CD44 is highly expressed on milk neutrophils in bovine mastitis and plays a role in their adhesion to matrix and mammary epithelium

Mastitis, inflammation of the mammary gland, is a common and economically important disease in dairy animals. Mammary pathogenic organisms, such as Escherichia coli, invade the teat canal,milk ducts, and mammary alveolar space, replicate in mammary secretions, and elicit a local inflammatory response characterized by massive recruitment of blood polymorphonuclear neutrophil leukocytes (PMN) into the alveoli and milk ducts. CD44 is a trans-membrane glycoprotein previously shown to play a role in mediation and control of blood PMN recruitment in response to inflammatory signals. Here we show, for the first time, increased expression of CD44 on recruited milk PMN in bovine mastitis and the expression of a CD44 variant, CD44v10, on these PMN. Furthermore, we demonstrate that CD44 mediates specific adhesion of bovine blood PMN to hyaluronic acid and mammary epithelial cells. Our results suggest that in mastitis CD44 plays a role in recruiting blood PMN into the mammary glands, the exact nature of this role needs to be elucidated.     

The bovine neutrophil: Structure and function in blood and milk

Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens. Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection. While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis. In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland. This will result in permanent scarring and reduced numbers of milk secretory cells. The life span of PMN is limited by the onset of apoptosis. To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis. Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue. The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria. One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14. Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-alpha and sepsis. Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection. Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants. Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis.     

Expression of potential lymphocyte trafficking mediator molecules in the mammary gland

The mammary gland performs a variety of immunological functions, including protecting itself from mastitis and protecting neonates from infectious agents. Several molecules that mediate lymphocyte trafficking in the immune system are also expressed in the mammary gland. This review is focused on the immunological function of these molecules, especially glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the mammary gland. GlyCAM-1 is expressed in the lactating mouse mammary gland. Endothelial cells produce this protein and secrete it into milk. The glycosylated modification of mammary gland GlyCAM-1 is different from that of the lymph nodes, and lacks the binding ability for L-selectin on lymphocytes. GlyCAM-1 in the mammary gland is not involved in lymphocyte migration, and probably has another function besides that of the lymph nodes. MAdCAM-1 is expressed on endothelial cells of small venules around mouse mammary lobules during lactation. This molecule has the ability to interact with alpha4beta7 integrin on lymphocytes and mediates lymphocyte recruitment to the mammary gland. The density of beta7+/CD3+ T-cells is correlated with the density of the MAdCAM-1-stained area, suggesting that MAdCAM-1 may mediate the migration of these cells. In contrast, there is no relationship between MAdCAM-1 expression and the number of beta7+/c-IgA+ B-cells, implying that some other factor is involved in lymphocyte migration to the mammary gland. Chemokines, such as IL-8, GRO-alpha, MCP-1, RANTES and MEC, have been detected in human and mouse mammary glands. Although little information is available, these molecules may contribute to lymphocyte migration to the mammary gland.     

Variations among unbred heifers in the activities of polymorphonuclear leucocytes from the mammary gland and blood

The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes NMN) obtained by either lavage of the cavity system of juvenile mammary glands stimulated with a synthetic muramyl dipeptide analogue or isolation from the peripheral blood. Attention was paid to the variability of characteristics and its sources, and to correlations among them. The following characteristics were investigated in 27 clinically healthy, unbred Bohemian Red Pied x Holstein heifers: migration activity in situ, number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and unstimulated and zymosan-stimulated luminol-dependent chemiluminescence. Considerable individual variation was found in the characteristics. Significant differences between blood PMN and PMN from lavages after influx induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A significant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r = 0.329; P < 0.01). Highly significant positive correlations (P < 0.01) were demonstrated between the number of phagocytosing PMN [a], phagocytotic index [b], and bactericidal activity [c] in both blood PMN (r(ab) = 0.602; r(ac) = 0.565; r(bc) = 0.529) and mammary gland PMN (r(ab) = 0.730, r(ac) = 0.618, r(bc) = 0.589). No significant correlation was demonstrated for non-stimulated (NS), zymosan-stimulated (ZS), or opsonized zymosan-stimulated (OZS) chemiluminescence with any of the other characteristics of phagocytotic activity, in either blood PMN or mammary gland PMN (P > 0.05). The animal was a highly significant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-significant source of variability for all the characteristics of phagocytotic activity except for NS chemiluminescence (P < 0.05) and ZS or OZS chemiluminescence (P < 0.01). However, udder quarter was a non-significant source of variability of chemiluminescence indices ZS/NS and OZS/NS (P > 0.05). It has been demonstrated that in situ migration activity, the number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and chemiluminescence indices of PMN collected from juvenile mammary glands of unbred heifers after influx induction can be regarded as candidate early markers of resistance to mammary infections.     

Effect of Intramammary Infusion of Tumour Necrosis Factor‐α on Milk Protein Composition and Induction of Acute‐Phase Protein in the Lactating Cow

The present study was designed to evaluate the effects of tumour necrosis factor‐α (TNF‐α) on lactating bovine mammary functions such as milk protein secretion and the integrity of the milk‐blood barrier. The effect on the induction of the systemic inflammatory response was also examined using concentrations of serum haptoglobin (Hp), a major inflammatory acute‐phase protein, as an index. One hundred micrograms per mammary gland of recombinant bovine (rBo) TNF‐α or placebo saline was individually infused into a rear mammary gland of each of four lactating cows, and milk and blood samples were collected before and 4, 8, 24, 32, 48, 96 and 168 h after infusion. In the rBoTNF‐α‐infused gland, increases of somatic cell counts were observed at 4–48 h. Although concentrations of total milk protein were not changed, compositions of milk proteins varied following rBoTNF‐α infusion. Concentrations of caseins, α‐lactalbumin and β‐lactoglobulin were significantly decreased at 4 and 8 h. Lactoferrin concentrations were significantly increased at 4 h. Significant infiltrations of serum albumin, immunoglobulin G1 (IgG1) and IgG2 were observed at 4 and 8 h. Elevations of the serum concentration of Hp were detected at 8‐32 h, but were very small in comparison with those reported in inflammatory diseases. Changes in rectal temperature and white blood cell counts were not significant. These results show that single rBoTNF‐α infusion into the lactating mammary gland suppresses the lactogenic function of the gland and influences the function of the milk‐blood barrier, with little effect on the generalized inflammatory response.     

High milk neutrophil chemiluminescence limits the severity of bovine coliform mastitis

Polymorphonuclear neutrophil (PMN) function changes during mastitis. To investigate the contribution of milk PMN to the severity of Escherichia coli (E. coli) mastitis, chemiluminescence (CL) of blood and milk PMN and their efficiency to destroy coliform bacteria in the mammary gland were examined following the induction of E. coli mastitis in early lactating cows. To better assess and define the degree of mastitis severity, cows were classified as moderate and severe responders according to milk production loss in the non-infected quarters at post-infection hour (PIH) 48. There was an inverse relationship between pre-infection milk PMN CL and colony-forming units at PIH 6. In moderate cows, the pre-infection blood and milk PMN CL was approximately 2-fold higher than that of severe cows. The probability of severe response increased with decreasing pre-infection PMN CL. At the beginning of the infection blood and milk PMN CL was consistently higher, and milk PMN CL increased faster after infection in moderate cows. At PIH > 48 milk PMN CL in severe cows exceeded that of moderate cows. The somatic cell count (SCC) in moderate cows increased faster than colony-forming units, whereas in severe cows the results were reversed. The kinetics of CL activity for blood and milk PMN before and during the early phase of infection confirmed an impairment in PMN CL activity for severe responding cows. High pre-infection blood and milk PMN CL and the immediate increase of milk PMN CL and SCC after infection limited bacterial growth thereby facilitating the recovery of E. coli mastitis in moderate cows. Our study strengthens the idea that pre-existing milk PMN (a static part of the udder's immune defense) functions as a "cellular antibiotic" before and during infection, and low milk PMN CL is a risk factor for bovine coliform mastitis.     

Chemotactic factors for bovine neutrophils in relation to mastitis

The chemotactic effect of a variety of agents for bovine blood polymorphonuclear neutrophils (PMN) was evaluated in vitro in assays of migration under agarose. Activated serum and leukotriene B4 showed significant chemotactic activity whereas various bacterial products, formyl peptides, casein and platelet activating factor failed to attract bovine PMN. In vitro cultures of bovine mammary gland macrophages released chemotactic activity for homologous PMN when stimulated by addition of opsonised, killed Staphylococcus aureus, Escherichia coli or Zymosan or sterile E. coli culture filtrates or endotoxin. No significant activity was produced by unstimulated macrophages. Pharmacological levels of various inhibitors or arachidonic acid oxygenation had no significant effect on the generation of PMN chemoattractants by mammary macrophages.     

牛乳腺上皮细胞体外分离培养模式、方法及其应用进展

乳腺由具有泌乳功能的腺泡组成,乳腺上皮细胞(MEC)以单层方式排列在腺泡外围,是乳腺对外界病原进行免疫保护的重要组分,负责将血液中的营养物质通过一系列复杂生化过程转化为乳汁.牛乳腺上皮细胞(BMECs)的体外分离培养在很大程度上解决了活体试验条件不可控、操作困难、成本高及个体差异大等诸多问题,还可以为体外研究乳腺组织生...     

奶牛乳腺免疫细胞防御机理研究进展

奶牛乳腺中的初级吞噬细胞、嗜中性粒细胞(PMN)和巨噬细胞构成了抵御病原体入侵的第1道防线。在健康奶牛的乳腺中,巨噬细胞占主导地位且充当哨兵的角色。当病原体侵入乳腺时,巨噬细胞及乳腺上皮细胞就会释放出直接将PMN迁移到该区域的趋化剂,使PMN从循环中迅速流入并吞噬和杀灭细菌,起到保护的作用。抵御病原体入侵的第2道防线是由记忆细胞和免疫球蛋白组成的网络,它们与第1道防线相互作用。随着分子生物学技术的发展,更好地了解炎症反应的调节机制可为研究和调节宿主与病原体的相互作用提供理论基础,因此本文就奶牛乳腺免疫细胞防御机理的研究进展进行了综述。     

Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46⁺/CD3⁻) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2 h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2 h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis.     

乳腺对氨基酸的摄取及其调控机制研究进展

乳蛋白是乳中重要的营养成分之一,超过90%的乳蛋白是乳腺利用从血液中摄取的氨基酸从头合成,因此在保证氨基酸充足供给的前提下,乳腺对氨基酸摄取率的高低是影响乳蛋白产量的关键因素。血液中的氨基酸不能自由扩散进出乳腺,需要由乳腺上皮细胞膜上特异的氨基酸转运载体(AAT)协助完成。而乳腺AAT活性受到营养物质和激素水平的调节,当乳腺感知到营养物质和激素水平变化的信号,能够通过激活或抑制以哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)和一般性调控阻遏蛋白激酶2(GCN2)为核心的2条信号通路的活性,进而影响AAT活性,调节乳腺对氨基酸的摄取。本文主要从乳腺AAT的分类和功能、影响乳腺摄取氨基酸的主要因素以及调控乳腺氨基酸摄取的信号通路机制3个方面作一综述,旨在从氨基酸摄取的角度为提高乳蛋白的合成提供参考。     

Immune cell differentiation in mammary gland tissues and milk of cows chronically infected with Staphylococcus aureus

This study identifies and compares the distribution of mononuclear cells in the mammary gland tissues and milk of healthy and chronically infected with Staphylococcus aureus cows. Somatic cell counts (SCCs) during the 3 months before the study were > 1 x 10(6) cell/ml in the infected quarters and < 1 x 10(5) cell/ml in the infection-free quarters. Immediately after slaughter, samples from the tissues above the gland cistern and supra-mammary lymph node were collected. No histological differences were found between the supra-mammary lymph nodes of the healthy and infected udders, and both appeared normal. In the milk of the healthy infection-free mammary glands, SCC was < 50,000 cells/ml) while epithelial cells were the predominant type. The percentage of CD18+ was low than 45%, of which over three-quarters were polymorphonuclear (PMN), and less than one- quarter were mononuclear cells. The later comprised CD4+ or CD8+ T-lymphocytes, macrophages (Mo) but not B-cells. In the tissues, there were few CD18+ leukocytes, and most of the cells were T-lymphocytes. The number of B-lymphocytes bearing CD21+ was similar to that of CD8+ and were localized in the connective tissue as clusters of 2-5 cells, mainly in areas with no alveoli, or as single cell having a dendritic like form. The number of Mos was negligible. In the milk of the infected glands, SCC exceeded 700,000 cells/ml, of which > 95% were CD18+ positive. The distribution of the leukocytes had two patterns: one presented (> 80%) of PMN cells and a small number of mononuclear cells; the second had less than 50% PMN and many mononuclear cells. The CD8+ cells in these infected sections were observed throughout the mammary epithelial cells (MEc) around the alveoli and in the alveolar lumen (AL). The numbers and the location of CD21+ B-lymphocytes were similar to those in the infection-free mammary glands. The number of CD5+ positive cells was lower than T and B- cells combined and were located throughout the mammary epithelial cells, around the alveoli and within the connective tissue. Mo numbers were high in most of those infected quarters, and were localized around the connective tissue and within the AL.     

Study on Expression of SYK in Dairy Cow Mammary Gland

To investigate the relationship between the expression of SYK and dairy cow mammary gland development and lactation, the expression of SYK in lactating dairy cow mammary gland with high or low quality milk and dry period Holstein dairy cow mammary gland was detected by Western blotting and laser confocal microscope.The results showed that SYK expression in dry period mammary gland was significant higher than that in lactating mammary gland (P<0.05).There was no SYK differential expression detected between lactating mammary gland with high quality milk and low quality milk (P>0.05).SYK was mainly located in the cytoplasm of ductal epithelial cells in dry period mammary gland.In lactating mammary gland, SYK was existed in acinar epithelial cells.All these results revealed that SYK was a regulator in mammary epithelial cell proliferation and differentiation.It participated in mammary gland reconstitution in dry period.     

脾源性酪氨酸激酶基因在奶牛乳腺中的表达研究

为探讨脾源性酪氨酸激酶(spleen tyrosine kinase,SYK)的表达与奶牛乳腺发育和泌乳功能之间的关系,试验采用Western blotting和激光共聚焦显微技术对泌乳期高乳品质、低乳品质及干乳期的中国荷斯坦奶牛乳腺组织中SYK的表达含量和表达部位的变化进行研究。结果表明,干乳期奶牛乳腺组织中SYK的表达显著高于泌乳期奶牛乳腺组织(P<0.05),泌乳期高乳品质、低乳品质奶牛乳腺组织中SYK的表达差异不显著(P>0.05);在干乳期SYK主要在乳腺导管上皮细胞的胞质中表达,而在泌乳期SYK在腺泡上皮细胞中表达。结果提示SYK是乳腺上皮细胞增殖与分化的调节因子,主要参与干乳期乳腺组织的重建过程。     

乳腺分泌物中体细胞的研究进展

不同物种的乳腺分泌物中含有的细胞成分被称为体细胞,其中包括淋巴细胞、白细胞、巨噬细胞和上皮细胞。物种、乳腺感染情况、不同生理阶段和饲养管理条件等因素均会影响乳中的体细胞数量和细胞类型。近年来,乳中体细胞得到了人们的关注和深入研究,显示出广阔的应用前景。人们利用从初乳和常乳中得到的乳腺上皮细胞已经成功进行了乳腺细胞的原代培养和建立了乳腺细胞系,为乳生成、被动免疫转移和乳腺癌的研究提供了良好平台。体细胞中提取的RNA代表了乳腺组织的基因表达,因此为研究乳腺组织的基因表达提供了方便、良好的来源。     

Effect of intramammary infusion of tumour necrosis factor-alpha on milk protein composition and induction of acute-phase protein in the lactating cow

The present study was designed to evaluate the effects of tumour necrosis factor-alpha (TNF-alpha) on lactating bovine mammary functions such as milk protein secretion and the integrity of the milk-blood barrier. The effect on the induction of the systemic inflammatory response was also examined using concentrations of serum haptoglobin (Hp), a major inflammatory acute-phase protein, as an index. One hundred micrograms per mammary gland of recombinant bovine (rBo) TNF-alpha or placebo saline was individually infused into a rear mammary gland of each of four lactating cows, and milk and blood samples were collected before and 4, 8, 24, 32, 48, 96 and 168 h after infusion. In the rBoTNF-alpha-infused gland, increases of somatic cell counts were observed at 4-48 h. Although concentrations of total milk protein were not changed, compositions of milk proteins varied following rBoTNF-alpha infusion. Concentrations of caseins, alpha-lactalbumin and beta-lactoglobulin were significantly decreased at 4 and 8 h. Lactoferrin concentrations were significantly increased at 4 h. Significant infiltrations of serum albumin, immunoglobulin G1 (IgG1) and IgG2 were observed at 4 and 8 h. Elevations of the serum concentration of Hp were detected at 8-32 h, but were very small in comparison with those reported in inflammatory diseases. Changes in rectal temperature and white blood cell counts were not significant. These results show that single rBoTNF-alpha infusion into the lactating mammary gland suppresses the lactogenic function of the gland and influences the function of the milk-blood barrier, with little effect on the generalized inflammatory response.     

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.     

Factors involved in the early pathogenesis of bovine Staphylococcus aureus mastitis with emphasis on bacterial adhesion and invasion. A review

Staphylococcus aureus is the most important and prevalent contagious mammary pathogen; it causes clinical and subclinical intramammary infection with serious economic loss and herd management problems in dairy cows. In vitro studies have shown that Staphylococcus aureus adheres to mammary epithelial cells and extracellular matrix components and invades into mammary epithelial as well as other mammary cells. Staphylococcus aureus strains from intramammary infection produce several cell surface-associated and extracellular secretory products. The exact pathogenic roles of most of the products and their effects on adhesion and invasion are not well evaluated. It is also known that mammary epithelial cell-associated molecules and extracellular matrix components interact with S. aureus during the pathogenesis of mastitis, but their roles on adhesion and invasion have not been characterized. The adhesion of S. aureus to epithelial cells may involve non-specific physicochemical interactions and/or specific interactions between bacterial cell-associated ligands and host cell surface receptors. In vitro adhesion depends on the S. aureus strain, the growth phase of the bacteria, the growth medium and the origin of the epithelial cells. Adhesion is hypothesized to be a prerequisite and crucial early step for mammary gland infection. Staphylococcus aureus invades mammary epithelial cells. It also invades other cells such as endothelial cells and fibroblasts. Bacteria are found enclosed in membrane bound vacuoles in the cytoplasm of mammary epithelial cells. Recent observations indicate that S. aureus escapes from the phagosome into the cytoplasm and induces apoptosis. The invasion into mammary epithelial cells may occur through an endocytic process that requires involvement of elements of the cytoskeleton or by direct binding of bacteria to epithelial cells through a process mediated by specific receptors that needs de novo protein synthesis by both cells. Thus, the recurrent subclinical infection may result from this intracellular existence of bacteria that are protected from host defenses and effects of antibiotics. This review emphasizes on recent findings on S. aureus adhesion to mammary epithelial cells and extracellular matrix components and invasion into mammary epithelial cells.     

Adhesion molecules and lymphocyte subsets in milk and blood of periparturient Holstein cows.

Migration of leukocytes into the mammary gland is an essential element of resistance to infection which is likely influenced by expression of adhesion molecules. The contribution of subsets to mammary gland resistance remains unclear. Mononuclear cells from milk and blood of dairy cows were examined for variation in CD4+, CD8+, and WC1+ (Workshop Cluster 1; marker for gammadelta T cells) lymphocyte phenotypes and expression of LFA-1 and L-selectin at several time points during the periparturient period and at Week 16 of lactation. Proportions of CD4+ T cells were higher (p < or = 10.05) in blood than milk at all times between Week 0 and Week 16 relative to calving; the inverse was true of CD8+ cells. Expression of L-selectin was lower (p < or = 0.05) on CD4+ cells and higher on CD8+ cells from milk. The WC1+ subset was more frequent in blood than in milk except at calving when the opposite was true. After calving, proportions of L-selectin+ WC1+ cells decreased steadily to Week 16. Expression of LFA-1 was examined on mononuclear cell populations and found to be lower on milk cells and did not vary over time. We conclude that proportions of T cells subsets differ significantly between blood and milk, particularly around calving. Corresponding variations in L-selectin expression may indicate a role for this molecule in regulating the movement of CD8+ and WC1+ T cells into the bovine mammary gland.     

牦牛乳腺上皮细胞的体外分离培养及鉴定

为提高牦牛乳腺上皮细胞体外培养成功率,实现高度可重复性,试验采用2.5 g/L(含0.5 g/L EDTA)胰酶和Ⅰ型胶原酶(1 mg/mL)分段消化牦牛乳腺组织块以分离细胞,原代培养时于DMEM/F12培养体系内添加氢化可的松(1 μg/mL)、表皮生长因子(50 ng/mL)及胰岛素-转铁蛋白-硒(5 μg/mL)...