CN117305218A - 一种暗纹东方鲀卵巢细胞系tov及其构建方法和应用 - Google Patents
一种暗纹东方鲀卵巢细胞系tov及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种暗纹东方鲀卵巢细胞系TOV及其构建方法和应用,所述暗纹东方鲀卵巢细胞系TOV保藏于中国典型培养物保藏中心(CCTCC),保藏日期为:2021年10月20日,保藏编号为CCTCC NO:C2021142,保藏地址:中国,武汉。所述暗纹东方鲀卵巢细胞系TOV为首次在体外成功培养,并可稳定连续传代,可提供大量的暗纹东方鲀卵巢来源细胞;且其呈现典型的上皮细胞形态,细胞生长温度为28℃,具有较强的增殖能力;所述暗纹东方鲀卵巢细胞显著表达河鲀毒素结合蛋白和有机毒物结合蛋白,可应用于河鲀毒素和有机毒物富集机制的体外研究平台;也可以应用于外源基因功能研究。
Description
技术领域
本发明涉及一种暗纹东方鲀卵巢细胞系TOV及其构建方法和应用,属于生物技术领域。
背景技术
暗纹东方鲀(Takifugu obscurus)俗称河鲀,是一种典型的江海洄游性鱼类,属于硬骨鱼纲、鲀形目(Tetrodontiformes)、鲀科(Tetradontidae)、东方鲀属(Takifugu)。野生河鲀的卵巢、肝、肾、血液等具有致命的河鲀毒素,而精巢、肌肉和皮肤无毒均可食用。随着自然环境的改变,目前野生河鲀数量迅速减少,人工养殖已经成为河鲀的主要产量来源。
目前,国内外关于暗纹东方鲀稳定细胞系建立的报道较为少见,除了本申请人建立的暗纹东方鲀精巢细胞系以外,尚未见其他能够稳定传代的暗纹东方鲀细胞系建立的报道。由于卵巢是河鲀毒素的主要富集组织之一,建立暗纹东方鲀卵巢细胞系为研究河豚毒素的富集过程提供了良好的体外研究平台,对于揭示河豚毒素在暗纹东方鲀体内的产生机制具有重要的意义。同时,卵巢细胞系可作为研究暗纹东方鲀性别发育和分化机理的细胞模型,也可以用于暗纹东方鲀免疫机制和基因功能的体外研究。因此,建立暗纹东方鲀卵巢细胞系有利于河鲀毒素,以及河鲀性别分化和发育相关领域的科学研究,在鱼类基因功能研究中也具有广泛的应用价值。
发明内容
发明目的:本发明所要解决的技术问题是提供了一种可稳定连续传代、具有较强增殖能力的暗纹东方鲀卵巢细胞系TOV及其应用。
技术方案:为解决上述技术问题,本发明提供了一种暗纹东方鲀卵巢细胞系TOV,所述暗纹东方鲀卵巢细胞系TOV保藏于中国典型培养物保藏中心(CCTCC),保藏日期为:2021年10月20日,保藏编号为CCTCC NO:C2021142,保藏地址:中国,武汉。
本发明还提供了一种所述暗纹东方鲀卵巢细胞系TOV的构建方法,包括以下步骤:
(1)原代培养:无菌条件下获取暗纹东方鲀卵巢组织,消化,加入暗纹东方鲀卵巢细胞培养液,进行原代培养;
(2)传代培养:当原代培养细胞长满瓶底时,吸弃旧培养液,加入胰酶消化细胞,消化完成后吸去胰酶,加入暗纹东方鲀卵巢细胞培养液重悬细胞,分瓶培养,直至建成可稳定传代的细胞系,即获得暗纹东方鲀卵巢细胞系TOV。
其中,所述暗纹东方鲀卵巢细胞培养液含有青霉素、链霉素、人碱性成纤维细胞生长因子、鼠表皮生长因子和胎牛血清。
本发明还提供了所述的暗纹东方鲀卵巢细胞系TOV在提高外源基因的表达量中的应用。
其中,所述外源基因包括EGFP基因。
本发明还提供了所述的暗纹东方鲀卵巢细胞系TOV在提高毒素表达量中的应用。
本发明还提供了所述的暗纹东方鲀卵巢细胞系TOV在提高毒素富集中的应用。
其中,所述毒素包括河鲀毒素TTX或有机毒物Tributyltin中的一种或两种。
有益效果:与现有技术相比,本发明具有如下显著优点:1、本发明提供的暗纹东方鲀卵巢细胞系TOV为首次在体外成功培养,并可以稳定连续传代,可提供大量的暗纹东方鲀卵巢来源细胞;
2、本发明提供暗纹东方鲀卵巢细胞呈现典型的上皮细胞形态,细胞生长温度为28℃,具有较强的增殖能力,已连续培养超过100代,细胞生长曲线正常、状态良好、特性稳定,可以冷冻保存;
3、本发明暗纹东方鲀卵巢细胞显著表达河鲀毒素结合蛋白和有机毒物结合蛋白,可以应用于河鲀毒素和有机毒物富集机制的体外研究平台;外源转染绿色荧光蛋白质粒可以观察到明显的绿色荧光,表明暗纹东方鲀卵巢细胞系可以应用于外源基因功能研究。
附图说明
图1为暗纹东方鲀卵巢细胞系传代培养不同代数后,在相差显微镜下观察的形态,呈现典型的上皮样细胞形态:A为暗纹东方鲀卵巢原代细胞;B为25代暗纹东方鲀卵巢细胞;C为65代暗纹东方鲀卵巢细胞;D为105代暗纹东方鲀卵巢细胞;
图2为第40代暗纹东方鲀卵巢细胞系的染色体:A为染色体数目分布;B为染色体分裂相;
图3为暗纹东方鲀卵巢细胞系在不同培养条件下的生长曲线图:A为不同的培养温度;B为不同血清浓度的培养基;
图4为暗纹东方鲀卵巢细胞系的性别SNP分子鉴定图;
图5为暗纹东方鲀卵巢细胞系转染绿色荧光质粒后的荧光显微图片;
图6为暗纹东方鲀卵巢细胞系中TBT-bp和PSTBP的PCR电泳图;
图7为暗纹东方鲀卵巢细胞系中河鲀毒素结合蛋白和有机毒物结合蛋白的基因表达图:A为卵巢和卵巢细胞中TBT-bp的相对表达量;B为卵巢和卵巢细胞中PSTBP的相对表达量。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1暗纹东方鲀卵巢细胞的原代培养和传代
(1)细胞的原代培养:取健康暗纹东方鲀鱼体(体重约100g),利用75%酒精进行体表消毒,随后放入提前紫外灭菌的超净台中,解剖取出卵巢组织,用含400U/ml青霉素、400μg/ml链霉素、10ng/ml的人碱性成纤维细胞生长因子(购自cell signaling technology公司)、20ng/ml的鼠表皮生长因子、15%胎牛血清的DFL细胞培养液(pH为7.2)漂洗3次。随后取出卵巢组织置于培养皿中,用灭菌手术剪将组织剪成约1mm3的小块,再加入1ml的0.25%胰酶,继续剪碎成糜状。室温静置15min后用细胞筛过滤,收集过滤后的细胞悬液离心弃上清,收集细胞沉淀,加入5ml DFL细胞培养液重悬细胞,转移至25cm2细胞培养瓶,置于28℃培养箱中培养48h后,在显微镜下观察细胞贴壁情况;吸弃培养瓶中的未贴壁细胞,更换新鲜培养基继续培养。
(2)细胞的传代培养:当原代培养细胞单层达培养瓶底面积90%时,吸弃旧培养液,加入PBS清洗细胞1次,吸出PBS后再加入1ml的0.25%胰酶室温(25℃)消化细胞15min;在显微镜下观察到细胞开始皱缩、大部分变成圆形时吸出胰酶终止消化,加入2ml新鲜细胞培养液,轻轻吸打使细胞脱离培养瓶底,按照1:1(v/v)比例分入两个25cm2细胞瓶,分别向每个细胞瓶补充细胞培养液至5ml,混匀后置于28℃培养箱中培养,此后每隔5~6天传代一次,分别在原代、第25代、65代和105代时进行细胞观察,结果如图1所示,原代分离的细胞形态大小不均一,随着传代次数的增加,细胞逐渐变得大小均一、形态稳定,呈现典型的上皮样细胞形态。
综上,本发明结合物理剪切和胰酶消化两种方法对卵巢组织细胞进行分离和原代培养,随后使用富营养培养基继续传代培养,继而首次成功构建了暗纹东方鲀卵巢细胞系TOV,可提供大量的暗纹东方鲀卵巢来源细胞。所述暗纹东方鲀卵巢细胞呈现典型的上皮细胞形态,细胞生长温度为28℃,已连续培养超过100代,增殖能力较强,生长曲线正常、状态良好、特性稳定,可以冷冻保存、复苏。本发明构建的暗纹东方鲀卵巢细胞系TOV保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2021年10月20日,保藏编号为CCTCC NO:C2021142,保藏地址:中国,武汉。
实施例2暗纹东方鲀卵巢细胞系的冻存复苏
(1)细胞的冻存:为保存细胞选取不同代数的细胞进行冻存,当细胞生长至铺满培养瓶底95%面积时,按实施例1的方法,吸弃旧培养基,加入1ml、0.25%胰酶消化液消化;观察到细胞收皱缩时吸出胰酶终止消化,再加入2ml新鲜细胞培养液洗吹使细胞脱离瓶底,随后将细胞移入5ml离心管,1000g离心5min后弃上清,加入1ml细胞冻存液重悬细胞,移入冻存管。将冻存管放入4℃预冷的程序降温盒中,-80℃放置24h,转移至液氮罐中长期保存。
(2)细胞的复苏:从液氮罐中取出冻存细胞,立即放入37℃水浴中,快速晃动冻存管使细胞融化,1000g离心5min后弃上清,加入1ml新鲜细胞培养液重悬细胞,将细胞悬液移入25cm2细胞培养瓶;补足培养基至5ml,置于28℃培养箱中培养。显微镜观察到细胞贴壁后,更换新鲜细胞培养液,5~6天后正常传代培养。复苏后的细胞存活率达65%,可传代培养,细胞形态和生长特性无改变。
实施例3暗纹东方鲀卵巢细胞的染色体分析
取第40代卵巢细胞,28℃培养48h后在培养基中加入5μL秋水仙素,使终浓度达到10μg/mL,继续培养6h后吸弃培养液,使用PBS冲洗后,加入胰酶消化,转移至15mL离心管中,1000g离心5min弃上清收集细胞,加入5mL、0.075mol/L的KCL溶液吹散沉淀,37℃水浴低渗处理30min,加入1mL预冷的卡诺固定液(甲醇:乙酸=3:1),预固定2min。1000g离心5min,弃上清,加入5mL预冷的卡诺固定液,37℃水浴固定20min,重复固定3次。将细胞重悬于0.5mL预冷的卡诺固定液中,用冷滴片法在预冷的载玻片上滴片,干燥后用10%的Giemsa染色10min,清水冲洗后自然晾干。显微镜下观察,选择100个分裂相进行分析和统计。从图2中可以看出,第40代暗纹东方鲀卵巢细胞的染色体众数为44条,染色体核型为2n=44t。表明暗纹东方鲀卵巢细胞的染色体数量确定,且数量符合二倍体鱼类的染色体数,也跟暗纹东方鲀精巢细胞染色体数一致,说明暗纹东方鲀卵巢细胞没有问题。
实施例4暗纹东方鲀卵巢细胞系生长特性测定
(1)最适培养温度的确定:取第45代卵巢细胞,设置20℃、24℃、28℃、32℃和37℃五个不同培养温度,按照1.5×105个细胞/mL的密度配制胎牛血清浓度为15%的细胞悬液,接种于12孔板,分别置于五种不同温度的培养箱中培养。此后6天每天同一时间,取每种温度的3孔细胞,胰酶消化后收集细胞,用细胞计数板计数。按照横坐标为培养时间,纵坐标为每ml培养液中的细胞数,绘制生长曲线。如图3A所示,细胞在24℃和28℃下生长状态良好,其中28℃下细胞生长速度相对快些。
(2)最适血清浓度的确定:
取第50代卵巢细胞,按照4×104个细胞/mL的密度分别配制胎牛血浓度为5%、10%、15%和20%的细胞悬液,接种于12孔板,置于28℃的培养箱中培养。此后6天每天同一时间,取每种血清浓度的3孔细胞,胰酶消化后收集细胞,用细胞计数板计数。按照横坐标为培养时间,纵坐标为每ml培养液中的细胞数,绘制生长曲线。如图3B所示,细胞在15%以上血清浓度的细胞培养液中生长状态比较稳定,细胞增殖速度随血清浓度上升而加快。
实施例5暗纹东方鲀卵巢细胞的分子鉴定
暗纹东方鲀amhr2基因的SNP位点检测:收集培养的暗纹东方鲀卵巢细胞,按照DNA提取试剂盒(Promega公司)的操作规程提取细胞总DNA。利用暗纹东方鲀amhr2基因的SNP差异位点设计引物进行PCR扩增。PCR反应体系为:Premix Taq 12.5μL,DNA模板0.5μL,上游引物(SEQ ID NO.1:5’-ACAAACCACCTGTGGCTCACG-3’)和下游引物(SEQ ID NO.2:5’-GAAATCAGAGCAGCACATCCA-3’)各1μL,补水至总体积25μL。PCR扩增条件为:预变性95℃,3min;变性PCR 95℃,30sec,退火55℃,30sec,延伸72℃,30sec,循环35次;再延伸72℃,5min,保存12℃,10min。PCR产物经纯化试剂盒(上海生工生物工程技术服务有限公司)纯化后测序,结果显示卵巢细胞amhr2基因的SNP位点为纯合基因型(C),符合暗纹东方鲀雌性遗传性状(雌性SNP位点为C纯合型,雄性SNP位点为C/G杂合型)(图4)。
实施例6暗纹东方鲀卵巢细胞的外源质粒转染实验
取生长良好的暗纹东方鲀卵巢细胞,常规方法消化传代,将细胞稀释至浓度为106个细胞/ml,接种于24孔板中,28℃培养过夜。待细胞铺板密度达85%时,用Lipofectamine2000(Invitrogen)进行转染,用50μl Opti-MEI培养基分别对0.8μg pEGFP-N3质粒(本身携带EGFP基因)和2μl Lipofectamine 2000进行稀释,室温孵育5min。再将稀释后的pEGFP-N3质粒和Lipofectamine 2000混合,移液枪吹打混匀后室温静置20min。将24孔板中的细胞培养液换成不含血清的DFL细胞培养液,每孔加入100μl转染液,28℃培养5h,再更换成含血清的DFL细胞培养液,继续培养18h后,在荧光显微镜下观察拍照。如图5所示,在转染了pEGFP-N3质粒的暗纹东方鲀卵巢细胞中可明显观察到绿色荧光,转染效率较高,表明暗纹东方鲀卵巢细胞可以高效表达外源基因,可以作为外源基因体外表达和功能研究的细胞模型。
实施例7河鲀毒素结合蛋白在暗纹东方鲀卵巢细胞中的检测
收集培养的暗纹东方鲀卵巢细胞,用Trizol方法提取细胞总RNA,按照TakaraPrimeScriptTM RT reagent Kit with gDNA Eraser(Perfect Real Time)试剂盒的操作步骤进行逆转录,获得cDNA模板。使用PCR技术检测河鲀毒素TTX结合蛋白PSTBP和有机毒物Tributyltin结合蛋白TBT-bp的表达,PCR反应体系为:Premix Taq 12.5μL,DNA模板0.5μL,上游引物和下游引物各1μL,补水至总体积25μL。所用引物分别为PSTBP(SEQ ID NO.3:5’-ATCTAACGAGTGGACGAAAC-3’和SEQ ID NO.4:5’-TCCATCTTGCCAGAAGTGTA-3’),TBT-bp(SEQID NO.5:5’-AGAATGGGGTTGTCACAGA-3’和SEQ ID NO.6:5’-CTTGGGCAGCCTTCAGTA-3’)。PCR扩增条件为:预变性95℃,3min;变性PCR 95℃,30sec,退火58℃,30sec,延伸72℃,30sec,循环35次;再延伸72℃,5min,保存12℃,10min。并利用real-time PCR的相对定量技术对比PSTBP和TBT-bp在卵巢组织(ovary)和卵巢细胞(ovary cell)中的表达量,结果显示TTX结合蛋白PSTBP和有机毒物结合蛋白TBT-bp的基因在暗纹东方鲀卵巢组织和卵巢细胞中均有表达(图6),在培养的卵巢细胞中表达量更高(图7),表明暗纹东方鲀卵巢细胞能够结合更大量的TTX和Tributyltin,可以作为河鲀毒素和有机毒物富集机制的体外研究平台。
Claims (8)
1.一种暗纹东方鲀卵巢细胞系TOV,其特征在于,所述暗纹东方鲀卵巢细胞系TOV保藏于中国典型培养物保藏中心(CCTCC),保藏日期为:2021年10月20日,保藏编号为CCTCC NO:C2021142,保藏地址:中国,武汉。
2.一种构建权利要求1所述暗纹东方鲀卵巢细胞系TOV的方法,其特征在于,包括以下步骤:
(1)原代培养:无菌条件下获取暗纹东方鲀卵巢组织,消化,加入暗纹东方鲀卵巢细胞培养液,进行原代培养;
(2)传代培养:当原代培养细胞长满瓶底时,吸弃旧培养液,加入胰酶消化细胞,消化完成后吸去胰酶,加入暗纹东方鲀卵巢细胞培养液重悬细胞,分瓶培养,直至建成可稳定传代的细胞系,即获得暗纹东方鲀卵巢细胞系TOV。
3.根据权利要求2所述的方法,其特征在于,所述暗纹东方鲀卵巢细胞培养液含有青霉素、链霉素、人碱性成纤维细胞生长因子、鼠表皮生长因子和胎牛血清。
4.权利要求1~3任一项所述的暗纹东方鲀卵巢细胞系TOV在提高外源基因的表达量中的应用。
5.根据权利要求4所述的应用,其特征在于,所述外源基因包括EGFP基因。
6.权利要求1~3任一项所述的暗纹东方鲀卵巢细胞系TOV在提高毒素表达量中的应用。
7.权利要求1~3任一项所述的暗纹东方鲀卵巢细胞系TOV在提高毒素富集量中的应用。
8.权利要求5~7任一项所述的应用,其特征在于,所述毒素包括河鲀毒素TTX结合蛋白PSTBP或有机毒物Tributyltin结合蛋白TBT-bp中的一种或两种。
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