团头鲂骨组织细胞系的建立及其生物学特性
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摘要
为研究鱼类骨组织细胞的特征,实验建立了团头鲂骨组织细胞的体外分离培养方法,并对其生物特性进行了鉴定。以3月龄团头鲂尾椎骨及肋骨为实验材料,利用组织块培养法和胰蛋白消化法分别进行骨组织细胞培养,确定最佳培养条件:采用组织块培养法,28°C培养,M-199培养基中加入体积分数为20%的胎牛血清、25 ng/m L表皮生长因子EGF和25 ng/m L碱性成纤维生长因子b FGF。细胞生物学特性鉴定结果显示:细胞碱性磷酸酶、钙化结节茜素红染色及矿化结节von Kossa氏法染色均呈阳性,表明所培养的细胞具有典型成骨细胞的生物学活性;鱼骨钙素ELISA试剂盒检测培养细胞中骨钙素含量为997.25 ng/L;荧光定量PCR结果显示特异性调节成骨细胞分化的转录因子runx2a、runx2b和osterix基因在团头鲂骨组织细胞系中均有表达,且runx2b和osterix的表达量显著高于团头鲂肌肉细胞系。利用本方法可获得增殖活性良好,生长速率快,且细胞生物学特性稳定的团头鲂骨组织细胞系(MBCs),为日后鱼类骨组织体外实验研究奠定了基础。
In order to study the characteristics of fish bone cells, methods for isolating and culturing bone cells from caudal vertebrae and rib of blunt snout bream Megalobrama amblycephala were explored in the present study, and the biological characters of bone cells were identified. The bone cells were cultured with tissue culture and trypsin digestion respectively by using 3-month-old M. amblycephala caudal vertebrae and ribs from tail part.The optimal conditions were as follows: 25 ng/m L epidermal growth factor(EGF) and 25 ng/m L basic fibroblast growth factor(b FGF) were added to the M-199 medium at 28 °C using tissue culture. The results of cell biology identification showed that the cultured cells had the biological activity of typical osteoblasts, with alkaline phosphatase staining, calcium nodules alizarin red staining and mineralized nodules von Kossa staining being positive.The osteocalcin content was 997.25 ng/L in the bone cells tested by the fish bone osteocalcin ELISA kit. The genes of runx2 a, runx2 b and osterix which regulated osteoblast differentiation, were expressed in bone cells, with the runx2 b and osterix expression levels significantly higher than that in muscle cells. Results indicated that bone cells of fish have good proliferation and faster growth rate as well as stable cell biological characteristics, which is suitable for related studies in vitro.
In order to study the characteristics of fish bone cells, methods for isolating and culturing bone cells from caudal vertebrae and rib of blunt snout bream Megalobrama amblycephala were explored in the present study, and the biological characters of bone cells were identified. The bone cells were cultured with tissue culture and trypsin digestion respectively by using 3-month-old M. amblycephala caudal vertebrae and ribs from tail part.The optimal conditions were as follows: 25 ng/m L epidermal growth factor(EGF) and 25 ng/m L basic fibroblast growth factor(b FGF) were added to the M-199 medium at 28 °C using tissue culture. The results of cell biology identification showed that the cultured cells had the biological activity of typical osteoblasts, with alkaline phosphatase staining, calcium nodules alizarin red staining and mineralized nodules von Kossa staining being positive.The osteocalcin content was 997.25 ng/L in the bone cells tested by the fish bone osteocalcin ELISA kit. The genes of runx2 a, runx2 b and osterix which regulated osteoblast differentiation, were expressed in bone cells, with the runx2 b and osterix expression levels significantly higher than that in muscle cells. Results indicated that bone cells of fish have good proliferation and faster growth rate as well as stable cell biological characteristics, which is suitable for related studies in vitro.
引文
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[22]韩云珍.大鼠成骨细胞分离培养及其生物学特性鉴定[D].福州:福建农林大学,2008.Han Y Z.Isolated culture and identification about bionomics in rat osteoblast[D].Fuzhou:Fujian Agricultural and Forestry University,2008(in Chinese).
[23]谢艳芳,陈克明,马小妮,等.大鼠颅骨成骨细胞和骨细胞的分离纯化与比较研究[J].解放军医药杂志,2015,27(3):1-5.Xie Y F,Chen K M,Ma X N,et al.Isolation and identification and comparative study of calvarial osteoblasts and osteocytes of rats[J].Medical&Pharmaceutical Journal of Chinese People's Liberation Army,2015,27(3):1-5(in Chinese).
[24]鄂征.组织培养和分子细胞学技术[M].北京:北京出版社,1995:202-206.E Z.Tissue culture and molecular cytology[M].Beijing:Beijing Publishing House,1995:202-206(in Chinese).
[25]尹美珍,李世普,刘琴.成骨细胞的数量对体外钙化结节形成的影响[J].解剖学杂志,2008,31(2):257-258.Yin M Z,Li S P,Liu Q.Effect of the number of osteoblasts on the formation of calcified nodules in vitro[J].Chinese Journal of Anatomy,2008,31(2):257-258(in Chinese).
[26]孟昭亨.骨钙素及其临床意义[J].中华内分泌代谢杂志,1992,8(1):41-44.Meng Z H.Osteocalcin and its clinical significance[J].Chinese Journal of Endocrinology and Metabolism,1992,8(1):41-44(in Chinese).
[27]姜利华,张瑾,韩翠华,等.不同年龄段的血清骨钙素含量检测的临床意义[J].放射免疫学杂志,2006,19(2):93-95.Jiang L H,Zhang J,Han C H,et al.Clinical significance of changes of serum osteocalcin(BGP)levels in subjects of different age-groups[J].Journal of Radioimmunology,2006,19(2):93-95(in Chinese).
[28]Ducy P,Starbuck M,Priemel M,et al.A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development[J].Gene&Development,1999,13:1025-1036.
[29]徐道志.成骨细胞分化与骨骼发育的转录因子Cbf-αl/Runx2[J].中医正骨,2006,18(11):61-63.Xu D Z.Osteoblast differentiation and bone development of the transcription factor Cbfαl/Runx2[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2006,18(11):61-63(in Chinese).
[30]Nakashima K,Zhou X,Kunkel G,et al.The novel zincfinger-containing transcription factor osterixis required for osteoblast differentiation and bone formation[J].Cell,2002,108(1):17-29.
[31]陈杨林.不同体外培养条件对小鼠早期胚胎发育及外胚层多能干细胞建立的影响[D].呼和浩特:内蒙古大学,2015.Chen Y L.Effect of in vitro culture conditions on early development and epiblaststem cell isolation of mouse embryos[D].Hohhot:Inner Mongolia University,2015(in Chinese).
[2]Zhou G Z,Gui L,Li Z Q,et al.Establishment of a Chinese sturgeon Acipenser sinensis tail-fin cell line and its susceptibility to frog iridovirus[J].Journal of Fish Biology,2008,73(8):2058-2067.
[3]孟凡华,尹洪滨,孙中武,等.鲤鱼(Cyprinus carpio)体细胞系的建立及其生物学特性分析(简报)[J].实验生物学报,2005,38(1):80-84.Meng F H,Yin H B,Sun Z W,et al.Construction and characteristics analysis of somatic cell lines from com-mon carp Cyprinus carpio[J].Acta Biologiae Experimentalis Sinica,2005,38(1):80-84(in Chinese).
[4]张铭,陈荪红,赵小立.淡水白鲳细胞建系及其生长温度特性的研究[J].生物工程学报,2001,17(1):105-108.Zhang M,Chen S H,Zhao X L.The establishment of four cell lines from Colossoma brachypomum and their growth characteristics under various temperatures[J].Chinese Journal of Biotechnology,2001,17(1):105-108(in Chinese).
[5]傅淑平,蔡宇.成骨细胞在破骨细胞性骨吸收中的调节作用[J].陕西医学杂志,2004,33(11):1040-1041.Fu S P,Cai Y.Regulatory effects of osteoblasts on osteoclastic bone resorption[J].Shaanxi Medical Journal,2004,33(11):1040-1041(in Chinese).
[6]Parfitt A M.Skeletal heterogeneity and the purposes of bone remodeling:Implications for the understanding of osteoporosis[J].Osteoporosis,2008,90(6):71-89.
[7]霍波,郭向东,龙勉.骨细胞网络中力致钙信号响应与传递的理论模型与数值分析[C]//2009中国力学学会学术大会.郑州:中国力学学会,2009.Huo B,Guo X D,Long M.Theoretical model and numerical analysis of calcium signal response and transmission in bone cell networks[C]//Chinese Conference Applied Mechanics Proceedings.Zhengzhou:Chinese Society of Mechanics,2009(in Chinese).
[8]Vijayakumar P,LaizéV,Cardeira J,et al.Development of an in vitro cell system from zebrafish suitable to study bone cell differentiation and extracellular matrix mineralization[J].Zebrafish,2013,10(4):500-509.
[9]Marques C L,Rafael M S,Cancela M L,et al.Establishment of primary cell cultures from fish calcified tissues[J].Cytotechnology,2007,55(1):9-13.
[10]Rafael M S,Marques C L,Parameswaran V,et al.Fish bone-derived cell lines:an alternative in vitro cell system to study bone biology[J].Journal of Applied Ichthyology,2010,26(2):230-234.
[11]高泽霞,王卫民,蒋恩明,等.团头鲂种质资源及遗传改良研究进展[J].华中农业大学学报,2014,33(3):138-144.Gao Z X,Wang W M,Jiang E M,et al.Research advances on germplasm resources and genetic improvement of blunt snout bream(Megalobrama amblycephala)[J].Journal of Huazhong Agricultural University,2014,33(3):138-144(in Chinese).
[12]王为民.团头鲂养殖产业现状[J].科学养鱼,2009(4):44-45.Wang W M.Present situation of aquaculture industry of blunt snout bream(Megalobrama amblycephala)[J].Scientific Fish Farming,2009(4):44-45(in Chinese).
[13]祝冬梅.团头鲂三种细胞系的建立、鉴定及其初步应用[D].武汉:华中农业大学,2013.Zhu D M.Establishment and characterization of three cell lines from blunt snout bream,Megalobrama amblycephala,and their preliminary applications studies[D].Wuhan:Huazhong Agricultural University,2013(in Chinese).
[14]中华人民共和国科学技术部.关于善待实验动物的指导性意见[OL].2006-09-30[2008-04-20].http://www.most.gov.cn/fggw/zfwj/zfwj2006/200609/t20060930_54389.htm.The Ministry of Science and Technology of the People’s Republic of China.Guidance suggestion of caring laboratory animals[OL].2006-09-30[2008-04-20].http://www.most.gov.cn/fggw/zfwj/zfwj2006/200609/t20060930_54389.htm(in Chinese).
[15]Levan A,Fredga K,Sandberg A A.Nomenclature for centromeric position on chromosomes[J].Hereditas,1964,52(2):201-220.
[16]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2?￠￠CTMethod[J].Methods,2001,25(4):402-408.
[17]肖艺,曾令兵,徐进,等.锦鲤鳍条组织细胞系的建立及其生物学特性[J].中国细胞生物学学报,2012,34(8):767-774.Xiao Y,Zeng L B,Xu J,et al.Establishment and characterization of a cell line derived from fin of Cryprinus carpiod[J].Chinese Journal of Cell Biology,2012,34(8):767-774(in Chinese).
[18]梁岳,马广智,方展强.剑尾鱼肝细胞原代培养[J].中国比较医学杂志,2006,16(3):185-187.Liang Y,Ma G Z,Fang Z Q.Primary culture of swordtail fish(Xiphophorus helleri)hepatocytes[J].Chinese Journal of Comparative Medicine,2006,16(3):185-187(in Chinese).
[19]Zellin G.Growth factors and bone regeneration:Implications of barrier membranes[J].Swedish Dental Journal Supplement,1998,129:7-65.
[20]Nishimura R,Harris E,Imamura K,et al.Expression oftranscription factor Cbfa1 is enhanced by the BMP-2 /SMAD signal transduction cascade in pluripotent mesenchymal cells[J].Journal of Bone and Mineral Research,1998,23:S150.
[21]St-Jacques B,Hammerschmidt M,Mc Mahon A P.Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation[J].Gene&Development,1999,13(16):2072-2086.
[22]韩云珍.大鼠成骨细胞分离培养及其生物学特性鉴定[D].福州:福建农林大学,2008.Han Y Z.Isolated culture and identification about bionomics in rat osteoblast[D].Fuzhou:Fujian Agricultural and Forestry University,2008(in Chinese).
[23]谢艳芳,陈克明,马小妮,等.大鼠颅骨成骨细胞和骨细胞的分离纯化与比较研究[J].解放军医药杂志,2015,27(3):1-5.Xie Y F,Chen K M,Ma X N,et al.Isolation and identification and comparative study of calvarial osteoblasts and osteocytes of rats[J].Medical&Pharmaceutical Journal of Chinese People's Liberation Army,2015,27(3):1-5(in Chinese).
[24]鄂征.组织培养和分子细胞学技术[M].北京:北京出版社,1995:202-206.E Z.Tissue culture and molecular cytology[M].Beijing:Beijing Publishing House,1995:202-206(in Chinese).
[25]尹美珍,李世普,刘琴.成骨细胞的数量对体外钙化结节形成的影响[J].解剖学杂志,2008,31(2):257-258.Yin M Z,Li S P,Liu Q.Effect of the number of osteoblasts on the formation of calcified nodules in vitro[J].Chinese Journal of Anatomy,2008,31(2):257-258(in Chinese).
[26]孟昭亨.骨钙素及其临床意义[J].中华内分泌代谢杂志,1992,8(1):41-44.Meng Z H.Osteocalcin and its clinical significance[J].Chinese Journal of Endocrinology and Metabolism,1992,8(1):41-44(in Chinese).
[27]姜利华,张瑾,韩翠华,等.不同年龄段的血清骨钙素含量检测的临床意义[J].放射免疫学杂志,2006,19(2):93-95.Jiang L H,Zhang J,Han C H,et al.Clinical significance of changes of serum osteocalcin(BGP)levels in subjects of different age-groups[J].Journal of Radioimmunology,2006,19(2):93-95(in Chinese).
[28]Ducy P,Starbuck M,Priemel M,et al.A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development[J].Gene&Development,1999,13:1025-1036.
[29]徐道志.成骨细胞分化与骨骼发育的转录因子Cbf-αl/Runx2[J].中医正骨,2006,18(11):61-63.Xu D Z.Osteoblast differentiation and bone development of the transcription factor Cbfαl/Runx2[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2006,18(11):61-63(in Chinese).
[30]Nakashima K,Zhou X,Kunkel G,et al.The novel zincfinger-containing transcription factor osterixis required for osteoblast differentiation and bone formation[J].Cell,2002,108(1):17-29.
[31]陈杨林.不同体外培养条件对小鼠早期胚胎发育及外胚层多能干细胞建立的影响[D].呼和浩特:内蒙古大学,2015.Chen Y L.Effect of in vitro culture conditions on early development and epiblaststem cell isolation of mouse embryos[D].Hohhot:Inner Mongolia University,2015(in Chinese).