本试验研究了二维和三维培养对奶牛乳腺上皮细胞(BMEC)形态与β-酪蛋白、κ-酪蛋白、αs1-酪蛋白基因表达的影响。试验采用内蒙古地区3头5岁泌乳期健康荷斯坦奶牛的乳腺组织,将BMEC经3代培养纯化后分别进行二维和三维培养,观察不同培养模式下细胞的生长形态,测定β-酪蛋白、κ-酪蛋白、αs1-酪蛋白基因的表达量。结果表明:2种培养模式下的BMEC呈现不同的细胞形态,二维培养的BMEC呈典型铺路石样细胞形态,三维培养的BMEC则形成了类腺泡的聚集物和管腔结构。利用三维培养的BMEC的αs1-酪蛋白基因的表达量显著地高于二维培养(P＜0.05),三维培养的BMEC的β-酪蛋白和κ-酪蛋白基因的表达量极显著地高于二维培养(P＜0.01)。结果提示,三维培养的BMEC 3种酪蛋白基因的表达量均高于二维培养,三维培养的BMEC在形态上呈现的特点与在体内更加接近。
This study was conducted to investigate the effects of two- and three-dimensional cultivation on cell morphology and gene expressions of β-casein, κ-casein and αs1-casein of bovine mammary epithelial cells (BMECs). Mammary parenchymal tissue was obtained from three healthy Holstein dairy cows aged 5 years. BMECs were purified after cultured for three generations, and then were cultured in two- and three-dimensional system, respectively. Cell morphology and gene expression levels of αs1-casein, β-casein and κ-casein of BMECs cultured in the two systems were compared. The results showed as follows: BMECs cultured in different systems displayed different cell morphologies, and those by two-dimensional cultivation displayed the classical cobblestone morphology, while those by three-dimensional cultivation formed mammospheres-forming aggregates and tubular structures. Gene expressions of αs1-casein (P＜0.05), β-casein (P＜0.01) and κ-casein (P＜0.01) of BMECs by three-dimensional cultivation were significantly higher than those by two-dimensional cultivation. The results indicate that gene expressions of the three caseins of BMECs by three-dimensional cultivation are higher than those by two-dimensional cultivation, and morphological characteristics of three-dimensional cultured BMECs are closely similar to those in vivo.
[1] MAILLEUX A A,OVERHOLTZER M,BURGGE J S.Lumen formation during mammary epithelial morphogenesis[J].The Journal of Landes Bioscience,2008,7(1):57-62.
[2] 弗雷谢尼R I.动物细胞培养——基本技术指南[M].张静波,徐存拴,译.北京:科学出版社,2008.
[3] DEBNATHA J,MUTHUSWAMY S K,BRUGGE J S.Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures[J].Methods,2003,30:256-268.
[4] BISSELL M J,RADISKY D.Putting tumours in context[J].Nature Reviews Cancer,2001,1(1):46-54.
[5] ANDERS M,HANSEN R,DING R X,et al.Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(4):1943-1948.
[6] BISSELL M J,RIZKI A,MIAN I S.Tissue architecture:the ultimate regulator of breast epithelial function[J].Current Opinion in Cell Biology,2003,15(6):753-762.
[7] WANG F,HONDA R K,RADISKY D,et al.Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts[J].Journal of the National Cancer Institute 2002,94(19):1494-1503.
[8] WANG F,VALERIE M W,OLE W P,et al.Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures:a different perspective in epithelial biology[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95:14821-14826.
[9] CUKIERMAN E,PANKOV R,STEVENS D R,et al.Taking cell-matrix adhesions to the third dimension[J].Science,2001,294:1708-1712.
[10] DEMOU Z N,MCINTIRE L V.Fully automated three-dimensional tracking of cancer cells in collagen gels:determination of motility phenotypes at the cellular level[J].Cancer Research,2002,62:5301-5307.
[11] YAMADA K M,CUKIERMAN E.Modeling tissue morphogenesis and cancer in 3D[J].Cell,2007,130(4):601-610.
[12] WEAVER V M,PETERSEN O W,WANG F,et al.Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies[J].Journal of Cell Biology,1997,137(1):231-245.
[13] JECHLINGER M,PODSYPANINA K,VARMUS H.Identifies cells that survive deinduction mouse mammary cells demonstrates oncogene dependence and Regulation of transgenes in three-dimensional cultures of primary[J].Genes Development,2009,23(14):1677-1688.
[14] LEE E Y,LEE W H,KAETZEL C S,et al.Interaction of mouse mammary epithelial cells with collagen substrata:Regulation of casein gene expression and secretion[J].Proceedings of the National Academy of Sciences of the United States of America,1985,82:1419-1423.
[15] CHRISTIAN S,BISSELL M J,CONNIE A M,et al.Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells[J].Proceedings of the National Academy of Sciences of the United States of America,1990,87:9118-9122.
[16] LIN C Q,DEMPSEY P J,COFFEY R J,et al.Extracellular matrix regulates whey acidic protein gene expression by suppression of TGF-α in mouse mammary epithelial cells:studies in culture and in transgenic mice[J].The Journal of Cell Biology,1995,129(4):1115-1126.
[17] 杨金勇,吴跃明,刘建新.蛋氨酸和蛋氨酸二肽对奶牛乳腺上皮细胞酪蛋白αs1基因表达的影响[J].农业生物技术学报,2007,15(1):24-27.
[18] 胡菡,王加启,李发弟,等.游离亚麻酸对奶牛乳腺上皮细胞脂肪酸代谢相关基因mRNA转录的影响[J].动物营养学报,2010,22(5):1342-1349.
[19] 王亨,孟霞,邱昌伟,等.脂多糖诱导奶牛乳腺上皮细胞先天性免疫反应[J].中国兽医学报,2010,30(3):398.
[20] 王秀美,考桂兰,高爱武,等.奶牛乳腺上皮细胞三维培养的形态观察[J].内蒙古农业大学学报,2009,30(4):9-13.
[21] KOZLOWSKI M,GAJEWSKA M,MAJEWSKA A,et al.Differences in growth and transcriptomic profile of bovine mammary epithelial monolayer and three-dimensional cell cultures[J].Journal of Physiology and Pharmacology,2009(Suppl.1):5-14.
[22] FRENCH M C,LITTLEJOHN R P,GREER G J,et al.Growth hormone and ghrelin receptor genes are differentially expressed between genetically lean and fat selection lines of sheep[J].Journal of Animal Science,2006,84:324-331.
[23] WARBURTON M J,MITCHELL D,ORMEROD E J,et al.Distribution of myoepithelial cells and basement membrane proteins in the resting,pregnant,lactating,and involuting rat mammary gland[J].Journal of Histochemistry and Cytochemistry,1982,30(7):667-676.
[24] WARBURTON M J,MONAGHAN P,FERNS S A,et al.Distribution of entactin in the basement membrane of the rat mammary gland:evidence for a non-epithelial origin[J].Experimental Cell Research,1984,152(1):240-254.
[25] HYNES R O.Integrins:a family of cell surface receptors[J].Cell,1987,48(4):549-554.
[26] BARCELLOS-HOFF M H,AGGELER J,RAM T G,et al.Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane[J].Development,1989,105:223-235.
[27] EMERMAN J T,BURWEN S J,PITELKA D R,et al.Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture[J].Tissue and Cell,1979,11(1):109-119.
[28] GORDON P,BETSEY C,CHARLOTTE S K,et al.Regulation of differentiation and polarized secretion mammary epithelial cells maintained in culture:extracellula matrix and membrane polarity influences[J].The Journal of Cell Biology,1987,105(5):2043-2051.
[29] BISSELL M J,HALLH G,PARRY G.How does the extracellular matrix direct gene expression?[J].Journal of Theoretical Biology,1982,99(1):31-68.
[30] BISSELL M J,AGGELER J.Dynamic reciprocity:how do extracellular matrix and hormones direct gene expression?[J].Progress in Clinical and Biological Research,1987,249:251-262.
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