荷斯坦奶牛乳腺干细胞的成骨诱导及鉴定
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摘要
背景:课题组已经完成乳腺干细胞向神经细胞和脂肪细胞的诱导,该实验诱导乳腺干细胞分化为成骨细胞,进一步丰富其分化潜能多样性,对乳腺干细胞的研究具有重要意义。目的:探讨荷斯坦奶牛乳腺干细胞定向诱导分化为成骨细胞的潜能。方法:分离纯化的荷斯坦奶牛乳腺干细胞系,由内蒙古生物制造重点实验室提供。取第4代乳腺干细胞加入成骨诱导液培养21 d,采用茜素红染色法和RT-PCR技术进行鉴定。结果与结论:荷斯坦奶牛乳腺干细胞经成骨诱导后,细胞边缘模糊,细胞中有颗粒状钙化结节出现,经茜素红染色呈阳性,诱导后成骨细胞标记性基因Alpl和Bglap呈阳性表达,表明了荷斯坦奶牛乳腺干细胞具有分化为成骨细胞的潜能。
BACKGROUND: The isolated and purified mammary stem cells from Holstein dairy cows have been induced to differentiate into nerve cells, adipocytes and osteoblasts, further enriching their multi-directional differentiation potential, which is of great significance for the study on mammary stem cells. OBJECTIVE: To investigate the osteogenic potential of mammary stem cells from Holstein dairy cows. METHODS: Mammary stem cells from Holstein dairy cows were isolated and purified, which were provided by the Key Laboratory of Biological Manufacturing of Inner Mongolia Autonomous Region. The fourth generation of mammary stem cells were cultured for 21 days in an osteogenic induction medium, and identified by alizarin red staining and RT-PCR. RESULTS AND CONCLUSION: After osteogenic induction, the cell edge was indistinct, and there were some granular calcified nodules in the cells. The induced cells were positive for alizarin red staining. The positive expressions of Alpl and Bglap in the induced osteoblasts were detected, indicating that the mammary stem cells from Holstein dairy cows have the potential to differentiate into osteoblasts.
BACKGROUND: The isolated and purified mammary stem cells from Holstein dairy cows have been induced to differentiate into nerve cells, adipocytes and osteoblasts, further enriching their multi-directional differentiation potential, which is of great significance for the study on mammary stem cells. OBJECTIVE: To investigate the osteogenic potential of mammary stem cells from Holstein dairy cows. METHODS: Mammary stem cells from Holstein dairy cows were isolated and purified, which were provided by the Key Laboratory of Biological Manufacturing of Inner Mongolia Autonomous Region. The fourth generation of mammary stem cells were cultured for 21 days in an osteogenic induction medium, and identified by alizarin red staining and RT-PCR. RESULTS AND CONCLUSION: After osteogenic induction, the cell edge was indistinct, and there were some granular calcified nodules in the cells. The induced cells were positive for alizarin red staining. The positive expressions of Alpl and Bglap in the induced osteoblasts were detected, indicating that the mammary stem cells from Holstein dairy cows have the potential to differentiate into osteoblasts.
引文
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[32]Capuco AV,Choudhary RK,Daniels KM,et al.Bovine mammary stem cells:cell biology meets production agriculture.Animal.2012;6(3):382-393.
[33]Hong D,Chen HX,Ge RS,et al.The biological roles of extracellular and intracytoplasmic glucocorticoids in skeletal cells.J Steroid Biochem Mol Biol.2008;111(3-5):164-170.
[34]雷栓虎,岳海源,汪静,等.骨髓间充质干细胞诱导分化成骨方法的研究与进展[J].中国组织工程研究,2013,17(6):1101-1106.
[35]刘迎春,陈钰萌,韩哲先,等.荷斯坦奶牛乳腺干细胞的成神经诱导鉴定及催乳素对其增殖活性的影响[J].畜牧兽医学报,2018,49(3):542-548.
[36]韩哲先,陈钰萌,刘迎春,等.荷斯坦奶牛乳腺干细胞的成脂诱导及鉴定[J].黑龙江畜牧兽医,2018(15):87-88,93,240.
[2]Deome KB,Faulkin LJ Jr,Bern HA,et al.Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice.Cancer Res.1959;19(5):515-520.
[3]Shackleton M,Vaillant F,Simpson KJ,et al.Generation of a functional mammary gland from a single stem cell.Nature.2006;439(7072):84-88.
[4]Inman JL,Robertson C,Mott JD,et al.Mammary gland development:cell fate specification,stem cells and the microenvironment.Development.2015;142(6):1028-1042.
[5]Dey D,Saxena M,Paranjape AN,et al.Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.PLoS One.2009;4(4):e5329.
[6]王代松,蔡车国,董小兵,等.多潜能乳腺干细胞的发现[J].中国细胞生物学学报,2015,37(1):1-5.
[7]Bai L,Rohrschneider LR.s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue.Genes Dev.2010;24(17):1882-1892.
[8]Amoh Y,Li L,Campillo R,et al.Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves.Proc Natl Acad Sci U S A.2005;102(49):17734-17738.
[9]Visvader JE.Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis.Genes Dev.2009;23(22):2563-2577.
[10]Richert MM,Schwertfeger KL,Ryder JW,et al.An atlas of mouse mammary gland development.J Mammary Gland Biol Neoplasia.2000;5(2):227-241.
[11]Watson CJ,Khaled WT.Mammary development in the embryo and adult:a journey of morphogenesis and commitment.Development.2008;135(6):995-1003.
[12]Smalley MJ.Isolation,culture and analysis of mouse mammary epithelial cells.Methods Mol Biol.2010;633:139-170.
[13]李庆章.奶牛乳腺发育与泌乳生物学[M].北京:科学出版社,2014:20-616.
[14]Jolicoeur F,Seemayer TA,Gabbiani G,et al.Multifocal,nascent,and invasive myoepithelial carcinoma(malignant myoepithelioma)of the breast:an immunohistochemical and ultrastructural study.Int J Surg Pathol.2002;10(4):281-291.
[15]Sopel M.The myoepithelial cell:its role in normal mammary glands and breast cancer.Folia Morphol(Warsz).2010;69(1):1-14.
[16]Capuco AV,Choudhary RK,Daniels KM,et al.Bovine mammary stem cells:cell biology meets production agriculture.Animal.2012;6(3):382-393.
[17]李吉霞,葛秀国,郭宏,等.牛乳腺干细胞的分离和生物学特征[J].西北农业学报,2013,22(6):1-5.
[18]Stingl J,Eirew P,Ricketson I,et al.Purification and unique properties of mammary epithelial stem cells.Nature.2006;439(7079):993-997.
[19]Fridriksdottir AJ,Petersen OW,R?nnov-Jessen L.Mammary gland stem cells:current status and future challenges.Int J Dev Biol.2011;55(7-9):719-729.
[20]李连宏,Xiao Gary Guishan,张众,等.乳腺干细胞研究及其再生医学[J].临床与实验病理学杂志,2013,29(9):933-937.
[21]Park BW,Kang EJ,Byun JH,et al.In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin,bone marrow and dental follicle tissues.Differentiation.2012;83(5):249-259.
[22]Cai R,Nakamoto T,Hoshiba T,et al.Matrices secreted during simultaneous osteogenesis and adipogenesis of mesenchymal stem cells affect stem cells differentiation.Acta Biomater.2016;35:185-193.
[23]Ehnes DD,Price FD,Shrive NG,et al.Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure.J Biomech.2015;48(10):1915-1921.
[24]李帅民.荷斯坦奶牛乳腺来源干细胞的初步分离及鉴定[D].呼和浩特:内蒙古农业大学,2014.
[25]Petersen OW,Polyak K.Stem cells in the human breast.Cold Spring Harb Perspect Biol.2010;2(5):a003160.
[26]宋嘉哲,李宁,薛恺.动物乳腺干细胞的研究进展[J].中国畜牧兽医,2011,40(11):110-114.
[27]Kordon EC,Smith GH.An entire functional mammary gland may comprise the progeny from a single cell.Development.1998;125(10):1921-1930.
[28]Shackleton M,Vaillant F,Simpson KJ,et al.Generation of a functional mammary gland from a single stem cell.Nature.2006;439(7072):84-88.
[29]Spike BT,Engle DD,Lin JC,et al.A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer.Cell Stem Cell.2012;10(2):183-197.
[30]Wansbury O,Mackay A,Kogata N,et al.Transcriptome analysis of embryonic mammary cells reveals insights into mammary lineage establishment.Breast Cancer Res.2011;13(4):R79.
[31]李吉霞,葛秀国,刘新峰,等.乳腺干细胞自我更新相关信号通路研究进展[J].天津农学院学报,2013,20(1):41-45.
[32]Capuco AV,Choudhary RK,Daniels KM,et al.Bovine mammary stem cells:cell biology meets production agriculture.Animal.2012;6(3):382-393.
[33]Hong D,Chen HX,Ge RS,et al.The biological roles of extracellular and intracytoplasmic glucocorticoids in skeletal cells.J Steroid Biochem Mol Biol.2008;111(3-5):164-170.
[34]雷栓虎,岳海源,汪静,等.骨髓间充质干细胞诱导分化成骨方法的研究与进展[J].中国组织工程研究,2013,17(6):1101-1106.
[35]刘迎春,陈钰萌,韩哲先,等.荷斯坦奶牛乳腺干细胞的成神经诱导鉴定及催乳素对其增殖活性的影响[J].畜牧兽医学报,2018,49(3):542-548.
[36]韩哲先,陈钰萌,刘迎春,等.荷斯坦奶牛乳腺干细胞的成脂诱导及鉴定[J].黑龙江畜牧兽医,2018(15):87-88,93,240.