CN113637632A - 一种银鲳肌肉组织细胞系 - Google Patents
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Abstract
本发明提供一种银鲳肌肉细胞系,所提供的银鲳肌肉组织细胞系,为银鲳肌肉细胞PaM,其保藏编号为CCTCC NO:C2021154。本发明所提供的银鲳肌肉组织细胞系可连续传代,迄今已传至50代并保持稳定,能够提供大量稳定的银鲳肌肉细胞,用于营养代谢、功能基因研究和育种研究等。所提供的细胞系性状优良,为成纤维细胞,细胞呈梭形或不规则三角形,胞体向外延伸出几个不规则突起,细胞间排列紧密。细胞传代时间短,可冻存和复苏,并可进行siRNA干扰、质粒转染等多类分子生物学实验。
Description
技术领域
本发明属于鱼类细胞培养技术领域,具体涉及一种银鲳肌肉组织细胞系。
背景技术
银鲳(Pampus argenteus)隶属于鲈形目(Perciformes)、鲳科 (Stromateidae)、鲳属(Pampus),其味道鲜美,营养丰富,分布广泛,是我国重要海水经济鱼类之一。近年来,在保护渔业资源和满足市场需求的双重要求下,我们团队展开银鲳人工繁育工作,并于近年来获得重大突破,逐步实现产业化。然而,在银鲳人工繁育工作迅速推进的同时,也面临着基础研究薄弱、病害频繁发生等突出问题,因此对银鲳病害防治、良种选育、遗传背景的研究极为迫切。
鱼类细胞培养最初始于20世纪60年代,由Wolf和Quimby在1962年建立了全球首个鱼类细胞系——虹鳟性腺细胞系RTG-2,而随着细胞培养技术的不断发展,迄今为止已经建立300余株鱼类细胞系,其中大部分鱼类细胞株系是从淡水鱼类和洄游性鱼类离体培养而来,而海水鱼类的细胞系占有 30%左右。相比于直接使用鱼类个体进行实验,利用鱼类细胞系进行实验有重复性高、操作简便、不受场地季节约束等优势。鱼类细胞系已经成为鱼类生理调控机制、功能基因组学和环境毒理学研究的重要工具,也是研究生物进化、发育和遗传的有效材料。
目前,鱼类细胞系主要来源于鱼类的鳍条、肝脏、性腺、大脑、脾脏和胚胎等,而对鱼类肌肉细胞的培养和建系相对较少,目前主要建系的肌肉细胞株系有大西洋鲑鱼、金头鲷和虹鳟等。肌肉参与脂肪累积、代谢调控、病原免疫等多种生理活动,对鱼类营养、发育及毒理病理等方面的研究具有重要意义。虽然鱼类细胞原代培养技术目前虽然是一种较为成熟的技术,但是要获得稳定传代、适用于实验模型构建的细胞系仍存在较大偶然性及困难性。
发明内容
本发明的目的在于提供一种银鲳肌肉细胞系及其构建方法,所保藏的细胞系传代稳定、存活率高;从而弥补现有技术的不足。
本发明所提供的银鲳肌肉组织细胞系,为银鲳肌肉细胞PaM,于2021 年6月8日保藏在中国典型培养物保藏中心(中国、武汉、武汉大学),保藏编号为CCTCC NO:C2021154;
本发明提供的银鲳肌肉细胞PaM是从银鲳幼鱼肌肉组织细胞构建后筛选获得的。
本发明所提供的银鲳肌肉细胞PaM,其建立方法包括如下的步骤:
一种上述银鲳肌肉组织细胞系的建立方法,包括以下步骤:
1)银鲳肌肉组织的获取:
取6月龄的银鲳幼鱼的肌肉组织,用包含有青霉素和链霉素的D-hanks 溶液漂洗后,移入基础培养基中;
2)原代培养:
将银鲳肌肉组织剪碎至1mm3大小的组织块,用胰蛋白酶消化,再加入完全培养基终止消化,用完全培养基重悬沉淀的细胞团,并加入组织块在后 26℃培养箱中培养;
3)传代培养:原代培养至贴壁细胞增殖至70-80%覆盖率时,移除旧培养基,并用含青霉素-链霉素双抗的D-hanks溶液清洗除去多余的血清和二价金属离子;然后用0.25%胰蛋白酶消化贴壁细胞,再进行传代培养获得银鲳肌肉组织细胞系。
在本发明的一个优选实施方案中,所述完全培养液为以Leibovitz’s L15为基础,包括终浓度分别为20%胎牛血清FBS,5‰β-巯基乙醇、50μg/ml N- 乙酰葡糖胺、50μg/ml羧甲基纤维素钠、10μg/l人FGF-basic、5μg/l人EGF、 10μg/l人GH、5μg/l人HGF、100IU/ml青霉素以及100μg/ml链霉素。
本发明的细胞系,选用添加20%FBS的完全培养基进行传代培养。
本发明所提供的银鲳肌肉组织细胞系用于营养代谢、功能基因研究和育种研究。
本发明所提供的银鲳肌肉组织细胞系可连续传代,迄今已传至50代并保持稳定,能够提供大量稳定的银鲳肌肉细胞,用于营养代谢、功能基因研究和育种研究等。所提供的细胞系性状优良,为成纤维细胞,细胞呈梭形或不规则三角形,胞体向外延伸出几个不规则突起,细胞间排列紧密。细胞传代时间短,可冻存和复苏,并可进行siRNA干扰、质粒转染等多类分子生物学实验。
附图说明
图1:银鲳肌肉细胞系原代培养第5天的显微镜照片图(10×);
图2:银鲳肌肉细胞系复苏后培养24小时的显微镜照片图(10×);
图3:银鲳肌肉细胞系复苏后培养72小时的显微镜照片图(10×);
图4:银鲳肌肉细胞系的染色体分析及核型分布图;
图5:银鲳肌肉细胞系在不同浓度胎牛血清中培养7天细胞活性检测图;
图6:银鲳肌肉细胞系在不同浓度胎牛血清中培养7天的细胞数量图;
图7:细胞系干扰0.5μl FAM-siRNA 6小时的显微镜照片图(10×)。
具体实施方式
本发明使用的完全培养液包含L15培养基、胎牛血清FBS、β-巯基乙醇、N-乙酰葡糖胺、羧甲基纤维素钠、人FGF-basic、人EGF、人GH、人HGF、青霉素和链霉素。其中L15培养基、胎牛血清FBS为细胞生长提供必须的营养物质;β-巯基乙醇、N-乙酰葡糖胺、羧甲基纤维素钠、人FGF-basic、人EGF、人GH、人HGF等为细胞提供稳定的生长环境,并刺激细胞增殖,提高细胞活性;青霉素和链霉素则扩大了抗菌谱,特别是在原代培养时能有效抑制细菌生长,防止污染。
下面结合附图及具体实施方式对本发明的技术方案进行进一步的说明和描述。
实施例1:细胞系的构建
本发明采用的银鲳鱼均来自浙江宁波市象山港湾水产苗种有限公司。培育温度为16-32℃,盐度26-30,pH:8.21±0.3,溶解氧:7.35±0.05mg/L。健康、生长旺盛,体重50g。
溶液配制
D-hanks溶液:50mlD-hanks溶液+1ml100X青霉素-链霉素双抗溶液(购自Gibco公司),4℃储存。
基础培养基:49ml L15培养基(Leibovitz’s L15培养基,购自Gibco公司) +1ml100X青霉素-链霉素双抗溶液(购自Gibco公司),4℃储存。
完全培养基:10ml胎牛血清(fetal bovine serum,FBS,购自Gibco公司) +0.5ml100X青霉素-链霉素双抗溶液及39.5ml L15培养基(Leibovitz’s L15培养基,购自Gibco公司),4℃储存。
细胞冻存液:5mlDMSO(购自invitrogen公司)+25ml胎牛血清(fetal bovineserum,FBS,购自Gibco公司)+20ml L15培养基(Leibovitz’s L15培养基,购自 Gibco公司),-20℃储存。
本发明银鲳肌肉细胞系的建立方法的步骤如下:
1)将6月龄的银鲳幼鱼(长约9-11cm,重约50g)在含双抗的灭菌海水中暂养1-2h。随后滴入2-3滴丁香酚,直至鱼停止游动,侧躺于桶底,鳃盖张合停止,继而停止呼吸。以浸有75%酒精的棉球擦拭鱼体表2次,用无菌手术剪分离组织。取出的肌肉组织用含青霉素-链霉素双抗的D-hanks溶液漂洗3遍,后立刻移入基础培养基中短暂储存。
2)超净工作台中,将上述银鲳肌肉组织用无菌手术剪剪碎。每剪5min静置5min,待组织块沉淀后更换培养基,反复剪5-6次,直至组织块至1mm3大小。剪碎后加入2ml 0.25%胰蛋白酶消化10min。反复消化3次后加入完全培养基终止消化,700rpm离心5min除去多余胰酶影响。用1ml完全培养基重悬离心管底部的细胞团块,将含有细胞的完全培养液移入T25培养瓶(购自康宁公司)中培养,并适当加入少量肌肉组织块,置于26℃培养箱培养,24h后补全培养基至4ml。适量添加的组织块能分泌细胞生长必须的生长因子,促进细胞特别是原代细胞贴壁增殖。
如图1所示,银鲳肌肉细胞启动原代培养后5天,贴壁细胞及组织块周围即有新细胞迁出,第15天左右有小片细胞簇出现。
3)当原代细胞在培养瓶覆盖率达到70%-80%时,可以进行传代培养。移除旧培养基,并用含青霉素-链霉素双抗的D-hanks溶液清洗培养瓶底部。然后,用0.25%胰蛋白酶消化1min(时间不宜过长,消化过度会损伤细胞,造成其难以贴壁)。吸弃胰酶并加入4ml完全培养基终止消化,用该培养基吹打培养瓶底部使贴壁细胞悬浮,以1:2进行传代。传代后置于26℃培养箱培养。此后每隔3-8天传代一次,直至50代。培养期间细胞状态稳定,传代速度快,适合作为实验工具细胞。
最终筛选获得的银鲳肌肉组织细胞系命名为银鲳肌肉细胞PaM,于2021 年6月8日保藏在中国典型培养物保藏中心(中国、武汉、武汉大学),保藏编号为CCTCC NO:C2021154。
实施例2:银鲳肌肉细胞PaM的冻存与复苏
1)冻存:取一瓶处于对数生长期的银鲳肌肉细胞系细胞,采取经典的胰酶消化法,用2ml 0.25%胰蛋白酶消化后离心收集细胞沉淀。用1ml配制好的细胞冻存液轻轻吹打细胞沉淀,使其均匀的分散在冻存液中。细胞系冻存遵循梯度降温原则,冻存液配制为90%FBS+10%DMSO。冻存管在装有异丙醇的梯度降温盒进行程序性降温,在-80℃放置24h,异丙醇能够保证细胞在冻存液中逐步降温,最后放入液氮中可以长期冻存。
2)复苏:细胞复苏遵循快融原则。将冻存管从液氮中取出,迅速放入 37℃水浴锅融化,为使其快速均匀解冻,可以轻轻晃动冻存管,解冻时间不超过1min。将融化的细胞悬液700rpm,离心5分钟,除去二甲亚砜的影响。用4ml完全培养基重悬离心管底部的细胞,将细胞悬液移入T25细胞培养瓶,置于26℃培养箱中培养。
如图2,银鲳肌肉细胞系复苏后24h贴壁率达到60%-80%,与冻存前细胞形态相似,状态良好。如图3,复苏后细胞72h可铺满培养瓶,并能够正常传代。
实施例3:银鲳肌肉细胞PaM的染色体核型分析
1)PaM细胞的细胞遗传学分析在第20代进行。取处于对数生长期的银鲳肌肉细胞,用最终浓度为10μg/ml的秋水仙碱,继续培养5h后消化下细胞,得到细胞悬液。
2)将收集到的细胞悬液1000rpm离心10min,轻轻吸弃上清,逐滴滴加预热(提前进行37℃水浴)的3mL低渗液,并将细胞均匀分布于悬液中,再逐滴滴加5ml低渗液,轻轻颠倒离心管至水平2次,放至37℃水浴作用30 分钟。
向离心管中加入0.5ml预冷的卡诺氏固定液(新鲜混合的甲醇∶冰醋酸=3∶1),固定10min。
3)将上述细胞悬液2000rpm离心10min,吸弃上清,再取1ml卡诺氏固定液重悬细胞沉淀,室温固定30min。重复该步骤反复固定5次。
4)经过5次固定后,将细胞悬浮在0.2毫升固定剂中,并从60cm高度滴在冷湿载玻片上。水平放置使细胞悬液延展,放入80℃恒温干燥箱烤片 15min。
5)将烤片结束的玻片放入盛有Giemsa染液的染缸中染色10min,用无酶无菌水轻轻冲洗玻片表面,干燥后中性树脂封片。
染色体的数目与核型是细胞遗传学的基础,是鉴定生物种属和性别的重要指标。在细胞培养中,染色体是鉴定细胞来源以及其在培养过程中是否发生转化的重要指标。如图4所示,对银鲳肌肉细胞系的染色体分析显示其核型呈近似正态分布;58%观察到的分裂相细胞染色体数目为48条;染色体数目均在单倍体和四倍体之间(24-96条)。综上证明,本发明获得的细胞系与银鲳个体染色体数相同,再传代50代后还能保持遗传学特征稳定。
实施例4:银鲳肌肉细胞PaM的最佳胎牛血清浓度筛选
1)取第50代银鲳肌肉细胞,前一天接种至六孔板使细胞覆盖率至 40-60%。
2)采取不同血清浓度的完全培养基进行培养(10%/15%/20%/25%FBS),连续培养7天后用CCK8检测细胞活和血球计数板统计细胞数量的方法来判断细胞的呼吸频率及增殖速度。
如图5和6的结果显示,20%FBS组和25%FBS组的肌肉细胞增殖速度相似,而10%FBS组细胞由于缺乏营养物质而增殖缓慢,在7天培养期内细胞活性及细胞数量没有出现明显的变化。综合以上结果,优选添加20%FBS的完全培养基,为银鲳肌肉细胞系最经济有效的培养基。
实施例5:银鲳肌肉细胞PaM的脂质体介导的FAM-siRNA干扰
1)提前一天将银鲳肌肉细胞种植在24孔板中,以转染时细胞汇合度在 40%-60%为宜。
2)FAM-siRNA干扰:分别取0.5/1/1.5μl FAM-siRNA(终浓度为 10/20/30μM)和1.5μl Lipofectamine 3000(购自invitrogen公司)用减血清培养基opti-MEM(购自Gibco公司)稀释至25μl,孵育5min。将 Lipofectamine 3000稀释液与FAM-siRNA稀释液充分混合,室温静置15min。将50μl干扰复合物滴加到含有0.95ml opti-MEM培养基的细胞上,前后移动培养皿使其混合均匀。
RNA干扰技术通过抑制转录翻译,以阻止某特定基因的表达,成为后基因组时代验证基因功能和药物靶向的重要工具。干扰6h以后,在荧光显微镜下镜检,如图7,观察到报告的绿色荧光。说明建立的银鲳肌肉组织细胞系能够适用于小RNA干扰实验。
以上所述,仅为本发明得较佳实施例,故不能依此限定本发明得实施范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,都应仍属本发明涵盖范围。
Claims (6)
1.一种银鲳肌肉组织细胞系,其特征在于,所述的细胞系的保藏编号为CCTCC NO:C2021154。
2.如权利要求1所述的细胞系,其特征在于,所述的细胞系是从银鲳幼鱼肌肉组织细胞构建的。
3.如权利要求1所述的细胞系,其特征在于,所述的细胞系的建立方法如下:
1)银鲳肌肉组织的获取:
取6月龄的银鲳幼鱼的肌肉组织,用包含有青霉素和链霉素的D-hanks溶液漂洗后,移入基础培养基中;
2)原代培养:
将银鲳肌肉组织剪碎至1mm3大小的组织块,用胰蛋白酶消化,再加入完全培养基终止消化,用完全培养基重悬沉淀的细胞团,并加入组织块在后26℃培养箱中培养;
3)传代培养:原代培养至贴壁细胞增殖至70-80%覆盖率时,移除旧培养基,并用含青霉素-链霉素双抗的D-hanks溶液清洗除去多余的血清和二价金属离子;然后用0.25%胰蛋白酶消化贴壁细胞,再进行传代培养获得银鲳肌肉组织细胞系。
4.如权利要求3所述的细胞系,其特征在于,所述的完全培养液为以Leibovitz’s L15为基础,包括终浓度分别为20%胎牛血清FBS,5‰β-巯基乙醇、50μg/mlN-乙酰葡糖胺、50μg/ml羧甲基纤维素钠、10μg/l人FGF-basic、5μg/l人EGF、10μg/l人GH、5μg/l人HGF、100IU/ml青霉素以及100μg/ml链霉素。
5.权利要求1所述的细胞系的传代培养方法,其特征在于,所述的方法是使用添加20%FBS的完全培养基进行传代培养。
6.权利要求1所述的细胞系在营养代谢、功能基因研究和育种研究中的应用。
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