奶牛乳腺上皮细胞培养体系中疑似干/祖细胞的鉴定

doi:10.16431/j.cnki.1671-7236.2016.08.023

摘要/Abstract

摘要：

为了揭示奶牛乳腺干细胞在体外泌乳调控中的潜在作用，本研究根据成体干细胞具有BrdU标记滞留的特点检测了奶牛乳腺上皮细胞（DCMECs）中疑似乳腺干/祖细胞，同时分析了两种新发现的乳腺干细胞分子标志（FNDC3B和PROCR）的表达定位，验证了它们指示DCMECs的可行性。通过实时荧光定量PCR方法检测分析DCMECs体外培养过程中催乳素、生长激素、胰岛素样生长因子Ⅰ及其受体的基因表达变化。结果显示，连续培养25 d后BrdU标记滞留细胞比例接近0.4%，极少数细胞呈FNDC3B或PROCR阳性，可以观察到不对称分裂细胞，原代和传代后DCMECs在激素、胰岛素样生长因子及其受体mRNA水平上差异极显著（P<0.01）。总之，在非诱导泌乳分化培养条件下，脱离泌乳生理环境的DCMECs自身具有的泌乳调控基因表达迅速受到抑制，但在一定培养时间内，DCMECs群体中能够保留一定比例疑似乳腺干/祖细胞，提示这些多能奶牛乳腺干/祖细胞在体外泌乳调控和乳腺腺泡重塑过程中可能发挥重要作用。

关键词: 奶牛; 乳腺干/祖细胞; 标记滞留; 泌乳

Abstract:

To develop the potential function of dairy cow mammary stem cells (DCMECs) in regulation of lactation,we identify putative DCMECs which were BrdU label retaining epithelial cells,at the same time,analysis the location of two new mammary stem cells molecular marks FNDC3B and PROCR to verify the feasibility of them to indicate DCMECs.The mRNA levels of prolactin,growth hormone,insulin-like growth factor-1 and their receptors were detected along with cell passage by Real-time quantitative PCR.The results showed that the proportion of BrdU label-retaining epithelial cells was nearly 0.4% after 25 d continuous culture (passaged 4 times) and few cells were positive for FNDC3B or PROCR.Moreover,we observed the BrdU labelled epithelial cells by asymmetric division.The mRNA levels of prolactin,growth hormone,insulin-like growth factor-Ⅰ and their receptors in primary and passage cells were extremely significant difference(P<0.01).DCMECs would rapidly lose some physiological characteristics and the ability of milk synthesis when not under the condition of induction of lactation differentiation,but a certain percentage of mammary stem/progenitor cells will be retained,whose potential effects on the regulation of lactation and mammary acinar remodeling were worthy of attention.

Key words: dairy cow; mammary stem/progenitor cell; label-retaining; lactation

中图分类号:

S813.1⁺1

郝雨蒙, 赵锋, 张娜, 陈丹, 李庆章. 奶牛乳腺上皮细胞培养体系中疑似干/祖细胞的鉴定[J]. 《中国畜牧兽医》, 2016, 43(8): 2095-2103.

HAO Yu-meng, ZHAO Feng, ZHANG Na, CHEN Dan, LI Qing-zhang. Identification of Suspected Stem/progenitor Cells in Bovine Mammary Epithelial Cells Culture System[J]. , 2016, 43(8): 2095-2103.

参考文献

[1] Deome K B,Faulkin L J,Bern H A,et al.Development of mammary tumors from hyper plastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice[J].Cancer Research,1959,19(5):515-520.
[2] Daniel C W,Young L J T,Medina D,et al.The influence of mammogenic hormones on serially transplanted mouse mammary gland[J].Experimental Gerontology,1971,6(1):95-101.
[3] Martignani E,Cravero D,Miretti S,et al.Bovine mammary stem cells:New perspective for dairy science[J].Vet Q,2014,34(1):52-58.
[4] Li J X,Zhang Y,Ma L B,et al.Isolation and culture of bovine mammary epithelial stem cells[J].Journal of Veterinary Medical Science,2009,71(1):15-19.
[5] Capuco A V,Ellis S E,Hale S A,et al.Lactation persistency:Insights from mammary cell proliferation studies[J].Journal of Animal Science,2003,81(suppl 3):18-31.
[6] Capuco A V,Choudhary R K,Daniels K M,et al.Bovine mammary stem cells:Cell biology meets production agriculture[J].Animal,2012,6(3):382-393.
[7] Shinin V,Gayraud-Morel B,Gomès D,et al.Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells[J]. Nature Cell Biology,2006,8(7):677-682.
[8] Smith G H.Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands[J]. Development,2005,132(4):681-687.
[9] Capuco A V,Evock-Clover C M,Minuti A,et al.In vivo expansion of the mammary stem/progenitor cell population by xanthosine infusion[J].Experimental Biology and Medicine,2009,234(4):475-482.
[10] Motyl T,Bier?a J B,Koz?owski M,et al.Identification,quantification and transcriptional profile of potential stem cells in bovine mammary gland[J]. Livestock Science,2011,136(2):136-149.
[11] Zeng Y A,Nusse R.Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture[J]. Cell Stem Cell,2010,6(6):568-577.
[12] Wang D,Cai C,Dong X,et al.Identification of multipotent mammary stem cells by protein C receptor expression[J].Nature,2014,517(7532):81-84.
[13] Nishizuka M,Kishimoto K,Kato A,et al.Disruption of the novel gene fad104 causes rapid postnatal death and attenuation of cell proliferation,adhesion,spreading and migration[J].Experimental Cell Research,2009,315(5):809-819.
[14] Fan X,Chen X,Deng W,et al.Up-regulated microRNA 143 in cancer stem cells differentiation promotes prostate cancer cells metastasis by modulating FNDC3B expression[J].BMC Cancer,2013,13(1):61.
[15] Choudhary R K.Identification and characterization of presumptive bovine mammary stem cells[D].The University of Maryland,Maryland,America,2011.
[16] Monzani P S,Bressan F F,Mesquita L G,et al.beta-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands[J].Genetics and Molecular Research,2011,10(2):604-614.
[17] Xu M N,Li S G,Zhao J Y,et al.The detection method of mammary gland bioreactor expression vector[J].Biotechnol Bull,2003,5:23.
[18] Ebner K E,Hoover C R,Hageman E C,et al.Cultivation and properties of bovine mammary cell cultures[J].Experimental Cell Research,1961,23(2):373-385.
[19] Sharma N,Kim J H,Sodhi S S,et al.Differentiation dynamics of mammary epithelial stem cells from Korean Holstein dairy cattle under ECM-free conditions[J].J Biomol Struct Dyn,2015,33(12):2633-2654.
[20] Capuco A V,Ellis S.Bovine mammary progenitor cells:Current concepts and future directions[J].Journal of Mammary Gland Biology and Neoplasia,2005,10(1):5-15.
[21] Zhou Y.Effects of growth hormone and insulin-like growth factor-1 on milk protein gene expression and nutrient uptake and cell proliferation in clonal bovine mammary epithelial cells[D].Virginia Polytechnic Institute and State University,America,2007.
[22] Sakamoto K,Komatsu T,Kobayashi T,et al.Growth hormone acts on the synthesis and secretion of alpha-casein in bovine mammary epithelial cells[J].J Dairy Res,2005,72(3):264-270.
[23] Fukudome K,Esmon C T.Molecular cloning and expression of murine and bovine endothelial cell protein C/activated protein C receptor (EPCR).The structural and functional conservation in human,bovine,and murine EPCR[J].J Biol Chem,1995,270(10):5571-5577.
[24] Schaffner F,Yokota N,Carneiro-Lobo T,et al.Endothelial protein C receptor function in murine and human breast cancer development[J].PLoS One,2013,8(4):e61071.
[25] Esmon C T.Structure and functions of the endothelial cell protein C receptor[J].Crit Care Med,2004,32(5):298-301.