镉对鲫外周血单个核细胞凋亡的诱导
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苏州大学农业科技学院

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科技部农业科技成果转化基金,江苏省科技发展计划,江苏省苏州市科技局资助项目,苏州大学校科研和教改项目


Cadmium-induced apoptosis to peripheral blood mononuclear cells in crussian carp, Carassius auratus
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College of Agricultural Science and Technology,Suzhou University,Suzhou215006,China

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    摘要:

    以Cd2 浓度1.25mg?kg-1腹腔注射染鲫,以富含T、B、NK细胞及单核细胞的鲫外周血单个核细胞(PBMC)为效应细胞,用亚显微形态分析、单细胞凝胶电泳(SCGE)、DNA凝胶电泳和流式细胞术(FCM)等方法进行Cd2 诱导鲫PBMC凋亡的研究.电镜显示染镉细胞的染色质边聚、中聚并向胞浆突出和断裂,胞浆内细胞器结构不清晰并见许多大小不等的空泡;对照细胞的核呈马蹄形,染色质分布均匀,胞浆内细胞器结构清晰可辨.SCGE显示染镉细胞呈现DNA边聚化的彗星小头及DNA片段移动形成的彗星尾,对照细胞的核骨架边缘清晰;染镉处理14、17和21d的细胞凋亡率分别为19.4%、16.4%和5.6%,明显高于对照组(1.1%)(P<0.05).DNA凝胶电泳显示染镉细胞出现180~220bp整数倍的DNA梯状带,14和17d的梯状带较21d明显;对照细胞的DNA为一条带.FCM显示染镉细胞的亚二倍体峰清晰可见,对照细胞的亚二倍体峰几乎看不见;染镉处理14、17和21d的亚二倍体峰细胞量分别为17.5%、23.6%和8.2%,明显高于对照组(1.4%)(P<0.05).因此,体内染镉可诱导鲫PBMC凋亡并在14~17d达到峰值,21d时则可能由于DNA的损伤修复而得以缓解,由此推测镉诱导鲫PBMC凋亡是其致细胞病变死亡的重要表现形式之一,低浓度镉致鲫免疫系统损伤,极有可能通过直接诱导免疫细胞凋亡而实现.

    Abstract:

    At present , cadmium2induced damage to fish immune system especially immune cells is hardly seen in references. In the paper , the auther studied for the first time the cadmiumly2induced apoptosis to peripheral blood mononuclear cell (PBMC) . In the paper , the auther studued for the first time the cadmiumly2induced apoptosis to peripheral blood mononuclear cell (PBMC) of crussian carp Carassius auratus (mainly T cell ,βcell and monocyte) by electron microscopy , single cell gel electrophoresis (SCGE) , DNA gel electrophoresis and flow cytometry (FCM) . At the beginning of our procedure , fish sample (400g even purchased from Suzhou Xiangcheng District aquafarm were randomly assigned to glass aquaria ( 100cm ?60cm ?50cm) at five of each. All the culture conditions were kept for 4 weeks including continuously aeration , twice diets daily (08 :00 and 14 :00) at the rate of 3 % body weight , half to two2thirds of daily water exchange , 23 to 27 ℃temperature range , and over 5mg?L -1dissolved oxygen content. Then , the test fish were intraperitonealy injected with Cd2 at 1. 25mg?kg 1 , and the control is Cd2 free. After injection on the 14th , 7th , 21st day , 5mL blood samples (within 150IU?mL -1 heparin) from the fish tail vein were mixed with equal volume PBS (0. 01mol?L-1, pH7. 2) , these duluted blood was gently covered by 5ml Ficoll2Paque , the band containing mainly PBMC between plasma and isolation solution was observed after being contrifuged at 1500r?min 1 for 10 min , then cellected the PBMC from this band , and washed twice in PBS , counted and adjusted to a density of 1 ?107 ind ?mL -1 in RPMI21640 plus 10 % FBS. Under electron microscope, the treated cells appeared chromatin marginating , condensating and extruding into cytoplasm , unclear structure of organelles and large vacuoles in cytoplasm , while the normal did evenly2distributed chromatin in U shape nuclei and intact organelles . By SCGE , the treated cells exhibited comet head of DNA margination and comet tails of DNA migration , while the normal did full nuclear skeleton ; the apoptotic rate was calculated according to the fraction of the apoptotic cells to the total cells at 14 , 17 and 21d were 19. 4 % , 16. 4 % , 5. 6 % respectively , which were significantly higher than that of the normal (1. 1 %) ( P < 0. 05) . By DNA gel electrophoresis , the treated cells had typical nucleosome bands (180 -220bp integer fold) 14 , 17 and 21d respectively , and these ladder bands at 14d and 17d were more apparent than those of 21d , while the normal had only an DNA band. In FCM , the treated cells showed aneuploid peaks , while the normal didn’t ; the nomber of cells in aneuploid peak were calculated aicording to the fraction of cells in aneuploid peak to the total cells at 14 , 17 and 21d were 17. 5 % , 23. 6 % and 8. 2 % respectively , which were significantly higher than that of the normal (1. 4 %) ( P < 0. 05) . The above evidences was suggested that cadmium could induce apoptosis of PBMC at this dose in vivo , and its apoptosis rate could reach summit valve in 14 -17d and then drop at 21d. So apoptosis of lower dose cadmium2stressed PBMC is likely to play a key role in the damage of cadmium to the immune system of C. auratus.

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丁磊.镉对鲫外周血单个核细胞凋亡的诱导[J].水产学报,2005,29(1):

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  • 收稿日期:2014-03-13
  • 最后修改日期:2014-03-13
  • 录用日期:2014-03-13
  • 在线发布日期: 2014-03-13
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