Percoll不连续密度梯度离心法分离红螯光壳螯虾血细胞
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摘要
建立了利用Percoll不连续密度梯度离心分离红螯光壳螯虾血细胞的方法,并对配制分离体系的缓冲液以及分离体系的密度组成进行了优化。结果显示,由20%、65%和100%的Percoll组成的分离体系分离效果最优。在转速为1 810 r/min的条件下离心20 min之后,可将红螯光壳螯虾血细胞分为SGC与GC 2个细胞层。经流式细胞术分析,发现细胞层纯度均在95%以上,细胞死亡比率低于1.5%,可用于后续的细胞功能分析。此方法简单有效,为后续研究螯虾的免疫防御机制及病害防治方法奠定了基础。
we developed a method to separate the GC and SGC of Cherax quadricarinatus by using discontinuous Percoll density gradient centrifugation. The dilution buffer and the combination of density gradient were optimized. We found that the density gradient should be diluted in anti-clotting buffer to prevent hemocytes aggregation, and the discontinuous Percoll density gradient that consisted of 20%, 65% and 100% of Percoll provided the best result compared with others. After centrifugation at 1 810 r/min for 20 min, the hemocytes were separated into two layers, corresponding to SGC and GC, respectively. Flow cytometry analysis showed that the purities of both fractions were >95% and the cell mortalities were <1.5%. The isolated cells were in good status and could be readily used for further analysis. This simple and effective method will facilitate the study on the function of different hemocytes and thus provide information for disease control.
we developed a method to separate the GC and SGC of Cherax quadricarinatus by using discontinuous Percoll density gradient centrifugation. The dilution buffer and the combination of density gradient were optimized. We found that the density gradient should be diluted in anti-clotting buffer to prevent hemocytes aggregation, and the discontinuous Percoll density gradient that consisted of 20%, 65% and 100% of Percoll provided the best result compared with others. After centrifugation at 1 810 r/min for 20 min, the hemocytes were separated into two layers, corresponding to SGC and GC, respectively. Flow cytometry analysis showed that the purities of both fractions were >95% and the cell mortalities were <1.5%. The isolated cells were in good status and could be readily used for further analysis. This simple and effective method will facilitate the study on the function of different hemocytes and thus provide information for disease control.
引文
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[12]Torres M A,Dangl J L.Functions of the respiratory burst oxidase in biotic interactions,abiotic stress and development[J].Current Opinion in Plant Biology,2005,8(4):397-403.
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[15]S?derh?ll K,Smith V J.Separation of the haemocyte populations of Carcinus maenas and other marine decapods,and prophenoloxidase distribution[J].Developmental&Comparative Immunology,1983,7(2):229-239.
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[17]Li C W,Shields J D.Primary culture of hemocytes from the Caribbean spiny lobster,Panulirus argus,and their susceptibility to Panulirus argus Virus 1(PaV1)[J].Journal of Invertebrate Pathology,2007,94(1):48-55.
[18]Van De Braak C B T,Faber R,Boon J H.Cellular and humoral characteristics of Penaeus monodon(Fabricius,1798)haemolymph[J].Comparative Haematology International,1996,6(4):194-203.
[19]Vargas-Albores F,Gollas-Galván T,Hernández-López J.Functional characterization of Farfantepenaeus californiensis,Litopenaeus vannamei and L.stylirostris haemocyte separated using density gradient centrifugation[J].Aquaculture Research,2005,36(4):352-360.
[20]Li F,Chang X F,Xu L M,et al.Different roles of crayfish hemocytes in the uptake of foreign particles[J].Fish&Shellfish Immunology,2018,77:112-119.
[21]Ratcliffe N A,Rowley A F,Fitzgerald S W,et al.Invertebrate immunity:basic concepts and recent advances[J].International Review of Cytology,1985,97:183-350.
[22]Johansson M W,S?derh?ll K.The prophenoloxidase activating system and associated proteins in invertebrates[M]//Rinkevich B,Müller W E G.Invertebrate Immunology.Berlin,Heidelberg:Springer,1996,15:46.
[23]S?derh?ll K,Cerenius L.Role of the prophenoloxidaseactivating system in invertebrate immunity[J].Current Opinion in Immunology,1998,10(1):23-28.
[24]Galko M J,Krasnow M A.Cellular and genetic analysis of wound healing in Drosophila larvae[J].PLoS Biology,2004,2(8):E239.
[25]Christensen B M,Li J Y,Chen C C,et al.Melanization immune responses in mosquito vectors[J].Trends in Parasitology,2005,21(4):192-199.
[26]Jiravanichpaisal P,Lee B L,S?derh?ll K.Cell-mediated immunity in arthropods:hematopoiesis,coagulation,melanization and opsonization[J].Immunobiology,2006,211(4):213-236.
[27]Lemaitre B,Hoffmann J.The host defense of Drosophila melanogaster[J].Annual Review of Immunology,2007,25:697-743.
[28]Cerenius L,Lee B L,S?derh?ll K.The proPO-system:pros and cons for its role in invertebrate immunity[J].Trends in Immunology,2008,29(6):263-271.
[29]Zhu L,Chang Y H,Xing J,et al.Comparative proteomic analysis between two haemocyte subpopulations in shrimp Fenneropenaeus chinensis[J].Fish&Shellfish Immunology,2017,72:325-333.
[30]Zhou Y L,Gu W B,Tu D D,et al.Hemocytes of the mud crab Scylla paramamosain:cytometric,morphological characterization and involvement in immune responses[J].Fish Shellfish Immunology,2018,72:459-469.
[31]Johansson M W,S?derh?ll K.Exocytosis of the prophenoloxidase activating system from crayfish haemocytes[J].Journal of Comparative Physiology B,1985,156(2):175-181.
[32]Koiwai K,Alenton R R R,Shiomi R,et al.Two hemocyte sub-populations of kuruma shrimp Marsupenaeus japonicus[J].Molecular Immunology,2017,85:1-8.
[33]Liu C H,Cheng W,Chen J C.The peroxinectin of white shrimp Litopenaeus vannamei is synthesised in the semigranular and granular cells,and its transcription is upregulated with Vibrio alginolyticus infection[J].Fish&Shellfish Immunology,2005,18(5):431-444.
[34]冼健安,王安利,苗玉涛.流式细胞术在克氏原螯虾血细胞的分类、活性和免疫功能研究中的应用[J].淡水渔业,2012,42(1):9-14.Xian J A,Wang A L,Miao Y T.Application of flow cytometry in studies of classification,viability and immune parameters of Procambarus clarkii Haemocytes[J].Freshwater Fisheries,2012,42(1):9-14(in Chinese).
[35]Smith V J,S?derh?ll K.A comparison of phenoloxidase activity in the blood of marine invertebrates[J].Developmental&Comparative Immunology,1991,15(4):251-261.
[36]Johansson M W,S?derh?ll K.Separation of crustacean blood cells by continuous density gradient centrifugation[M]//Stolen J S,Fletcher T C,Smith S A,et al.Techniques in Fish Immunology:Fish Immunology Technical Communication 4.New Jersey:SOS Publications,1995:141-145.
[37]Mangkalanan S,Sanguanrat P,Utairangsri T,et al.Characterization of the circulating hemocytes in mud crab(Scylla olivacea)revealed phenoloxidase activity[J].Developmental&Comparative Immunology,2014,44(1):116-123.
[38]Ding Z F,Du J,Ou J T,et al.Classification of circulating hemocytes from the red swamp crayfish Procambarus clarkii and their susceptibility to the novel pathogen Spiroplasma eriocheiris in vitro[J].Aquaculture,2012,356-357:371-380.
[39]Durliat M,Vranckx R.Action of various anticoagulants on hemolymphs of lobsters and spiny lobsters[J].The Biological Bulletin,1981,160(1):55-68.
[40]S?derh?ll I.Crustacean hematopoiesis[J].Developmental&Comparative Immunology,2016,58:129-141.
[41]Jiravanichpaisal P,Sricharoen S,S?derh?ll I,et al.White spot syndrome virus(WSSV)interaction with crayfish haemocytes[J].Fish Shellfish Immunology,2006,20(5):718-727.
[2]Tropea C,Piazza Y,Greco L S L.Effect of long-term exposure to high temperature on survival,growth and reproductive parameters of the“redclaw”crayfish Cherax quadricarinatus[J].Aquaculture,2010,302(1-2):49-56.
[3]蒋琦辰,张文逸,谭红月,等.不同光周期下红螯螯虾幼虾摄食节律的研究[J].淡水渔业,2012,42(5):89-91.Jiang Q C,Zhang W Y,Tan H Y,et al.A study on the feeding rhythm of juvenile Australia red claw crayfish(Cherax quadricarinatus)under different photoperiods[J].Freshwater Fisheries,2012,42(5):89-91(in Chinese).
[4]Bachère E.Shrimp immunity and disease control[J].Aquaculture,2000,191(1-3):3-11.
[5]Lightner D V,Poulos B T,Tang-Nelson K F,et al.Application of molecular diagnostic methods to penaeid shrimp diseases:advances of the past 10 years for control of viral diseases in farmed shrimp[J].Developments in Biologicals,2006,126:117-22;discussion 325-6.
[6]Lightner D V,Redman R M.Opportunities for training in shrimp diseases[J].Developments in Biologicals,2007,129:137-146.
[7]Kurtz J,Franz K.Evidence for memory in invertebrate immunity[J].Nature,2003,425(6953):37-38.
[8]马寨璞,张繁霜,佟霁坤.甲壳动物免疫系统概述及其图论表示[J].安徽农业科学,2011,39(1):291-294.Ma Z P,Zhang F S,Tong J K.Overview and graph theory of the immune system of Crustacean[J].Journal of Anhui Agricultural Sciences,2011,39(1):291-294(in Chinese).
[9]姚翠鸾,王志勇,相建海.甲壳动物血细胞及其在免疫防御中的功能[J].动物学研究,2006,27(5):549-557.Yao C L,Wang Z Y,Xiang J H.Crustacean haemocytes and their function in immune responses[J].Zoological Research,2006,27(5):549-557(in Chinese).
[10]Th?rnqvist P O,Johansson M W,S?derh?ll K.Opsonic activity of cell adhesion proteins andβ-1,3-glucan binding proteins from two crustaceans[J].Developmental&Comparative Immunology,1994,18(1):3-12.
[11]Hoffmann J A,Kafatos F C,Janeway Jr C A,et al.Phylogenetic perspectives in innate immunity[J].Science,1999,284(5418):1313-1318.
[12]Torres M A,Dangl J L.Functions of the respiratory burst oxidase in biotic interactions,abiotic stress and development[J].Current Opinion in Plant Biology,2005,8(4):397-403.
[13]Johansson M W,Keyser P,Sritunyalucksana K,et al.Crustacean haemocytes and haematopoiesis[J].Aquaculture,2000,191(1-3):45-52.
[14]邹广众,孙虎山.水产甲壳动物免疫学研究进展与前景展望[J].生命科学仪器,2009,7(6):17-21.Zou G Z,Sun H S.Research advances and prospect in aquatic crustaceans immunology[J].Life Science Instruments,2009,7(6):17-21(in Chinese).
[15]S?derh?ll K,Smith V J.Separation of the haemocyte populations of Carcinus maenas and other marine decapods,and prophenoloxidase distribution[J].Developmental&Comparative Immunology,1983,7(2):229-239.
[16]Van De Braak C B T,Botterblom M H A,Liu W,et al.The role of the haematopoietic tissue in haemocyte production and maturation in the black tiger shrimp(Penaeus monodon)[J].Fish&Shellfish Immunology,2002,12(3):253-272.
[17]Li C W,Shields J D.Primary culture of hemocytes from the Caribbean spiny lobster,Panulirus argus,and their susceptibility to Panulirus argus Virus 1(PaV1)[J].Journal of Invertebrate Pathology,2007,94(1):48-55.
[18]Van De Braak C B T,Faber R,Boon J H.Cellular and humoral characteristics of Penaeus monodon(Fabricius,1798)haemolymph[J].Comparative Haematology International,1996,6(4):194-203.
[19]Vargas-Albores F,Gollas-Galván T,Hernández-López J.Functional characterization of Farfantepenaeus californiensis,Litopenaeus vannamei and L.stylirostris haemocyte separated using density gradient centrifugation[J].Aquaculture Research,2005,36(4):352-360.
[20]Li F,Chang X F,Xu L M,et al.Different roles of crayfish hemocytes in the uptake of foreign particles[J].Fish&Shellfish Immunology,2018,77:112-119.
[21]Ratcliffe N A,Rowley A F,Fitzgerald S W,et al.Invertebrate immunity:basic concepts and recent advances[J].International Review of Cytology,1985,97:183-350.
[22]Johansson M W,S?derh?ll K.The prophenoloxidase activating system and associated proteins in invertebrates[M]//Rinkevich B,Müller W E G.Invertebrate Immunology.Berlin,Heidelberg:Springer,1996,15:46.
[23]S?derh?ll K,Cerenius L.Role of the prophenoloxidaseactivating system in invertebrate immunity[J].Current Opinion in Immunology,1998,10(1):23-28.
[24]Galko M J,Krasnow M A.Cellular and genetic analysis of wound healing in Drosophila larvae[J].PLoS Biology,2004,2(8):E239.
[25]Christensen B M,Li J Y,Chen C C,et al.Melanization immune responses in mosquito vectors[J].Trends in Parasitology,2005,21(4):192-199.
[26]Jiravanichpaisal P,Lee B L,S?derh?ll K.Cell-mediated immunity in arthropods:hematopoiesis,coagulation,melanization and opsonization[J].Immunobiology,2006,211(4):213-236.
[27]Lemaitre B,Hoffmann J.The host defense of Drosophila melanogaster[J].Annual Review of Immunology,2007,25:697-743.
[28]Cerenius L,Lee B L,S?derh?ll K.The proPO-system:pros and cons for its role in invertebrate immunity[J].Trends in Immunology,2008,29(6):263-271.
[29]Zhu L,Chang Y H,Xing J,et al.Comparative proteomic analysis between two haemocyte subpopulations in shrimp Fenneropenaeus chinensis[J].Fish&Shellfish Immunology,2017,72:325-333.
[30]Zhou Y L,Gu W B,Tu D D,et al.Hemocytes of the mud crab Scylla paramamosain:cytometric,morphological characterization and involvement in immune responses[J].Fish Shellfish Immunology,2018,72:459-469.
[31]Johansson M W,S?derh?ll K.Exocytosis of the prophenoloxidase activating system from crayfish haemocytes[J].Journal of Comparative Physiology B,1985,156(2):175-181.
[32]Koiwai K,Alenton R R R,Shiomi R,et al.Two hemocyte sub-populations of kuruma shrimp Marsupenaeus japonicus[J].Molecular Immunology,2017,85:1-8.
[33]Liu C H,Cheng W,Chen J C.The peroxinectin of white shrimp Litopenaeus vannamei is synthesised in the semigranular and granular cells,and its transcription is upregulated with Vibrio alginolyticus infection[J].Fish&Shellfish Immunology,2005,18(5):431-444.
[34]冼健安,王安利,苗玉涛.流式细胞术在克氏原螯虾血细胞的分类、活性和免疫功能研究中的应用[J].淡水渔业,2012,42(1):9-14.Xian J A,Wang A L,Miao Y T.Application of flow cytometry in studies of classification,viability and immune parameters of Procambarus clarkii Haemocytes[J].Freshwater Fisheries,2012,42(1):9-14(in Chinese).
[35]Smith V J,S?derh?ll K.A comparison of phenoloxidase activity in the blood of marine invertebrates[J].Developmental&Comparative Immunology,1991,15(4):251-261.
[36]Johansson M W,S?derh?ll K.Separation of crustacean blood cells by continuous density gradient centrifugation[M]//Stolen J S,Fletcher T C,Smith S A,et al.Techniques in Fish Immunology:Fish Immunology Technical Communication 4.New Jersey:SOS Publications,1995:141-145.
[37]Mangkalanan S,Sanguanrat P,Utairangsri T,et al.Characterization of the circulating hemocytes in mud crab(Scylla olivacea)revealed phenoloxidase activity[J].Developmental&Comparative Immunology,2014,44(1):116-123.
[38]Ding Z F,Du J,Ou J T,et al.Classification of circulating hemocytes from the red swamp crayfish Procambarus clarkii and their susceptibility to the novel pathogen Spiroplasma eriocheiris in vitro[J].Aquaculture,2012,356-357:371-380.
[39]Durliat M,Vranckx R.Action of various anticoagulants on hemolymphs of lobsters and spiny lobsters[J].The Biological Bulletin,1981,160(1):55-68.
[40]S?derh?ll I.Crustacean hematopoiesis[J].Developmental&Comparative Immunology,2016,58:129-141.
[41]Jiravanichpaisal P,Sricharoen S,S?derh?ll I,et al.White spot syndrome virus(WSSV)interaction with crayfish haemocytes[J].Fish Shellfish Immunology,2006,20(5):718-727.