Fin cell line from isogeneic ginbuna crucian carp
Fin cell line from isogeneic ginbuna crucian carp
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同基因银鲫鱼鳍细胞系
DOI：
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发表时间：
1997
期刊：
影响因子：
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通讯作者：
Y. Ikeda
中科院分区：
文献类型：
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作者：
S. Hasegawa;C. Nakayasu;N. Okamoto;T. Nakanishi;Y. Ikeda
尊敬的编辑：对于T细胞介导的免疫的研究，近交动物和/或来自它们的细胞系是必不可少的，因为T淋巴细胞的抗原识别在遗传上受到主要组织相容性复合体（MHC）的限制。然而，在鱼类中，来自近交鱼类的细胞系是罕见的。我们尝试建立近交鱼类细胞系，并成功地将等基因的芥菜耳Carass ius aura tus langsdorf l i细胞系传至第83代。同基因银鲫是一种天然雌核发育的鱼类，目前已建立了几个异基因的银鲫无性系。这些克隆鱼已被用于研究免疫的过继转移（Nakanishi，1987 a，1987 b）和移植排斥（Nakanishi，1987 e）。这些报告表明，近交系银纹鲤，像近交系小鼠，是足够的免疫学研究等基因。已经报道了硬骨鱼中的MHC序列（Hashimoto等人，1990;狄克逊等人，1995），并且在鲤鱼中也发现了针对（2，4，6-三硝基苯基）TNP修饰的自体细胞的特异性细胞毒性（Verlhac等人，1990年）。因此，来自细长鱼的细胞系可用于研究介导的细胞毒性（例如，细胞毒性T细胞（CTL）或自然杀伤（NK）细胞。从国立水产研究所获得了来自Suwa湖的雌性和一岁等亲鱼鲫鱼Carass ius aura tu s langsdorf ii。从鱼上切下尾鳍，在1%次氯酸钠溶液中浸泡1分钟，然后在70%乙醇中冲洗几秒钟。用无菌剪刀将翅片切成约1-mm的立方体，并将立方体转移到25 cm 2的长颈烧瓶（Corning Labs，Inc.，Coming Labs，Inc. Coming，NY），并使其保持2小时，以使它们粘附在烧瓶的内表面上。培养基（MEM-IO）由Eagle最低必需培养基（MEM; Nissui Pharmaceutical Co.，Tokyo，Japan），补充有10%胎牛血清（FBS; Commonweahh Serum Laboratories，墨尔本，维多利亚，澳大利亚）、4.5 mM碳酸氢钠（NaHCO 3）、4 mM L-谷氨酰胺和14 mM N-[2-羟乙基]哌嗪-N '-[2-乙磺酸]（HEPES; Sigma Chemical Co.，圣路易斯，密苏里州）。将含有鳍块的烧瓶在20 ° C下与MEM-10-起孵育，并使用在含有0.02%乙二胺四乙酸二钠盐（EDTA）的磷酸盐缓冲盐水（PBS）中的0.1%胰蛋白酶（DIFCO Laboratories，Detroit，MI）的分散剂将细胞在汇合处传代培养。这些细胞被命名为CFS（来自Suwa湖的鲫鱼鳍）细胞。将已传代52次的CFS细胞用于确定最佳生长温度。将1 ml的等分试样（5.0 × 10细胞/ml）接种到12孔培养板（Coming Labs）的3个威尔斯孔中。在4、10、15、20、25、30和37 ° C下孵育7天后，除去每个孔的上清液，用分散剂分散细胞，并在4 ° C下以800 × g离心5分钟，如图1所示。一个融合的单层成纤维细胞样细胞'的鳍银鲫'的同基因型'的Suwa湖。该细胞系被命名为CFS。酒吧100 ~上午。
Dear Editor: For the study of T cell-mediated immunity, inbred animals and/or cell lines derived from them are essential because antigen recognition by T lymphocytes is genetically restricted by the major histocompatibility complex (MHC). In fish, however, cell lines from inbred fishes are rare. We tried to establish a cell line from inbred fish and have succeeded in passaging and maintaining a cell line of isogeneic ginbuna erucian earp Carass ius aura tus langsdorf l i through the 83rd passage. Isogeneic ginbuna crucian carp is a naturally oecun'ing gynogenetic fish and several clones that are allogeneie to one another have been established. These clonal fishes have been used to investigate adoptive transfer of immunity (Nakanishi, 1987a, 1987b) and graft rejection (Nakanishi, 1987e). These reports have shown that inbred ginbuna crueian carp, like inbred mice, are sufficiently isogeneic for immunological studies. MHC sequences in teleosts have been reported (Hashimoto et al., 1990; Dixon et al., 1995), and specific cellular eytotoxieity against (2,4,6-trinitrophenyl) TNP-modified autologous cells has also been found in carp (Verlhac et al., 1990). Therefore, cell lines from elonal fish are useful for studying cellmediated eytotoxicity (e.g., eytotoxic T cells (CTL) or natural killer (NK) cells. Female and yearling isogeneie ginbuna erueian carp, Carass ius aura tu s langsdorf i i , which originated from Lake Suwa, were obtained from the National Research Institute of Aquaculture. Caudal fins were cut fi'om the fish, bathed in 1% sodium hypochlorite solution for 1 min, and then rinsed in 70% ethyl alcohol for a few seconds. The fins were cut into approximately 1-mm cubes with sterile scissors, and the cubes were transferred to 25 cm 2 euhure flasks (Corning Labs, Inc., Coming Labs, Inc., Coming, NY), and allowed to do~ for 2 h so that they would stick to the inside surface of the flasks. Culture medium (MEM-IO) was prepared from Eagle's minimum essential medium (MEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Commonweahh Serum Laboratories, Melbourne, Victoria, Australia), 4.5 mM sodium bicarbonate (NaHC03), 4 mM L-glutamine, and 14 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES; Sigma Chemical Co., St. Louis, MO). Flasks containing the fin cubes were incubated at 20 ° C with MEM-10 and the cells were subcultured at confluency using a dispersant of 0.1% trypsin (DIFCO Laboratories, Detroit, MI) in phosphate-buffered saline (PBS) containing 0.02% ethylenediaminetetraacetic acid disodium salt (EDTA). These cells were designated CFS (Carassius fin from Lake Suwa) cells. CFS cells that had been passaged 52 times were used to determine the optimal growth temperature. Aliquots of 1 ml (5.0 X 1@ eells/ml) were plated into 3 wells of 12-well culture plates (Coming Labs). After 7 d of incubation at 4, 10, 15, 20, 25, 30, and 37 ° C, the supernatant of each well was removed and cells were dispersed with the dispersant and centrifuged at 800 × g for 5 min at 4 ° C to FIG. l. A confluent monolayer of fibroblastlike cells fi'om fins of an isogeneic ginbuna crucian carp fi'om Lake Suwa. This cell line was designated CFS. Bar 100 ~am.