中国水产科学  2022, Vol. 29 Issue (10): 1407-1416  DOI: 10.12264/JFSC2022-0112
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引用本文 

巩志宏, 王娜, 刘娟娟, 胡国斌, 陈松林. 齐口裂腹鱼鳃丝细胞系的建立、鉴定及免疫研究[J]. 中国水产科学, 2022, 29(10): 1407-1416. DOI: 10.12264/JFSC2022-0112.
GONG Zhihong, WANG Na, LIU Juanjuan, HU Guobin, CHEN Songlin. Establishment, identification, and immunological study of gill filament cell line of Schizothorax prenanti[J]. Journal of Fishery Sciences of China, 2022, 29(10): 1407-1416. DOI: 10.12264/JFSC2022-0112.

基金项目

国家海洋水产种质资源库项目；中国水产科学研究院创新团队专项（2020TD20）；山东省泰山学者攀登计划项目；三峡工程鱼类资源保护湖北省重点实验室开放课题项目（191164740001）.

作者简介

巩志宏（1984-），男，博士研究生，研究方向为鱼类种质保存和抗病分子育种. E-mail: gongzhihong@stu.ouc.edu.cn

通信作者

通信作者：陈松林，研究员，研究方向为鱼类种质保存、性别控制与抗病分子育种. E-mail: chensl@ysfri.ac.cn

文章历史

收稿日期：2022-03-28
修改日期：2022-04-06
Contents              Abstract              Full text             Figures/Tables             PDF
齐口裂腹鱼鳃丝细胞系的建立、鉴定及免疫研究
巩志宏1,2,王娜2,刘娟娟3,胡国斌1,陈松林2,     
1. 中国海洋大学海洋生命学院，山东 青岛266071
2. 中国水产科学研究院黄海水产研究所，山东 青岛266071
3. 中国长江三峡集团有限公司中华鲟研究所，湖北 宜昌 443100
收稿日期：2022-03-28；修改日期：2022-04-06
基金项目：国家海洋水产种质资源库项目；中国水产科学研究院创新团队专项（2020TD20）；山东省泰山学者攀登计划项目；三峡工程鱼类资源保护湖北省重点实验室开放课题项目（191164740001）.
作者简介：巩志宏（1984-），男，博士研究生，研究方向为鱼类种质保存和抗病分子育种. E-mail: gongzhihong@stu.ouc.edu.cn.
摘要：齐口裂腹鱼（Schizothorax prenanti）是长江上游水域的珍稀鱼类，近年来由于环境变化和疾病爆发野外齐口裂腹鱼数量急剧减少，现已被列入长江上游二级急切保护的特有鱼类，因此，对齐口裂腹鱼的保护刻不容缓。建立齐口裂腹鱼细胞系是保护其种质资源的有效手段，也可以在不伤害现有鱼群的条件下进行多种齐口裂腹鱼相关生物学研究。本研究建立了首个来源于齐口裂腹鱼的细胞系，SPG细胞系。原代细胞分离自齐口裂腹鱼鳃丝组织，呈均一的上皮状，使用含15%血清的L-15培养，在15个月时间里成功传至55代。线粒体COI基因鉴定，证明该细胞来源于齐口裂腹鱼，核型检测细胞染色体数目为2n=96。在液氮中保存12个月的细胞，复苏后能保持75%以上活力。EGFP-N3质粒转染SPG细胞后观察到明显的绿色荧光蛋白表达。病毒类似物poly（I:C）和大肠杆菌脂多糖LPS可引起细胞IL-1β、IL-8、TNFa和TLR22等免疫相关基因表达量升高。表明本研究建立的齐口裂腹鱼鳃丝细胞系可用于免疫学研究。此外，此细胞系还将在种质保存，外源蛋白表达和齐口裂腹鱼体外生物学研究中发挥重要作用。
关键词：齐口裂腹鱼    SPG    细胞系    免疫研究    
Establishment, identification, and immunological study of gill filament cell line of Schizothorax prenanti
GONG Zhihong1,2,WANG Na2,LIU Juanjuan3,HU Guobin1,CHEN Songlin,2    
1. College of Marine Life, Ocean University of China, Qingdao 266071, China
2. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
3. Research Institute of Chinese Sturgeon, China Three Gorges Corporation, Yichang 443100, China
Abstract: Schizothorax prenanti is a rare fish in the upper reaches of the Yangtze River and used to be an important economic fish in its production area. However, in recent years, due to diseases and environmental changes, the number of Schizothorax prenanti in the wild has decreased sharply. Thus, there is an urgent need to protect this fish species. The establishment of Schizothorax cell lines is a cost-effective method to protect its germplasm resources. Since the establishment of the fish cell line RTG-2 in 1962, research on the establishment of fish cell lines has developed rapidly. A total of 783 cell lines have been established from various fish tissues, including embryos. However, thus far, reports on the establishment of cell lines from Schizothorax prenanti are lacking. The establishment of the Schizothorax prenanti cell line not only enables the protection of its germplasm resources but also facilitates research on its living environment toxicology, nutrition, immunology, and disease control without harming the existing population of this rare fish. Schizothorax prenanti can be infected with Streptococcus agalactiae in the natural environment, resulting in exophthalmos, surface hemorrhage, and neurological symptoms, as well as marked degeneration, necrosis, and inflammatory cell infiltration in various internal organs. Mucosal immunity is an important part of the immune system of this fish, wherein the gills, skin, and intestinal mucosa constitute the first line of defense against pathogenic microorganisms. Thus, the establishment of a stable growth of the gill filament cell line of Schizothorax prenanti will enable the study of immune mechanism and disease control of Schizothorax prenanti. The most commonly used media for fish cell culture are MEM and L-15, where L-15 is an amino acid-rich medium that does not require CO2 to maintain the pH of the medium. Among the 280 newly established fish cell lines in the world from 2010 to 2020, 180 use L-15 medium, accounting for 64% of the total. In this study, L-15 was used as the basis to prepare the Schizothorax prenanti culture medium. The primary cell medium was based on L-15 medium supplemented with 5 ng/mL bFGF, 5 ng/mL EGF, 27.5 μmol/L beta-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B, and 20% fetal bovine serum (FBS). The cell subculture medium was based on L-15 supplemented with 5 ng/mL bFGF, 5 ng/mL EGF, 27.5 μmol/L beta-mercaptoethanol, and 15% fetal bovine serum. The cell cryopreservation solution was made of 50% L-15 medium supplemented with 40% FBS and 10% DMSO. Schizothorax prenanti with a body length of about 11 cm was taken and placed in purified water containing 500 U/mL penicillin, 500 μg/mL streptomycin, and 1.25/mL amphotericin B overnight. On the second day, anesthesia was used, and the fish was wiped with 75% alcohol and moved to an ultra-clean bench. Ophthalmic scissors were used to cut the gill arches and gill filaments, which were transferred to a Petri dish containing PBS. The gill filaments were cut along the gill arch with a scalpel before rinsing five times in PBS containing 500 U/mL penicillin, 500 μg/mL streptomycin, and 1.25 μg/mL amphotericin B, changing the PBS wash solution each time. After washing, the gill filaments were placed in a petri dish containing PBS. Then, a scalpel was used to cut the gill filaments into 1-2 mm3 sections. A disposable Pasteur pipette was used to transfer the gill filament sections into a 15-mL centrifuge tube, to which PBS washing solution was added three times to remove surface debris and mucus. Then, the tissue sections were inoculated into T25 culture flasks, and as little medium as possible to cover the tissue blocks was added, without causing the tissue blocks to float or move. The cells were placed in a 24 ℃ incubator for static culture, to which 1 mL of fresh primary medium was replaced after 48 h. Cell migration was observed daily. When the cells migrated from the tissue section, 3 mL of medium was added to continue the culture, which was changed every three days to remove non-adherent cells and impurities. In this study, a cell line derived from Schizothorax prenanti (SPG strain) was established for the first time. The cells were isolated from the gill filament tissue of Schizothorax prenanti with a uniform epithelium. These were then cultured with L-15 containing 15% serum and successfully passed to 55 passages in 15 months. Mitochondrial COI gene identification proved that this cell was derived from Schizothorax prenanti, wherein the number of chromosomes in the karyotype was 2n = 96. Cells stored in liquid nitrogen for 12 months can maintain over 75% viability after resuscitation. EGFP-N3 plasmid was transfected into SPG cells, and marked green fluorescent protein expression was observed. The viral analog poly(I:C) and Escherichia coli lipopolysaccharide LPS increased the expression of immune-related genes, such as IL-1β, IL-8, TNFa, and TLR22, indicating that the SPG strain can be used for immunological and toxicological studies. In addition, this cell line also plays an important role in germplasm preservation, exogenous protein expression, and in vitro biological studies of Schizothorax prenanti. Although over 783 fish cell lines have been established in the world today, many of these have not been fully explored. Furthermore, a major problem in the field of fish cell line development is that many of the developed cell lines are not deposited in user-friendly cell banks. To facilitate the acquisition of this cell by researchers, the cell line established in this study has been stored in the National Marine Aquatic Germplasm Bank (YSFRI-C-2020-SPG).
Key words： Schizothorax prenanti     SPG    cell line    immunological    

长江流域鱼类资源丰富，但是长期以来由于过度捕捞、外来生物入侵、气候变化、环境污染和疾病爆发等原因，导致了长江流域多种鱼类的濒危和灭绝[1-2]。齐口裂腹鱼(Schizothorax prenanti)是长江上游水域的冷水底栖巡游鱼类，隶属鲤科(Cyprinidae)，裂腹鱼亚科(Schizothoracinae)，裂腹鱼属(Schizothorax)[3]，曾是产区重要的经济鱼类。近年来由于野外齐口裂腹鱼数量的急剧减少，现已被列入长江上游二级急切保护的特有鱼类[4-5]。因此，对齐口裂腹鱼的保护刻不容缓。建立齐口裂腹鱼细胞系是一种保护其种质资源的经济有效的方法。自1962年鱼类细胞系RTG-2建立以来，鱼类细胞建系研究迅速发展，至2020年已使用肝、脾、肾、性腺、皮肤、鳍条、鳃丝、肌肉、脑、眼、胚胎等多种鱼类组织建立783株细胞，但至今没有齐口裂腹鱼细胞建系的相关报道[6-9]。体外培养的细胞生长快速、成本低、结果容易重复，可轻易地在不同实验室操作。建立齐口裂腹鱼细胞系不仅可以保护其种质资源，还能在不伤害现有珍稀鱼群的前提下，进行其生存环境毒理学、营养学、免疫学及病害防治等研究。

2011—2020年间建立的280个细胞系中有8%来自于鳃组织[7]。使用卡特拉鲏(Catla catla)鳃组织建立的细胞系进行重金属毒理学检测其结果与活体有相同趋势[10]。露斯塔野鲮(Labeo rohita)鳃组织建立的细胞系可表达外源蛋白，用于毒理学检测的结果与活体呈正相关趋势[11]。来源于绿腹丽鱼(Etroplus suratensis)鳃丝的细胞系可表达外源蛋白，对野田病毒(nodavirus)敏感[12]。齐口裂腹鱼在自然环境中会感染无乳链球菌引起发病。感染无乳链球菌后会出现凸眼、体表出血和神经症状，多种内脏器官出现明显的变性、坏死以及炎症细胞浸润[13]。齐口裂腹鱼感染嗜水气单胞菌后鱼体表多处出现充血出血、溃疡，肝脏、肾脏和肌肉组织出现不同程度的损伤、变性和局部坏死[14-15]。齐口裂腹鱼在细菌类似物LPS刺激后多种炎症因子出现显著上调[16]。黏膜免疫是机体免疫系统的重要组成部分，鱼类的鳃、皮肤、肠的黏膜构成抵御病原微生物侵害的第一道防线[17]。建立齐口裂腹鱼鳃丝细胞系将为齐口裂腹鱼免疫机制研究和病害防治研究提供良好细胞载体。使用细胞进行免疫研究的优点包括节省时间、低成本、易于操作、不伤害现有鱼群数量，而且实验结果不受鱼体自身调节和实验应激的影响。

本研究使用鳃丝组织建立了来自齐口裂腹鱼的细胞系，命名为SPG细胞系。并对此细胞系进行了来源鉴定、细胞核型检测、冻存复苏验证、外源基因表达和在细菌提取物LPS及病毒类似物poly(I:C)刺激下免疫相关基因表达变化的研究。实验结果表明此细胞生长稳定，可用于齐口裂腹鱼种质保存，能表达外源蛋白和进行体外免疫学等研究，作为齐口裂腹鱼首个细胞系将在齐口裂腹鱼体外生物学研究中发挥重要作用。

1　材料与方法 1.1　实验材料 1.1.1　实验鱼

体长11 cm左右的齐口裂腹鱼，购自四川雅安。在实验室饲养2周以上，每日喂食1次。

1.1.2　主要试剂

二甲基亚砜(DMSO)、L-15培养基、PBS溶液、0.25%胰酶含EDTA、青霉素、链霉素和两性霉素B均购自索莱宝生物科技有限公司；碱性成纤维细胞生长因子bFGF和上皮细胞生长因子EGF购自碧云天生物技术有限公司；beta-巯基乙醇(2-Me) (VWR Life Science，美国); FBS胎牛血清(GIBCO，澳大利亚)；反转录试剂、PCR试剂和荧光定量PCR试剂购自宝日医生物技术(北京)有限公司；细胞培养瓶、细胞培养板、移液管、15 mL离心管、50 mL离心管均为Corning公司产品。

1.1.3　主要仪器设备

生物安全柜(力康HFsafe- 1200，中国)；细胞培养箱(SANYO MIR-153，日本)；细胞冻存液氮罐(金凤YDS-120-216，中国)；荧光倒置显微镜(Nikon TE2000-U，日本)；低温高速离心机(Eppendorf Centrifuge 5424 R，德国)；荧光定量PCR仪器(ABI 7500，美国)。

1.2　实验方法 1.2.1　培养基和细胞冻存液

鱼类细胞培养最常用的培养基是MEM和L-15，其中L-15是一种富含氨基酸的培养基，而且不需要二氧化碳来维持培养基的pH。2010―2020年近十年全球新建立的280个鱼类细胞系，有180个使用L-15培养基，占总数的64%[7-9]。本实验以L-15为基础，配制齐口裂腹鱼细胞培养基。原代培养基以L-15培养基为基础添加5 ng/mL bFGF, 5 ng/mL EGF, 27.5 μmol/L beta-巯基乙醇，100 U/mL青霉素，100 μg/mL链霉素，0.25 μg/mL两性霉素B和20%胎牛血清[18]。传代培养以L-15培养基为基础添加5 ng/mL bFGF, 5 ng/ml EGF, 27.5 μmol/L beta-巯基乙醇，15%胎牛血清。细胞冻存液为50% L-15培养基加入40% FBS和10% DMSO制成。

1.2.2　原代培养

取齐口裂腹鱼放置含500 U/mL青霉素、500 μg/mL链霉素和1.25 μg/mL两性霉素B的纯化水中过夜。第2天使用麻醉剂麻醉，用75%酒精擦拭鱼体，移入超净台中。使用眼科手术剪在超净台内剪下鳃弓和鳃丝，移入盛有PBS的培养皿中。使用解剖刀沿腮弓切下鳃丝，放入盛有含500 U/mL青霉素、500 μg/mL链霉素和1.25 μg/mL两性霉素B的PBS中漂洗5次，每次更换PBS洗液。洗完后将鳃丝移入盛有PBS的培养皿中，使用解剖刀将鳃丝切至1~2 mm3，使用一次性巴氏吸管将鳃丝组织块转移到15 mL离心管中，加入PBS洗液冲洗3次，洗掉表面碎屑和黏液。随后将冲洗干净的组织块接种于T25培养瓶，加入尽量少的培养基以没过组织块，又不至于让组织块漂浮移动。放入24 ℃培养箱中静置培养，细胞接种后48 h更换1 mL新鲜原代培养基，以除去散落未贴壁细胞，每日观察细胞外迁情况。当有细胞从组织块迁出后，添加培养基至3 mL继续培养，此后每3 d更换1次培养基以除去未贴壁细胞和杂质。

1.2.3　传代培养

原代细胞逐渐从组织块中迁出，待细胞铺满后进行传代培养。细胞传代时弃去培养液，加入3 mL PBS冲洗细胞，加入1mL胰酶消化30 s，用吸管吸走多余胰酶保留0.5 mL继续消化。待细胞整体圆缩脱落后，加入培养基轻轻吸打混匀后按1∶1分成2瓶传代培养[19]。

1.2.4　冻存与复苏

取对数生长期的SPG细胞，弃去培养液，加入3 mL PBS冲洗细胞，加入1mL胰酶消化，待细胞整体圆缩脱落后，加入1 mL培养基轻轻吹打将培养瓶中细胞全部转移到15 mL离心管中，1000 r/min 离心5 min。在生物安全柜内吸掉上清，向沉淀中加入1 mL细胞冻存液。将细胞悬液吸打均匀后转移到细胞冻存管中，放入梯度降温盒，放置-80 ℃冰箱过夜，第二天转至液氮中长期保存。细胞复苏时，从液氮中取出冻存的细胞，用镊子夹紧瓶盖，将瓶体没入37 ℃温水中轻轻摇动，使细胞尽快融化，当冻存的细胞大部分融化只剩少部分结冰未融化时，从37 ℃水浴中取出细胞。1000 r/min，离心5 min，弃去上清液，加入培养基重悬细胞。

细胞活力计算：复苏的细胞经台盼蓝染色后，使用倒置显微镜计数，其中染蓝色为死细胞，未被染色为活细胞。使用血球计数板，计数2个对角线大格的细胞，计算细胞活力。细胞活力=(活细胞数÷总细胞数)×100%。

1.2.5　最适血清浓度

取第40代细胞按细胞数为1×105 cell/well的密度将细胞接种到24孔细胞培养板。每12孔为一组，培养6 h后每组分别更换血清浓度为5%、10%、15%和20%的培养基。培养至24 h、48 h、72 h、96 h、120 h、144 h时，不同血清浓度组细胞经胰蛋白酶消化后，使用血球计数板计数。计算细胞在不同浓度血清中的增值速度。根据公式计算细胞倍增时间(doubling time, T2)：T2=Δt/log2(ΔN/N0+1) 在公式中N0为观察开始时的细胞数，Δt为观察的时间长度，∆N为时间长度Δt期间细胞数的增加量[20]。

1.2.6　细胞分子特征鉴定

根据NCBI齐口裂腹鱼粒体细胞色素氧化酶亚基1(cytochrome oxidase subunit 1, COI)序列(GenBank: KM364601.1)设计线粒体COI基因引物，SP-COI-F: GTATTTGGTG- CCTGAGCCGGAA; SP-COI-R: GCAGCCGTGA- AGTCATTCTACG。Trizol法提取第30代细胞RNA。反转录后PCR扩增SPG细胞线粒体COI基因片段。使用40 μL反应体系进行PCR扩增，体系中包括2.0 μL模板，2.0 μL SP-COI-F引物，2.0 μL SP-COI-R引物，14 μL H2O, 20 μL Taq DNA聚合酶2×MIX。反应条件为：94 ℃ 3 min, 94 ℃ 30 s, 50 ℃ 30 s, 72 ℃ 1 min, 2~4步33个循环，72 ℃延伸10 min, 4 ℃ 30 min。PCR产物使用1%琼脂糖凝胶电泳检测，将有目的条带的PCR产物送擎科生物有限公司测序。测序结果通过BLAST进行比对分析。

1.2.7　细胞核型检测

使用第40代SPG细胞进行核型分析。细胞传代后第48小时，在培养基中加入终浓度为0.1 μg/mL的秋水仙素，放置24 ℃培养箱中培养6 h。胰酶消化后，1000 r/min离心 5 min 收集细胞，弃上清，加入5 mL 0.075 mol/L的KCl低渗溶液作用30 min。加入5.0 mL预冷的卡诺固定液(甲醇∶冰乙酸=3∶1，体积比)固定 2 min。1000 r/min离心5 min，弃上清，离心管底部可见细胞团块明显变小，加入1 mL预冷的卡诺固定液室温固定30 min。1000 r/min离心5 min，用适当卡诺固定液重悬细胞，将细胞放入冰上预冷，在30 cm左右的高度处，用冷滴片法滴片，每个载玻片上滴加2~3滴细胞悬液，尽量使细胞均匀分散。待载玻片略微干燥后，将载玻片过火微烤，使载玻片进一步干燥。用浓度为5%的吉姆萨染液染色20 min。用自来水缓慢地冲掉吉姆萨染液，待干燥后用显微镜观察细胞核染色情况，选择细胞分裂中期染色体进行拍照统计[19]。

1.2.8　外源蛋白表达

按细胞数为4×105 cell/well的密度将细胞接种到12孔细胞培养板，过夜培养，第2天把12孔板中的培养基更换为含5% FBS的新鲜L-15培养基。将1 μg的pEGFP-N3质粒与2.5 μL lipo8000脂质体转染试剂分别稀释到50 μL的L-15中，室温静置5 min后将质粒溶液加入到脂质体中，轻轻混匀在室温下静置15 min，将质粒-脂质体混合物加入细胞。转染后24 h观察荧光蛋白表达情况。

1.2.9　LPS刺激

使用PBS溶解大肠杆菌脂多糖(lipopolysaccharide, LPS)配制成20 mg/mL备用。细胞从培养瓶中消化后使用培养基稀释成8×105 cell/mL, 1 mL每孔接种12孔板。培养过夜后按照1.5 μg/mL、5 μg/mL、15 μg/mL、50 μg/mL、150 μg/mL、500 μg/mL的终浓度在孔板中加入LPS，每3个孔为一组，对照孔加入等量PBS作为对照组[21-23]。加入LPS刺激12 h后，收细胞放置-80 ℃冰箱以备检测。

1.2.10　Poly(I:C)刺激

使用PBS将聚肌苷酸胞苷酸(polyinosinic-polycytidylic acid, poly(I:C))配制成20 mg/mL备用。细胞从培养瓶中消化后使用培养基稀释成8×105 cell/mL, 1 mL每孔接种12孔板。培养过夜后按照1.5 μg/mL、5 μg/mL、15 μg/mL、50 μg/mL、150 μg/mL、500 μg/mL的终浓度在孔板中加入poly(I:C)，每3个孔为一组，对照孔加入等量PBS作为对照组[24]。加入LPS刺激12 h后，收细胞放置-80 ℃冰箱以备检测。

1.2.11　荧光定量PCR

LPS和poly (I:C)刺激后样品使用Trizol法提取RNA，反转录成cDNA。使用荧光定量PCR法(quantitative real-time PCR, qPCR)检测IL-1β、IL-8、TNFa、TLR22等免疫相关基因表达情况[14,25](表1)。在Excel中使用2-ΔΔCT法计算基因相对表达量[26]，使用Prism 9制图。

表1 　荧光定量PCR检测相关引物 Tab. 1 　Quantitative real-time PCR detection related primers
2　结果与分析 2.1　细胞原代培养和传代培养

组织块接种后24 h周围有上皮状细胞迁出，视野内有部分散落的未贴壁细胞(图1 A)。使用吸管吸走未贴壁细胞加入新的培养基，此后每3 d换液1次，培养到第21天时细胞铺满细胞瓶80%(图1 B)，细胞汇合度不再增加，胰酶消化后传代培养。在前10代使用含20%胎牛血清的原代培养基，10代以后使用含15%胎牛血清的传代培养基，每5~7 d，按1∶2比例进行传代培养。细胞传代后贴壁良好生长稳定，现已传至55代以上，细胞低密度时呈现均匀分散的上皮状(图1 C)，细胞铺满后呈现上皮状细胞组成的致密单层(图1 D)。

图1 　倒置显微镜下观察的原代和传代培养的细胞形态A：组织贴片法原代培养24 h的细胞；B：培养21 d的原代细胞；C：第55代SPG细胞低密度形态；D：第54代SPG细胞高密度形态. Fig. 1 　Morphology of primary cell and subculture cell observed under an inverted microscopeA: Primary cell cultured for 24 h; B: Primary cell cultured for 21 d; C: Low-density SPG cells at passage 55; D: High-density SPG cells at passage 54.
2.2　细胞冻存复苏检测

取在液氮中分别冻存3个月、6个月、12个月的P15代细胞各2支，复苏后使用台盼蓝染色计算活力。结果显示复苏的细胞活力均大于75%。细胞复苏后能贴壁生长，生长状态良好，形态与冻存前无差异，可见SPG细胞可以在液氮中长期保存。现已将细胞系保存至国家海洋水产种质资源库，编号：YSFRI-C-2020-SPG。

2.3　细胞生长特性

SPG细胞在5%血清浓度培养基中几乎停止增长，随着血清浓度增高细胞生长速度变快。在血清浓度为20%的培养基中生长速度最快，接种后第7天细胞密度达到2.2×105 cell/well (图2)，细胞群体倍增时间最短为153.7 h (表2)。血清浓度为15%时细胞生长曲线与20%时相近。从细胞生长速度和血清消耗两方面考虑，我们选择含15%血清浓度的培养基作为细胞传代培养用培养基。

图2 　SPG细胞在不同血清浓度培养基中的生长情况 Fig. 2 　Growth of SPG cells in medium with different serum concentrations
表2 　细胞在不同血清浓度培养基中的倍增时间 Tab. 2 　The doubling time of cells in different serum concentration media
2.4　细胞特征鉴定

COI基因PCR产物电泳显示在1500 bp处有阳性条带(图3)，与设计引物时的预期长度相近。测序后blast结果显示扩增序列与NCBI中齐口裂腹鱼线粒体COI基因序列一致性为99.9%。

图3 　齐口裂腹鱼线粒体COI鉴定电泳图 Fig. 3 　COI identification electropherogram of Schizothorax prenanti
2.5　细胞核型检测

选择拍照清晰的100个分裂相细胞进行染色体计数，染色体最少为86条，最多为190条，其中52%的细胞染色体数目为96条(图4, 图5)。

图4 　齐口裂腹鱼细胞核型 Fig. 4 　Cell karyotype of Schizothorax prenanti
图5 　齐口裂腹鱼细胞染色体计数 Fig. 5 　Chromosome count of Schizothorax prenanti
2.6　荧光蛋白表达

使用Lipo8000脂质体转染试剂成功将pEGFP-N3质粒转入细胞中，转染后48 h可在细胞中观察到清晰的绿色荧光(图6)。

2.7　LPS刺激后几种免疫相关基因的表达情况

大肠杆菌脂多糖LPS刺激后细胞IL-1β、IL-8、TNFa和TLR22等4种免疫相关基因随LPS浓度的增加，表达量逐渐升高。IL-8在LPS浓度为500 μg/mL时相对表达量远高于低浓度时的表达量(图7)。

2.8　poly(I:C)刺激后几种免疫相关基因的表达情况

聚肌苷酸胞苷酸poly(I:C)刺激后细胞IL-1β、IL-8、TNFa和TLR22等4种免疫相关基因表达量均有升高，特别是当poly(I:C)浓度达到500 μg/mL时4种基因的表达量相比低浓度时有大幅度的升高(图8)。

图6 　pEGFP-N3质粒转染后48 h的荧光蛋白表达A：绿色荧光观察，B：明场观察，红色箭头指示发出绿色荧光的细胞位置. Fig. 6 　Fluorescent protein expression 48 h after pEGFP-N3 plasmid transfectionA: green fluorescence observation; B: bright field observation. Red arrows indicate the location of cells that emit green fluorescence.
图7 　LPS刺激后qPCR检测IL-8 (A)、TNFa (B)、TLR22 (C)和IL-1β (D)基因表达情况 Fig. 7 　Gene expression of IL-8 (A), TNFa (B), TLR22 (C) and IL-1β (D) detected by qPCR after LPS stimulation
图8 　poly(I:C)刺激后qPCR检测细胞IL-1β (A)、IL-8 (B)、TLR22 (C)和TNFa (D)的基因表达 Fig. 8 　Gene expression of IL-1β (A), IL-8 (B), TLR22 (C) and TNFa (D) detected by qPCR after poly(I:C) stimulation
3　讨论

齐口裂腹鱼作为曾经的重要经济鱼类，近年来由于野外齐口裂腹鱼数量的急剧减少，现已被列入长江上游二级急切保护的特有鱼类[6]，因此，对齐口裂腹鱼的保护刻不容缓。建立齐口裂腹鱼细胞系是保护其种质资源的有效手段，但至今没有齐口裂腹鱼细胞建系的相关报道。本研究于2019年10月从齐口裂腹鱼鳃丝组织中分离建立了SPG细胞系，已成功传至第55代，细胞生长稳定，液氮保存的复苏活性高于75%。SPG细胞呈均一上皮状，形态与纹鳢和卡特拉鱼分离的鳃丝细胞系相似[10,27]。

细胞系排除了体内复杂生理环境的影响，是体外研究疾病作用机制的良好工具。齐口裂腹鱼在自然环境中会感染无乳链球菌引起发病[13]。使用嗜水气单胞菌人工感染齐口裂腹鱼，导致其在 24 h全身充血出血，48 h明显出现眼球突出、溃疡等症状，甚至死亡[15]。齐口裂腹鱼感染嗜水气单胞菌后肠和肾脏IL-1β、TNFa和TLR22表达量都有显著升高[15]。齐口裂腹鱼在细菌类似物LPS刺激后多种炎症因子显著上调，包括TNFa, IL-1β和IL-8等[16]。本研究使用LPS刺激SPG细胞后IL-1β、IL-8、TNFa和TLR22等4种免疫相关基因表达量都有升高，并与LPS浓度的增加呈正相关。poly(I:C)可模拟病毒感染[28], poly(I:C)刺激下齐口裂腹鱼脾脏IL-1β、TLR22和TNFa等免疫表达明显量升高[29]，本研究建立的SPG细胞在poly(I:C)诱导下4个免疫相关基因IL-1β、IL-8、TNFa和TLR22的表达均有不同程度升高。说明此细胞系可以进行细菌和病毒相关的免疫学研究。

基因过表达和敲除是研究基因功能的常用方法。本研究使用脂质体转染试剂成功将EGFP-N3质粒导入细胞，并在细胞中成功表达了绿色荧光蛋白。说明此细胞系可转入和表达外源基因蛋白，可作为基因敲降或过表达研究的目标细胞系，也有作为蛋白表达工程细胞株的潜力。

尽管如今全球已建立了783 种以上的鱼类细胞系。然而，人们对许多已经建立的细胞系尚未充分探索研究。而且鱼类细胞系开发领域的一个主要问题是，许多已开发的细胞系并未存放在方便使用者获取的细胞库中[7]。为方便有需要的科研工作者获取本细胞，本研究建立的细胞系已保存至国家海洋水产种质资源库，编号：YSFRI- C-2020-SPG。

总之，本研究使用长江珍稀鱼种齐口裂腹鱼鳃丝组织建立了SPG细胞系，并对细胞系进行鉴定和应用研究。此细胞可用于齐口裂腹鱼种质保存，能代替活体进行免疫学和毒理学相关研究，有表达外源蛋白的能力，作为齐口裂腹鱼首个细胞系将在齐口裂腹鱼体外生物学研究中发挥重要作用。

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