中华鲟幼鱼鳃上氯细胞的免疫定位研究
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摘要
为了研究氯细胞(CCs)在中华鲟(Acipenser sinensis Gray,1835)幼鱼鳃上的分布状况及形态特征,通过石蜡切片-HE染色法及冰冻切片免疫荧光染色法对氯细胞进行了定位研究。研究表明:利用棋盘法,确定一抗和二抗的最佳实验浓度分别为1∶1 500和1∶500时定位其鳃上的Na+/K+-ATPase免疫阳性细胞(NKA-IR细胞,即氯细胞)效果较好,可用于中华鲟幼鱼鳃上氯细胞的免疫定位研究。鳃丝与鳃小片上氯细胞数量分别为(4.72±0.34)个/100μm和(0.64±0.19)个/100μm,差异极显著(P<0.001);鳃丝与鳃小片上氯细胞平均面积无显著性差异(P>0.05),分别为(187.28±18.30)μm2和(159.53±16.72)μm2;鳃丝与鳃小片上氯细胞的细胞形状因子分别为0.72±0.33和0.68±0.46,差异不显著(P>0.05)。由此可见,中华鲟幼鱼鳃上氯细胞呈卵圆形,主要分布在鳃丝上,尤其是鳃丝及鳃小片基部区域,少量分布在鳃小片上,鳃丝与鳃小片上氯细胞大小基本一致。
In order to study the distribution pattern of chloride cells(CCs) on the gills of the juvenile Acipenser sinensis, immune localization of CCs was studied by using the methods of HE and immunofluorescence staining in this paper. The results indicated that the optimal dilution of the first antibodyα5 and the fluorescent second antibody Alexa 488 donkey-anti-mouse was 1∶ 1 500 and 1∶ 500,respectively,which had good effect on the location of Na+-K+-ATPase immunoreactive cells(NKA-IR cells,i. e.,CCs)and could be used to locate the CCs on the gills of juvenile A. sinensis. The number of the CCs on gill filaments(4. 72 ± 0. 34 ind /100 μm) was significantly(P < 0. 001) greater than that on gill lamella(0. 64 ± 0. 19 ind /100μm). The mean area of the CCs had no significant difference(P > 0. 05) between gill filaments(187. 28 ± 18. 30 μm2) and lamella(159. 53 ± 16. 72 μm2). The shape factor of the CCs in the gill filaments and gill lamella was 0. 72 ± 0. 33 and 0. 68 ± 0. 46,respectively,which showed no significant difference(P > 0. 05) between them. Thus,the CCs of juvenile A. sinensis were oval and mostly found on the gill filament,especially at the base,with a small amount on gill lamella. The CCs on the gill filament and lamella were of the same size.
In order to study the distribution pattern of chloride cells(CCs) on the gills of the juvenile Acipenser sinensis, immune localization of CCs was studied by using the methods of HE and immunofluorescence staining in this paper. The results indicated that the optimal dilution of the first antibodyα5 and the fluorescent second antibody Alexa 488 donkey-anti-mouse was 1∶ 1 500 and 1∶ 500,respectively,which had good effect on the location of Na+-K+-ATPase immunoreactive cells(NKA-IR cells,i. e.,CCs)and could be used to locate the CCs on the gills of juvenile A. sinensis. The number of the CCs on gill filaments(4. 72 ± 0. 34 ind /100 μm) was significantly(P < 0. 001) greater than that on gill lamella(0. 64 ± 0. 19 ind /100μm). The mean area of the CCs had no significant difference(P > 0. 05) between gill filaments(187. 28 ± 18. 30 μm2) and lamella(159. 53 ± 16. 72 μm2). The shape factor of the CCs in the gill filaments and gill lamella was 0. 72 ± 0. 33 and 0. 68 ± 0. 46,respectively,which showed no significant difference(P > 0. 05) between them. Thus,the CCs of juvenile A. sinensis were oval and mostly found on the gill filament,especially at the base,with a small amount on gill lamella. The CCs on the gill filament and lamella were of the same size.
引文
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[13]ALLEN P J,CECH J J,KULTZ D.Mechanisms of seawater acclimation in a primitive,anadromous fish,the green sturgeon[J].Journal of Comparative Physiology,2009,179(7):903-920.
[14]TAKEYASUK,TAMKUN M M,RENAUD K J,et al.Ouabain-sensitive Na+-K+-ATPase activity expressed in mouse L cells by transfection with DNA encoding the alphasubunit of an avian sodium pump[J].Journal of Biological Chemistry,1988,263(9):4347-4354.
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[17]TSE W K F,AU D W T,WONG C K C.Characterization of ion channel and transporter mRNA expressions in isolated gill chloride and pavement cells of seawater acclimating eels[J].Biochemicaland Biophysical Research Communications,2006,346(4):1181-1190.
[18]龚志锦,詹锫洲.病理组织制片和染色技术[M].上海:上海科学技术出版社,1994:5GONG Z J,ZHAN P Z.Preparing tissue sections and staining technology[M].Shanghai:Shanghai Science and Technology Press,1994:5.
[19]郑辉,罗红英,颜亚辉.免疫组织化学技术常见问题分析及对策[J].中国组织化学与细胞化学杂志,2007,16(1):126-127.ZHENG H,LUO H Y,YAN Y H.Analysis and solution for normal problems in the immunohistochemistry technique[J].Chinese Journal of Histochemistry and Cytochemistry,2007,16(1):126-127.
[20]赵云转,李英敏,张英怀.一种消除免疫组织化学非特异性染色的简易方法[J].河北医科大学学报,2003,24(4):227-227.ZHAO Y Z,LI Y M,ZHANG Y H.A method of eliminating unspecific staining in imunohischemistry[J].Journal of Hebei Medical University,2003,24(4):227-227.
[21]何新明,罗颖洁,杨通,等.免疫组织化学染色技术常见问题的研究与探讨[J].中国免疫学杂志,2011(s1):1188-1190,1194.HE X M,LUO Y J,YANG T,et al.Research and analysis on normal problems in the immunohischemistry technique[J].Chinese Journal of Immunology,2011(s1):1188-1190,1194.
[22]KANG C K,LIU F C,CHANG W B,et al.Effects of low environmental salinity on the cellular profiles and expression of Na+-K+-ATPase and Na+,K+,2Cl-cotransporter 1 of branchial mitochondrion-rich cells in the juvenile marine fish Monodactylus argenteus[J].Fish Physiology and Biochemistry,2012,38(3):665-678.
[23]OLSON K R,FROMM P O.A scanning electron microscopestudy of secondary lamellae and chloride cells of rainbow trout(Salmo gairdneri)[J].Zeitschrift für Zellforschung und Mikroskopische Anatomie,1973,143(4):439-449.
[24]LIN Y M,CHEN C N,LEE T H.The expression of gill Na+-K+-ATPase in milkfish,Chanos chanos,acclimated to seawater,brackish water and fresh water[J].Comparative Biochemistry and Physiological,2003,135:489-497.
[25]SHIKANO T,FUJIO Y.Immunolocalization of Na+-K+-ATPase in branchial epithelium of chum salmon fry during seawater and freshwater acclimation[J].Journal of Experimental Biology,1998,201(22):3031-3040.
[26]CINOI C,MERICH D,CATALDI E,et al.Fine structure of chloride cells in freshwaterand seawater adapted Oreochromis niloticus(Linnaeus)and Oreochromis mossambicus(Peters)[J].Journal of Fish Biology,1991,39(2):197-209.
[27]ALTIONOK I,GALLI S M,CHAPMAN F A.Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon,Acipenser oxyrinchusde sotoi[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,1998,120(4):609-616.
[28]ZHAO F,ZHUANG P,ZHANG L,et al.Changes in growth and osmoregulation during acclimation to saltwater in juvenile Amur sturgeon Acipenser schrenckii[J].Chinese Journal of Oceanology and Limnology,2010,28(3):603-608.
[2]HE X,ZHUANG P,ZHANG L,et al.Osmoregulation in juvenile Chinese sturgeon(Acipenser sinensis Gray)during brackish water adaptation[J].Fish Physiology and Biochemistry,2009,35(2):223-230.
[3]ZHAO F,QU L,ZHUANG P,et al.Salinity tolerance as well as osmotic and ionic regulation in juvenile Chinese sturgeon(Acipenser sinensis Gray,1835)exposed to different salinities[J].Journal of Applied Ichthyology,2011,27(2):231-234.
[4]赵峰,张涛,侯俊利,等.长江口中华鲟幼鱼血液水分渗透压及离子浓度的变化规律[J].水产学报,2013,37(12):1795-1800.ZHAO F,ZHANG T,HOU J L,et al.Changes in blood moisture,osmolality and ion concentration of juvenile Chinese sturgeon(Acipenser sinensis)in the Yangtze Estuary[J].Journal of Fisheries of China,2013,37(12):1795-1800.
[5]封苏娅,赵峰,庄平,等.中华鲟幼鱼鳃丝Na+-K+-ATPaseα亚基渗透调节的分子机制初步研究[J].水产学报,2012,36(9):1386-1391.FENG S Y,ZHAO F,ZHUANG P,et al.Preliminary studies on molecular mechanism of salinity regulation of Na+,K+-ATPaseα-subunit in gills of juvenile Chinese sturgeon(Acipenser sinensis)[J].Journal of Fisheries of China,2012,36(9):1386-1391.
[6]HIROSE S,KANEKO T,NAITO N,et al.Molecular biology of major components of chloride cells[J].Comparative Biochemistry and Physiology,2003,136(4):593-620.
[7]HWANG P P,LEE T H.New insights into fish ion regulationand mitochondrion-rich cells[J].Comparative Biochemistry and Physiology,2007,148A:479-497.
[8]林浩然.鱼类生理学[M].广州:中山大学出版社,2011:205-221.LIN H R.Fish Physiology[M].Guangzhou:Sun Yat-sen University Press,2011:205-221.
[9]HEIJDEN A,VERBOST P M,EYGENSTEYN J,et al.Mitochondria-rich cells in gills of tilapia(Oreochromis mossambicus)adapted to fresh water or sea water:quantification by confocal laser scanning microscopy[J].Journal of Experimental Biology,1997,200(1):55-64.
[10]RICHMAN N H,DIAZ S T,NISHIOKA R S,et al.Osmoregulatory and endocrine relationships with chloride cell morphology and density during smoltification in coho salmon(Onchorhynchus kisutch)[J].Aquaculture,1987,60(3/4):265-285.
[11]SHIRAISHI K,KANEKO T,HASEGAWA S,et al.Development of multicellular complexes of chloride cells in the yolk-sac membrane of tilapia(Oreochromis mossambicus)embryos and larvae in seawater[J].Cell Tissue Research,1997,288(3):583-590.
[12]CARMONA R,GALLEGO G M,SANZ A,et al.,Chloride cells and pavement cells in gill epithelia of Acipenser naccarii:ultrastructural modifications in seawater-acclimated specimens[J].Journal of Fish Biology,2004,64(2):553-566.
[13]ALLEN P J,CECH J J,KULTZ D.Mechanisms of seawater acclimation in a primitive,anadromous fish,the green sturgeon[J].Journal of Comparative Physiology,2009,179(7):903-920.
[14]TAKEYASUK,TAMKUN M M,RENAUD K J,et al.Ouabain-sensitive Na+-K+-ATPase activity expressed in mouse L cells by transfection with DNA encoding the alphasubunit of an avian sodium pump[J].Journal of Biological Chemistry,1988,263(9):4347-4354.
[15]PELIS R M,ZYDLEWSKI J,MCCORMICK S D.Gill Na+-K+-2Cl-cotransporter abundance and location in Atlantic salmon:effects of seawater and smolting[J].American Journal of Physiology-Regulatory,Integrative and Comparative Physiology,2001,280(6):1844-1852.
[16]EVANS D H,PIERMARINI P M,CHOE K P.The multifunctional fish gill:dominant site of gas exchange,osmoregulation,acid-base regulation,and excretion of nitrogenous waste[J].Physiological Reviews,2005,85(1):97-177.
[17]TSE W K F,AU D W T,WONG C K C.Characterization of ion channel and transporter mRNA expressions in isolated gill chloride and pavement cells of seawater acclimating eels[J].Biochemicaland Biophysical Research Communications,2006,346(4):1181-1190.
[18]龚志锦,詹锫洲.病理组织制片和染色技术[M].上海:上海科学技术出版社,1994:5GONG Z J,ZHAN P Z.Preparing tissue sections and staining technology[M].Shanghai:Shanghai Science and Technology Press,1994:5.
[19]郑辉,罗红英,颜亚辉.免疫组织化学技术常见问题分析及对策[J].中国组织化学与细胞化学杂志,2007,16(1):126-127.ZHENG H,LUO H Y,YAN Y H.Analysis and solution for normal problems in the immunohistochemistry technique[J].Chinese Journal of Histochemistry and Cytochemistry,2007,16(1):126-127.
[20]赵云转,李英敏,张英怀.一种消除免疫组织化学非特异性染色的简易方法[J].河北医科大学学报,2003,24(4):227-227.ZHAO Y Z,LI Y M,ZHANG Y H.A method of eliminating unspecific staining in imunohischemistry[J].Journal of Hebei Medical University,2003,24(4):227-227.
[21]何新明,罗颖洁,杨通,等.免疫组织化学染色技术常见问题的研究与探讨[J].中国免疫学杂志,2011(s1):1188-1190,1194.HE X M,LUO Y J,YANG T,et al.Research and analysis on normal problems in the immunohischemistry technique[J].Chinese Journal of Immunology,2011(s1):1188-1190,1194.
[22]KANG C K,LIU F C,CHANG W B,et al.Effects of low environmental salinity on the cellular profiles and expression of Na+-K+-ATPase and Na+,K+,2Cl-cotransporter 1 of branchial mitochondrion-rich cells in the juvenile marine fish Monodactylus argenteus[J].Fish Physiology and Biochemistry,2012,38(3):665-678.
[23]OLSON K R,FROMM P O.A scanning electron microscopestudy of secondary lamellae and chloride cells of rainbow trout(Salmo gairdneri)[J].Zeitschrift für Zellforschung und Mikroskopische Anatomie,1973,143(4):439-449.
[24]LIN Y M,CHEN C N,LEE T H.The expression of gill Na+-K+-ATPase in milkfish,Chanos chanos,acclimated to seawater,brackish water and fresh water[J].Comparative Biochemistry and Physiological,2003,135:489-497.
[25]SHIKANO T,FUJIO Y.Immunolocalization of Na+-K+-ATPase in branchial epithelium of chum salmon fry during seawater and freshwater acclimation[J].Journal of Experimental Biology,1998,201(22):3031-3040.
[26]CINOI C,MERICH D,CATALDI E,et al.Fine structure of chloride cells in freshwaterand seawater adapted Oreochromis niloticus(Linnaeus)and Oreochromis mossambicus(Peters)[J].Journal of Fish Biology,1991,39(2):197-209.
[27]ALTIONOK I,GALLI S M,CHAPMAN F A.Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon,Acipenser oxyrinchusde sotoi[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,1998,120(4):609-616.
[28]ZHAO F,ZHUANG P,ZHANG L,et al.Changes in growth and osmoregulation during acclimation to saltwater in juvenile Amur sturgeon Acipenser schrenckii[J].Chinese Journal of Oceanology and Limnology,2010,28(3):603-608.