureus Small Colony Variants on Mammary Epithelial Cells in Vitro

In order to study the situation of S. aureus small colony variants (SASCVs) and the normal strains hitting the mammary epithelial cells of primary culture, we observed and compared the degree of damage to the cellular structure and the effects on mammary epithelial cells. This test was carried out with a strain of SASCV which was separated by Yunnan Agricultural University Animal Medical Experimental Teaching Center Laboratory via infecting mice mammary epithelial cells in vitro. We put the SASCVs and quality control strains ATCC 25923 respectively into the mammary epithelial cells cultivated in vitro in the laboratory, and observed the morphological change of mammary epithelial cell apoptosis induced by quality control strains ATCC 25923 and SASCVs using ordinary light microscope and scanning electron microscope; At the same time, we also observed the mammary gland epithelial cells apoptosis induced by quality control strains ATCC 25923 and SASCVs using flow cytometry (Annexin V-FITC/PI double staining). The results showed that 3 h after quality control strains and small colony mutant strains infecting the mammary epithelial cells, the mammary epithelial cells could be induced to apoptosis. It showed some typical characteristics of apoptosis such as the nucleus shrivelled, chromatin marginalized, mammary gland epithelial cells could be induced apoptosis, expression was shrinking nucleus, chromatin marginalized, the increase of cell intracytoplasmic vacuoles and so on. After testing using the Annexin V-FITC/PI double staining, we found that compared with the control group, the apoptosis rate of infection group was obviously higher, and the apoptosis rate of the positive control group was obviously higher than that of the experimental group. The results showed that in the process of infecting cells, the SASCVs reached a certain number and then stopped; The apoptosis rate of SASCVs was obviously lower than the normal of S.aureus. Therefore, SASCVs could also induce mammary epithelial cells apoptosis. It had the typical morphological characteristics of apoptosis. It provided experimental basis for the prevention and control of SASCVs chronic mastitis cows.     

原代奶牛子宫内膜上皮细胞体外培养方法的改进

目前利用奶牛子宫内膜上皮细胞体外培养技术，建立奶牛子宫内膜炎模型从而减少不可控因素来研究奶牛子宫内膜炎疾病已成为国内外普遍使用的一类方法，因此获得高纯度、同一性的子宫内膜上皮细胞是体外研究的关键环节，国内外所使用方法主要是酶消化法、组织块贴壁法和组织块消化贴壁法。本文旨对先前的方法进行改良，建立一种简便易行、培养周期短、细胞活性及形态良好的原代奶牛子宫内膜上皮细胞体外培养技术。采用健康未孕奶牛子宫为试验材料，取下子宫内膜组织，加入5 mL含2%双抗、40%胰酶的DMEM/F12培养基，4 ℃消化12 h，再将组织移至25 cm²细胞培养瓶中贴壁培养。获得原代细胞后，应用角蛋白-18抗体对细胞进行免疫组化荧光鉴定，并对第3代细胞采用CCK-8法测定不同时间点的D_{450 nm}值，绘制细胞生长曲线。结果显示，培养至第8天，原代细胞基本铺满细胞培养瓶瓶底，有效地缩短了常规组织块贴壁法的周期。通过角蛋白-18免疫组化荧光鉴定，原代上皮细胞的阳性率可达98%以上，相比常规组织块贴壁法来说，纯度明显提高，省去了细胞纯化的操作步骤。细胞增殖状态，符合正常的分裂生长特性。试验结果表明将常规组织块贴壁法进行了优化改良，不仅缩短了培养周期，同时提高纯度，保持细胞活性，为原代奶牛子宫内膜上皮细胞体外培养技术的改良提供一定的参考。     

荷斯坦奶牛乳腺上皮细胞的分离培养

目的：建立荷斯坦奶牛乳腺上皮细胞的分离培养方法。方法：采用组织块种植法培养奶牛乳腺上皮细胞,利用胰蛋白酶差时消化法分离、纯化上皮细胞。结果：成功培养出奶牛乳腺上皮细胞,显微镜下观察,纯化的乳腺上皮细胞呈典型上皮细胞形态,细胞之间排列紧密,呈鹅卵石铺路样,形态均一,多角形的单层聚集。通过荧光免疫细胞染色方法对细胞骨架蛋白-角蛋白18进行鉴定,呈现阳性反应。乳腺上皮细胞增殖旺盛,经25次以上传代后长势仍然良好。结论：采用组织块种植法结合胰酶差时消化法成功获得纯化的奶牛乳腺上皮细胞。     

敲低SQLE基因对奶牛乳腺上皮细胞增殖及凋亡的影响

为了探讨角鲨烯环氧酶（squalene epoxidase,SQLE）基因对体外培养奶牛乳腺细胞凋亡及增殖的影响,本研究用RNAi技术敲低奶牛乳腺上皮细胞中SQLE基因;用实时荧光定量PCR方法检测奶牛乳腺上皮细胞中SQLE基因及凋亡和周期相关基因的表达;用CCK-8法检测其对细胞增殖的影响;用周期和凋亡检测试剂盒筛选细胞,用流式细胞术准确计数处于不同周期的细胞数和凋亡细胞数。结果显示,奶牛乳腺上皮细胞转染siRNA后,SQLE基因相对表达量显著下降（P<0.05）,细胞增殖受到极显著抑制（P<0.01）;G1期细胞数量显著下降（P<0.05）,G2期细胞数量没有显著性改变,S期细胞数量显著上升（P<0.05）;肿瘤坏死因子超家族成员6（又称Fas或CD5）的相对表达量显著上升（P<0.05）,细胞周期蛋白依赖性激酶抑制剂1B（cyclin dependent kinase inhibitor 1B,P27）相对表达量显著上升（P<0.05）,而细胞周期素D1（Cyclin D1）、B淋巴细胞瘤因子2（B-cell lymphoma-2,Bcl-2）、细胞周期蛋白依赖性激酶抑制剂1A（cyclin dependent kinase inhibitor 1A,P21）、Bcl-2相关X蛋白（Bcl-2 associated X,apoptosis regulator,Bax）显著下降（P<0.05）。综上所述,敲低SQLE基因通过调节相关基因的表达抑制乳腺上皮细胞的增殖。     

Culture of Piglet Intestinal Epithelial Cells in vitro and the Infectivity of Porcine Epidemic Diarrhea Virus on the Cells

The study was aimed to establish a simple in vitro culture system for piglet small intestinal epithelial cells,and to provide materials for researches on porcine epidemic diarrhea virus (PEDV).In this study,newborn piglets that did not eat colostrum were used as the initial donors,and the primary cells were separated in vitro by scraping the intestinal mucosa from the intestinal lumen and mechanical separation and dispersion.The piglet intestinal epithelial cells were purified using 0.1% trypsin differential digestion method.Compared the effects of newborn weak piglets and normal piglets as donors on the activity of primary intestinal epithelial cells.MTT method was used to compare the proliferation activity of primary cells of different generations.Immunofluorescence and Real-time RT-PCR were used to detect the infection and proliferation of PEDV strain CV777 in primary intestinal epithelial cells.The results showed that the isolation and culture method in vitro used in this study could obtain primary intestinal epithelial cells with good proliferation activity,with obvious S-type cell proliferation curves.The cells with high purity and single morphology could be obtained by differential digestion,and had good proliferation activity after five consecutive passages.The proliferative activity of intestinal epithelial cells isolated from weak piglets and normal piglets had no obvious difference,and provided new way for reducing the cost of primary cell culture.The results of immunofluorescence and Real-time RT-PCR showed that PEDV could infect the primary intestinal epithelial cells,and replicated and proliferated in them.In this study,we established a simple,practical and low-cost method for in vitro culture of piglet primary small intestinal epithelial cells.The primary cells cultured by this method could be used as basic materials for the isolation and culture of PEDV and related research.     

仔猪小肠上皮细胞的体外培养及猪流行性腹泻病毒对其感染性研究

本研究旨在建立一种操作简单的仔猪小肠上皮细胞体外培养体系，为猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）的相关研究提供材料。研究以未吃初乳的新生仔猪作为肠道供体，采用肠腔面刮取肠黏膜和机械分离分散的方式进行原代细胞的体外分离。采用0.1%胰蛋白酶差速消化法进行仔猪小肠上皮细胞的纯化；比较新生弱仔猪和正常仔猪作为供体对原代小肠上皮细胞活性的影响；MTT法比较不同代次原代细胞的增殖活性；免疫荧光和实时荧光定量RT-PCR方法检测PEDV毒株CV777在原代小肠上皮细胞感染和增殖情况。结果显示，本研究建立的体外分离培养方法能获得增殖活性良好的原代小肠上皮细胞，具有明显的"S"型细胞增殖曲线。通过胰酶差速消化可得到纯度高、形态单一的小肠上皮细胞，同时细胞连续传代5次仍保持良好的增殖活性。弱仔猪和正常仔猪分离培养的小肠上皮细胞的增殖活性比较显示，两者并没有明显区别，这为降低原代细胞培养的成本提供新的思路。免疫荧光和实时荧光定量RT-PCR结果显示，PEDV可感染本方法分离培养的仔猪原代小肠上皮细胞，并在其中进行复制增殖。本研究建立了一种操作简单、实用性强、成本较低的仔猪原代小肠上皮细胞体外培养方法，该方法培养的原代细胞可作为PEDV分离培养和相关研究的基础材料。     

Role of extracellular matrix and prolactin in functional differentiation of bovine BME-UV1 mammary epithelial cells

Interactions between extracellular matrix (ECM) and epithelial cells are necessary for proper organisation and function of the epithelium. In the present study we show that bovine mammary epithelial cell line BME-UV1 cultured on ECM components, commercially available as Matrigel, constitutes a good model for studying mechanisms controlling functional differentiation of the bovine mammary gland. In contact with Matrigel BME-UV1 cells induce apicobasal polarity, and within 16 days form three dimensional (3D) acinar structures with a centrally localized hollow lumen, which structurally resemble mammary alveoli present in the functionally active mammary gland. We have shown that the 3D culture system enables a high expression and proper localisation of integrin receptors and tight junction proteins in BME-UV1 cells to be induced. This effect was not obtained in cells grown in the classical 2D culture system on plastic. Moreover, ECM highly stimulated the synthesis of one of the major milk proteins, beta-casein, even in the absence of prolactin. Our results show that contact with ECM plays an important role in the lactogenic activity of bovine MECs, however, prolactin is necessary for the efficient secretion of milk proteins.