鳜胚胎细胞系的建立与应用
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摘要
为更好地开展鳜基因功能研究和药物筛选工作,提高基因转染效率,本研究以鳜囊胚期胚胎为材料,采用含有20%胎牛血清的DMEM培养基进行培养,建立了生长稳定的鳜胚胎细胞系MFE。在此基础上,采用绿色荧光蛋白(GFP)作为标记物,在HEK293T细胞中体外包装逆转录病毒,再感染MFE细胞系。MTT法分析表明传代后细胞培养96 h内,细胞生长率变化也经历增殖、降低到达稳定期。而且MFE细胞能稳定表达GFP基因,感染效率为20%±5%,而脂质体转染效率为3%±2%。可见包装病毒感染细胞不仅能获得稳转细胞系,效率也远高于脂质体瞬时转染。荧光定量PCR分析表明,MFE细胞系能表达Irf1、Irf3和Irf7基因,Irf1基因表达量最高。MFE细胞系受到poly I:C刺激后,Irf1、Irf3和Irf7的表达量分别升高3.5,2.3和2.1倍。因此,MFE细胞通过病毒感染可以获得较高的转染效率,该细胞可作为鳜免疫相关基因功能研究的工具。
There are only a few cell lines of Siniperca chuatsi(mandarin fish) that could be widely used in scientific research. For mandarin fish, cell culture technique is an important aspect of the quality control program for the National Wild Fish Health Survey. Healthy, sensitive and mycoplasma-free cells are essential for detection of fish viruses in free-ranging fish populations. S. chuatsi is native to China and an important commercial species. In addition, the transfection efficiency is quite low in most fish cell lines. That is an obstacle in the basic research on fishes. In this study, a continuous cell(MFE) culture derived from the mandarin fish embryo was developed and has been subcultured over 20 passages in DMEM cell culture medium. MFE consists predominantly of epitheliallike cells and grows well in DMEM supplemented with 20% fetal bovine serum. Cell proliferation was assessed by MTT assay. The absorbance of the sample was read directly in the wells at an optimal wavelength of 570 nm. The results showed that MFE cell experienced proliferation, decrease and stable phases during 96 h. Meanwhile, we used GFP-expressing retrovirus produced by HEK293 T cells to test the transfection efficiency in MFE cell line.The result showed that the transfection efficiency reached 20%±5% without affecting the growth of these cells.Subsequently, the expression of three genes of Irf1, Irf2 and Irf7 was examined in MFE cell line by q RT-PCR. The results indicated that Irf1 is the highest one, and all increased to 3.5, 2.3 and 2.1 folds after poly I:C stimulation.The results of this study showed that MFE is the first cell line originated from embryo of mandarin fish. It could be used as a tool for gene function research and genetic modification.
There are only a few cell lines of Siniperca chuatsi(mandarin fish) that could be widely used in scientific research. For mandarin fish, cell culture technique is an important aspect of the quality control program for the National Wild Fish Health Survey. Healthy, sensitive and mycoplasma-free cells are essential for detection of fish viruses in free-ranging fish populations. S. chuatsi is native to China and an important commercial species. In addition, the transfection efficiency is quite low in most fish cell lines. That is an obstacle in the basic research on fishes. In this study, a continuous cell(MFE) culture derived from the mandarin fish embryo was developed and has been subcultured over 20 passages in DMEM cell culture medium. MFE consists predominantly of epitheliallike cells and grows well in DMEM supplemented with 20% fetal bovine serum. Cell proliferation was assessed by MTT assay. The absorbance of the sample was read directly in the wells at an optimal wavelength of 570 nm. The results showed that MFE cell experienced proliferation, decrease and stable phases during 96 h. Meanwhile, we used GFP-expressing retrovirus produced by HEK293 T cells to test the transfection efficiency in MFE cell line.The result showed that the transfection efficiency reached 20%±5% without affecting the growth of these cells.Subsequently, the expression of three genes of Irf1, Irf2 and Irf7 was examined in MFE cell line by q RT-PCR. The results indicated that Irf1 is the highest one, and all increased to 3.5, 2.3 and 2.1 folds after poly I:C stimulation.The results of this study showed that MFE is the first cell line originated from embryo of mandarin fish. It could be used as a tool for gene function research and genetic modification.
引文
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[29]叶湘辉,刘汉勤,王铁辉.草鱼囊胚细胞系的生物学特性[J].上海水产大学学报,2000,9(4):362-365.Ye X H,Liu H Q,Wang T H.The biological characteristics of grass carp blastula cell line[J].Journal of Shanghai Fisheries University,2000,9(4):362-365(in Chinese).
[30]任国诚,陈松林,沙珍霞.漠斑牙鲆胚胎细胞系的建立与鉴定[J].中国水产科学,2007,14(4):579-583.Ren G C,Chen S L,Sha Z X.Development and characterization of a continuous embryonic cell line from southern flounder(Paralichthys lethostigma)[J].Journal of Fishery Sciences of China,2007,14(4):579-583(in Chinese).
[31]王亚军,吴淑勤,林文辉,等.鳜塘浮游生物DNA序列多样性、水质和疾病的关系[J].应用生态学报,2007,18(1):163-168.Wang Y J,Wu S Q,Lin W H,et al.Relationships among planktons DNA sequence diversity,water quality and fish diseases in Siniperca chuatsi ponds[J].Chinese Journal of Applied Ecology,2007,18(1):163-168.
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[2]李传阳,许淼洋,Thammaratsuntorn J,等.3种鳜鱼生长与摄食量、胃蛋白酶活性和胃蛋白酶原基因表达相关分析[J].上海海洋大学学报,2016,25(1):1-7.Li C Y,Xu M Y,Thammaratsuntorn J,et al.Comparison of growth,food intake,pepsin activity and pepsinogen genes expression among Siniperca species[J].Journal of Shanghai Ocean University,2016,25(1):1-7(in Chinese).
[3]许淼洋,赵金良,李传阳,等.三种鳜鱼骨骼肌生长及相关调控基因表达比较[J].生物技术通报,2014,30(12):184-189.Xu M Y,Zhao J L,Li C Y,et al.Comparison on muscle growth and expression of muscle growth-related genes among three mandarin fishes[J].Biotechnology Bulletin,2014,30(12):184-189(in Chinese).
[4]王伟伟,赵金良,李思发,等.斑鳜不同地理群体遗传变异的AFLP分析[J].水生生物学报,2009,33(2):304-309.Wang W W,Zhao J L,Li S F,et al.Genetic variation of Siniperca scherzeri steindachner from different geographical populations by AFLP analysis[J].Acta Hydrobiologica Sinica,2009,33(2):304-309(in Chinese).
[5]邓燕飞,薛洋,赵金良,等.斑鳜(Siniperca scherzeri)胃蛋白酶原A、胃质子泵基因的克隆与组织表达分析[J].海洋与湖沼,2013,44(3):618-625.Deng Y F,Xue Y,Zhao J L,et al.Cloning and tissue expression analysis of the pepsinogen a and gastric proton pump genes from Siniperca scherzeri[J].Oceanologia et Limnologia Sinica,2013,44(3):618-625(in Chinese).
[6]Wise J S,Winn R N,Renfro J L.Generating new marine cell lines and transgenic species-conference summary[J].Journal of Experimental Zoology,2002,292(3):217-220.
[7]Wolf K,Quimby M C.Established eurythermic line of fish cells in vitro[J].Science,1962,135(3508):1065-1066.
[8]Lakra W S,Swaminathan T R,Joy K P.Development,characterization,conservation and storage of fish cell lines:a review[J].Fish Physiology and Biochemistry,2011,37(1):1-20.
[9]Dong C F,Weng S P,Shi X J,et al.Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus(ISKNV)[J].Virus Research,2008,135(2):273-281.
[10]Dong C F,Shuang F,Weng S P,et al.Cloning of a new fibroblast cell line from an early primary culture from mandarin fish(Siniperca chuatsi)fry for efficient proliferation of megalocytiviruses[J].Cytotechnology,2014,66(6):883-890.
[11]赖迎迢.鳜脑细胞系培养条件及传染性脾肾坏死病毒灭活工艺的研究[D].广州:华南农业大学,2016.Lai Y T.The study of optimal culture conditions and the methods of inactivating infectious spleen and kidney necrosis virus in Siniperca chuatsi[D].Guangzhou:South China Agricultural University,2016(in Chinese).
[12]Zhang X,Guan G J,Chen J B,et al.Parameters and efficiency of direct gene disruption by zinc finger nucleases in medaka embryos[J].Marine Biotechnology(NY),2014,16(2):125-134.
[13]Zheng H X,Tian H,Jin Y,et al.Development of a hamster kidney cell line expressing stably T7 RNA polymerase using retroviral gene transfer technology for efficient rescue of infectious foot-and-mouth disease virus[J].Journal of Virological Methods,2009,156(1-2):129-137.
[14]Vesterlund L,Jiao H,Unneberg P,et al.The zebrafish transcriptome during early development[J].BMC Developmental Biology,2011,11(1):30.
[15]Miyamoto M,Fujita T,Kimura Y,et al.Regulated expression of a gene encoding a nuclear factor,IRF-1,that specifically binds to IFN-βgene regulatory elements[J].Cell,1988,54(6):903-913.
[16]Mamane Y,Heylbroeck C,Génin P,et al.Interferon regulatory factors:the next generation[J].Gene,1999,237(1):1-14.
[17]Nguyen H,Hiscott J,Pitha P M.The growing family of interferon regulatory factors[J].Cytokine&Growth Factor Reviews,1997,8(4):293-312.
[18]Gu Y F,Wei Q,Tang S J,et al.Molecular characterization and functional analysis of IRF3 in tilapia(Oreochromis niloticus)[J].Developmental&Comparative Immunology,2016,55:130-137.
[19]Liu Q Z,Wang Y Z,Lin F,et al.Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells[J].PLo S One,2015,10(6):e0127961.
[20]Sun B J,Chang M X,Song Y,et al.Gene structure and transcription of IRF-1 and IRF-7 in the mandarin fish Siniperca chuatsi[J].Veterinary Immunology and Immunopathology,2007,116(1-2):26-36.
[21]左文功,钱华鑫,许映芳,等.草鱼肾组织细胞系CIK的建立及其生物学特性[J].水产学报,1986,10(1):11-17.Zuo W G,Qian H X,Xu Y F,et al.A cell line derived from the kidney of grass carp(Ctenopharyngodon idellus)[J].Journal of fisheries of China,1986,10(1):11-17(in Chinese).
[22]张君.河川沙塘鳢细胞培养及胚胎发育研究[D].苏州:苏州大学,2010.Zhang J.The study of the embryonic development and ell culture of Odontobutis potamophila[D].Suzhou:Soochow University,2010(in Chinese).
[23]Zheng Y,Wang N,Xie M S,et al.Establishment and characterization of a new fish cell line from head kidney of half-smooth tongue sole(Cynoglossus semilaevis)[J].Fish Physiology and Biochemistry,2012,38(6):1635-1643.
[24]耿龙武,鲁翠云,姜海峰,等.一种大鳞鲃鱼肾细胞体内培养制备染色体的方法:中国,103091144 A[P].2013-05-08.Geng L W,Lu C Y,Jiang H F,et al.Method for preparing chromosomes through large-scale barbel fish nephrocyte in vivo culture:CN,103091144A[P].2013-05-08(in Chinese).
[25]骆源,张春晓,王玲,等.斜带石斑鱼肝细胞分离及原代培养方法的建立[J].水产学报,2016,40(4):558-565.Luo Y,Zhang C X,Wang L,et al.Study on the isolation and primary culture of hepatocytes from liver of grouper(Epinephelus coioides)[J].Journal of Fisheries of China,2016,40(4):558-565(in Chinese).
[26]董丹丹,孙燕侠,郭华荣.斑马鱼性腺组织的原代细胞培养技术的建立[J].安徽农业科学,2016,44(15):130-134.Dong D D,Sun Y X,Guo H R.Development of primary cell culture from the gonad tissues of zebrafish[J].Journal of Anhui Agricultural Sciences,2016,44(15):130-134(in Chinese).
[27]洪锡钧.南方鲶胚胎细胞培养及其细胞周期特性检测[J].水产学报,1997,21(3):240-245.Hong X J.Subculture and observation of cell cycle characteristics of embryonic cell from Siluroidei meridionalis meridionalis[J].Journal of Fisheries of China,1997,21(3):240-245(in Chinese).
[28]胡洋.尼罗罗非鱼及斑马鱼胚胎细胞分离培养的研究[D].湛江:广东海洋大学,2010.Hu Y.Study on isolation and culture of Nile tilapia and zebrafish embryonic cells[D].Zhanjiang:Guangdong Ocean University,2010(in Chinese).
[29]叶湘辉,刘汉勤,王铁辉.草鱼囊胚细胞系的生物学特性[J].上海水产大学学报,2000,9(4):362-365.Ye X H,Liu H Q,Wang T H.The biological characteristics of grass carp blastula cell line[J].Journal of Shanghai Fisheries University,2000,9(4):362-365(in Chinese).
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