CN107189986B - 一种基于内质网应激的报告基因细胞株构建方法及其应用 - Google Patents
一种基于内质网应激的报告基因细胞株构建方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于内质网应激的报告基因细胞株构建方法,该方法选用与内质网应激相关的CHOP基因作为特异性诱导基因构建CHOP启动子,再将CHOP启动子与便于检测的SEAP基因连接,构建慢病毒CHOP‑SEAP质粒载体;最后将CHOP‑SEAP质粒转染至Hela细胞中。本发明还公开了上述基于内质网应激的报告基因细胞株构建方法构建的报告基因细胞株的应用。本发明方法构建的报告基因细胞株能够指示早期细胞毒性损伤,大大提高细胞株的检测敏感性和降低检测限值,从而表征毒害污染物的早期毒性,且适用于大多数毒害污染物;同时还可表征实际水体的综合毒性。细胞株选用的报告基因SEAP基因具有便于检测、可动态监测的优点。
Description
技术领域
本发明涉及一种基于内质网应激的报告基因细胞株的构建方法,还涉及上述基于内质网应激的报告基因细胞株构建方法构建的报告基因细胞株的应用,属于污染物毒性细胞检测领域。
背景技术
据不完全统计,目前世界上已登记的化学物质约400万种,销售、生产、使用的化学物质约有5-6万种,经常使用的约4000多种,而且每年以2万种的速度增加。它们主要包括:化学药物、化学试剂、食品添加剂、农药、装饰材料、建筑材料、化妆品及军工产品等。化学品与人们的饮食、工作、生活及周围环境息息相关,密不可分。同时,化学物对人类健康和生物环境的影响及其危害,也日益受到人们的密切关注。新型的人体细胞毒性试验相较于传统毒性试验而言,可以更直接地体现毒害污染物对于人类健康的影响,但其敏感性仍存在局限性。报告基因法因其敏感、迅速、重现性好的特点,被广泛应用于毒性评价中。研究表明报告基因法可显著降低毒性检出限、提高灵敏度并且缩短响应时间。目前已有的基于报告基因的研究主要是通过特异性的诱导基因和报告基因组合,监测特异性目标物质,例如DNA损伤类物质、内分泌干扰物、二噁英类物质等。但由于污染物毒性效应的复杂性,不同污染物具有不同的毒性效应机制。已有的研究表明内质网应激及损伤是污染物毒性的常见效应之一,因此,构建一株可以指示早期内质网应激及损伤的报告基因细胞株,可以更加灵敏地检测污染物的早期毒性效应。
水是生命的第一要素,水质优劣与人体健康密切相关。水污染正在严重地威胁着人类健康。而现有水质安全性评价方法主要为理化指标检测(COD、BOD、氨氮、总氮、总磷等)与生物测试(指示生物法、发光菌急性毒性法、大型蚤毒性测试等)相结合。新型的人体细胞毒性试验相较于传统毒性试验而言,可以更直接地体现污染水体对于人类健康的影响,但其敏感性仍存在局限性,目前常用的测定细胞存活率、氧化损伤和遗传损伤等细胞损伤水平的毒性评估实验耗时长、操作复杂,且仅能分析环境污染物对细胞或生物体的某一类毒性效应。因此,亟须建立一种操作简便的毒性评估方法来综合评价水体的综合毒性。近年来,基于细胞应激基因启动子调控的报告基因实验已成为最具有潜力的毒性评估方法之一,报告基因法因其敏感、迅速、重现性好的特点,被广泛应用于毒性评价中。研究表明报告基因法可显著降低毒性检出限、提高灵敏度并且缩短响应时间。目前已有的基于报告基因的研究主要是通过特异性的诱导基因和报告基因组合,监测特异性目标物质,例如内分泌干扰物受体基因和报告基因组合可用于检测内分泌干扰物的存在;Gadd153和报告基因组合可以监测环境中的DNA损伤类物质;HSE和报告基因组合可以监测重金属。但由于实际水体的复杂性,不同污染物具有不同的毒性效应机制,简单的监测水体中的某几类特异性目标物质并不能全面表征水体的综合毒性,且不能指示水体可能造成的早期毒性损伤。因此,构建一株可以指示早期细胞应激及损伤的报告基因细胞株,可以灵敏地检测污染物的早期毒性效应,并表征水体综合毒性的检测细胞株将可以更敏感地评价水质安全。
发明内容
本发明所要解决的技术问题是提供一种基于内质网应激的报告基因细胞株构建方法,该构建方法得到的细胞株不仅可以指示毒害污染物早期的内质网应激效应,表征毒害污染物的毒性效应,而且还可以对水体综合毒性进行表征。
本发明还要解决的技术问题是提供上述基于内质网应激的报告基因细胞株构建方法构建的报告基因细胞株在表征毒害污染物毒性方面以及在表征实际水体综合毒性方面的应用。
为解决上述技术问题,本发明所采用的技术方案如下:
一种基于内质网应激的报告基因细胞株构建方法,该方法选用与内质网应激相关的CHOP基因作为特异性诱导基因构建CHOP启动子,再将CHOP启动子与便于检测的SEAP基因连接,构建慢病毒CHOP-SEAP质粒载体;最后将CHOP-SEAP质粒转染至Hela细胞中得到所需的检测细胞株。
其中,内质网应激会诱导三条信号通路,而每条信号通路都会诱导CHOP基因的表达,即内质网应激必然诱导CHOP基因的表达。
上述基于内质网应激的检测细胞株的构建方法,具体包括如下步骤:
步骤1,构建慢病毒CHOP-SEAP质粒载体:将pHBLV-CMVIE-ZsGreen-Puro载体用EcoRI和XhoI酶切,载体酶切完成后进行胶回收;同时根据SEAP基因和CHOP基因序列设计引物并进行PCR扩增,然后将处理好的目的片段与载体进行连接反应,再转化至DH5a感受态细胞中;转化后进行平板挑菌,37℃250转/分钟摇菌14h,用菌液进行PCR鉴定,得到阳性克隆菌液,将阳性克隆菌液送测序公司测序;
步骤2,慢病毒CHOP-SEAP质粒载体抽提:从步骤1获得的阳性克隆菌液中抽提慢病毒CHOP-SEAP质粒载体,具体步骤按照质粒大量提取纯化试剂盒说明书进行,当慢病毒CHOP-SEAP质粒载体浓度大于1μg/μL,A260/280在1.7~1.8之间方可进行下一步病毒包装;
步骤3,慢病毒CHOP-SEAP载体包装:铺板293T细胞用于转染,操作完毕后置于37℃、5%CO2培养箱中培养,细胞汇合率至70~80%时进行脂质体转染,每培养皿100mm的转染体系如下:
转染后更换含10%胎牛血清的新鲜完全培养基,转染后48h和72h分别两次收集病毒上清,将收集的病毒上清于50mL离心管中在4℃,2000×g下离心10min,去除细胞碎片;再将收集的病毒原液上清置于超速离心管中,于4℃,82700×g下离心120min;最后将慢病毒超离液分装到灭菌处理的病毒管中,分装标记好后于-80℃冰箱保存;
步骤4,稳转细胞株构建:10cm大培养皿中待细胞长满后用胰酶消化稀释至细胞密度为3.0×105/mL,接种于六孔板,每孔接种体积为2mL,使得第二天细胞的融合率在60%左右,利于感染,感染时弃原有培养基,每孔加入2mL 5%FBS的DMEM+1%双抗,并加入适量病毒悬液;感染24h后,弃去病毒感染液,换新鲜完全培养基;病毒感染后待细胞的汇合率达到90%,将细胞传至10cm的大培养皿中;
步骤5,稳转细胞株筛选:因病毒载体上带有嘌呤霉素抗性基因,细胞感染上病毒后则会获得嘌呤霉素抗性,根据这一特性,利用嘌呤霉素多次重复处理病毒感染后的细胞即可从中筛选出成功感染病毒的细胞,随后对细胞进行扩大培养和冻存。
上述基于内质网应激的报告基因细胞株构建方法构建的细胞株在表征毒害污染物毒性方面的应用,尤其是针对重金属污染物,如镉、铅、铬、砷等毒害污染物的毒性表征,反应灵敏,检测限值低,能够快速反应出毒害污染物的早期毒性。
用砷作为典型毒害污染物,稀释浓度梯度:0、0.05、0.1、0.15、0.2、0.5、1μM,进行七组平行对比试验,将各个浓度砷分别对转染后的Hela细胞染毒24h;取每组试验中的Hela细胞上清液,于65℃灭活30min后,用SEAP Reporter Gene Assay试剂盒检测Hela细胞的SEAP酶活(SEAP酶活具有指示发光的作用,便于监测);按说明书稀释标曲,将标曲和样品每孔10μL加入到96孔白板中,每孔加入50μL底物,室温孵育10~30min后检测其化学发光。毒性越高,Hela细胞的SEAP酶活越高,化学发光越强。
上述基于内质网应激的报告基因细胞株构建方法构建的细胞株在表征实际水体综合毒性方面的应用,实际水体中,毒性污染物的成分很复杂多样,有持久性有机污染物,内分泌干扰物,重金属等,本发明检测细胞株针对复杂多样的水体毒性污染物能全面进行表征,可灵敏地检测污染物的早期毒性效应,即当水体中含有任何一种或两种以上毒性污染物时,本发明的检测细胞株均能产生应激反应,指示水体造成的早期毒性损伤,且检测限值低。
用化工园区水样作为染毒物质,5L水样通过固相萃取浓缩后用2%DMSO定容至1mL,分别稀释至浓度梯度:0、0.02%、1%和2%(分别对应原水浓度的0、1、50、100倍),进行平行对比试验,将各个浓度水样分别对转染后的Hela细胞染毒24h;取每组试验中的Hela细胞上清液,于65℃灭活30min后,用SEAP Reporter Gene Assay试剂盒检测Hela细胞的SEAP酶活(SEAP酶活具有指示发光的作用,便于监测);按说明书稀释标曲,将标曲和样品每孔10μL加入到96孔白板中,每孔加入50μL底物,室温孵育10~30min后检测其化学发光。毒性越高,Hela细胞的SEAP酶活越高,化学发光越强。
与现有技术相比,本发明技术方案具有的有益效果为:
本发明方法构建的检测细胞株一方面能够指示早期细胞毒性损伤,从而能够表征毒害污染物的早期毒性,大大提高细胞毒性损伤的检测敏感性和降低检测限值,且适用于大多数毒害污染物;本发明方法构建的检测细胞株不仅可以指示毒害污染物的早期的内质网应激效应,表征毒害污染物的毒性效应,而且还可以对水体综合毒性进行表征;构建的检测细胞株选用的报告基因SEAP基因具有便于检测、可动态监测的优点。
附图说明
图1为实施例1不同浓度砷(μM)染毒后SEAP酶活,其中,0-表示未经转染的Hela细胞且未经染毒的SEAP酶活;0+表示转染后Hela细胞未经染毒的SEAP酶活;
图2为对比实施例1不同浓度砷(μM)染毒后的细胞活性;
图3为对比实施例2不同浓度砷(μM)染毒后细胞内活性氧(ROS)水平;
图4为对比实施例3不同浓度砷(μM)染毒后细胞线粒体膜电位变化;
图5为实施例2不同浓度水样染毒后SEAP酶活,其中,0-表示未经转染的Hela细胞且未经染毒的对照组;0+表示转染后Hela细胞未经染毒的对照组;0.02%+表示转染后Hela细胞用0.02%浓度的水样染毒的实验组;
图6为对比实施例4不同浓度水样染毒后的细胞活性;
图7为对比实施例5不同浓度水样染毒后细胞内活性氧(ROS)水平;
图8为对比实施例6不同浓度水样染毒后细胞线粒体膜电位变化。
具体实施方式
下面结合附图对本发明技术方案作进一步说明。
实施例1
本发明基于内质网应激的新型报告基因细胞株的构建方法,具体包括如下步骤:
步骤1,构建慢病毒CHOP-SEAP质粒载体:将pHBLV-CMVIE-ZsGreen-Puro载体用EcoRI和XhoI酶切,载体酶切完成后进行胶回收;同时根据SEAP基因和CHOP基因序列设计引物并进行PCR扩增,然后将处理好的目的片段与载体进行连接反应,然后转化至DH5a感受态细胞中;转化后进行平板挑菌,37℃下250转/分钟摇菌14小时,用菌液进行PCR鉴定,得到阳性克隆菌液,将阳性克隆菌液送测序公司测序;
步骤2,慢病毒CHOP-SEAP质粒载体抽提:从步骤1获得的阳性克隆菌液中抽提出慢病毒CHOP-SEAP质粒载体,具体步骤按照质粒大量提取纯化试剂盒说明书进行即可,在浓度大于1μg/μL,A260/280在1.7~1.8之间方可进行下一步病毒包装;
步骤3,慢病毒CHOP-SEAP载体包装:铺板293T细胞用于转染,操作完毕后置于37℃、5%CO2培养箱中培养,细胞汇合率至70~80%时进行脂质体转染,每培养皿100mm的转染体系如下:
转染后更换含10%胎牛血清的新鲜完全培养基,转染后48h和72h分别两次收集病毒上清,将收集的病毒上清于50mL离心管中在4℃,2000×g离心10min,去除细胞碎片;再将收集的病毒原液上清置于超速离心管中,于4℃,82700×g离心120min;最后将慢病毒超离液分装到灭菌处理的病毒管中,分装标记好后于-80℃冰箱保存;
步骤4,稳转细胞株构建:10cm大培养皿中待Hela细胞长满后用胰酶消化稀释至细胞密度为3.0×105/mL,接种于六孔板,每孔接种体积为2mL,使得第二天Hela细胞的融合率在60%左右,利于感染;感染时弃原有培养基,每孔加入2mL 5%FBS的DMEM+1%双抗,并加入适量病毒悬液,感染24h后,弃去病毒感染液,换新鲜完全培养基,病毒感染后待Hela细胞的汇合率达到90%,将细胞传至10cm的大培养皿中;
步骤5,稳转细胞株筛选:因病毒载体上带有嘌呤霉素抗性基因,Hela细胞感染上病毒后则会获得嘌呤霉素抗性,根据这一特性,利用嘌呤霉素多次重复处理病毒感染后的Hela细胞即可从中筛选出成功感染病毒的细胞,随后对细胞进行扩大培养和冻存。
用砷作为典型毒害污染物,稀释浓度梯度:0、0.05、0.1、0.15、0.2、0.5、1μM,进行七组平行对比试验,将各个浓度的砷毒液分别对各个试验组转染后的稳转Hela细胞染毒24h;取每组试验中的Hela细胞上清液,于65℃灭活30min后,用SEAP Reporter Gene AssayKit试剂盒检测Hela细胞的SEAP酶活;按说明书稀释标曲,将标曲和样品每孔10μL加入到96孔白板中,每孔加入50μL底物,室温孵育10~30min后检测其化学发光;实验结果如图1所示。
通过图1可知,不同染毒组与对照组之间具有显著性差异(p<0.05)。砷在0.05μM浓度时即可产生显著性差异,说明本发明方法构建的细胞株能够表征细胞早期损伤,且具有低的毒性物浓度检测下限,结果显示出一定的剂量效应关系。
实施例2
用化工园区水样作为染毒物质,5L水样通过固相萃取浓缩后用2%DMSO定容至1mL,分别稀释至浓度梯度:0、0.02%、1%和2%(分别对应原水浓度的0、1、50、100倍),进行平行对比试验,将各个浓度水样分别对转染后的Hela细胞染毒24h;取每组试验中的Hela细胞上清液,于65℃灭活30min后,用SEAP Reporter Gene Assay试剂盒检测Hela细胞的SEAP酶活(SEAP酶活具有指示发光的作用,便于监测);按说明书稀释标曲,将标曲和样品每孔10μL加入到96孔白板中,每孔加入50μL底物,室温孵育10~30min后检测其化学发光。毒性越高,Hela细胞的SEAP酶活越高,化学发光越强。实验结果如图5所示。
通过图5可知,不同染毒组与对照组之间具有显著性差异(p<0.05)。水样在0.02%浓度时即可产生显著性差异,说明本发明方法构建的细胞株能够表征细胞早期损伤,且结果显示出一定的剂量效应关系。
对比实施例1
细胞毒性试验-细胞活性CCK-8检测
步骤1,96孔板中,Hela细胞以10000个/孔的密度种板;
步骤2,种板24h后,用砷作为染毒物质,稀释至浓度梯度:0、0.5、1、2、5μM,对细胞染毒24h;
步骤3,每孔加入10μL CCK-8试剂,孵育1~4h后,用酶标仪测定其450nm处OD值。
如图2所示,对比实施例1的检测结果显示,砷在0~2μM浓度时未引起显著的的细胞活性抑制,5μM浓度时则出现了显著的细胞活性抑制。
对比实施例2
细胞毒性试验-胞内活性氧水平ROS检测
步骤1,96孔板中,Hela细胞以10000个/孔的密度种板;
步骤2,种板24h后,用砷作为染毒物质,稀释至浓度梯度:0、0.5、1、2、5μM,对细胞进行染毒24h;
步骤3,吸去孔中培养基,用Hanks缓冲液清洗一遍,用Hanks缓冲液将DCF探针和Hoechst 33342探针分别稀释至10μM和2.5μg/mL,每孔加入100μL稀释后探针,孵育20min后,Hanks缓冲液清洗一遍,用酶标仪测定分别检测其488/530nm处和350/460nm处荧光值。
如图3所示,对比实施例2的检测结果显示,砷在0~0.5μM浓度时均未产生显著的胞内ROS水平升高,1μM浓度时开始出现显著的ROS水平升高,表征氧化应激的产生。
对比实施例3
细胞毒性试验-线粒体膜电位JC-1检测
步骤1,96孔板中,Hela细胞以10000个/孔的密度种板;
步骤2,种板24h后,用砷作为染毒物质,稀释至浓度梯度:0、0.5、1、2、5μM,对细胞进行染毒24h;
步骤3,用ddH2O将10x孵育液稀释成1x孵育液,每500μL孵育液加入1μL JC-1,涡旋混匀配成JC-1工作液,10000rpm离心1min后取上清液;
步骤4,吸去孔中培养基,用Hanks缓冲液清洗一遍,每孔加入100μL JC-1工作液,孵育20min后,用酶标仪测定分别检测其488/530nm处和530/590nm处荧光值。
如图4所示,对比实施例3的检测结果表明,砷在0~0.5μM浓度时均未引起显著的线粒体膜电位变化,1μM浓度时则开始出现了显著的线粒体膜电位变化,表征早期细胞凋亡。
对比实施例4
细胞毒性试验-细胞活性CCK-8检测
步骤1,96孔板中,Hela细胞以10000个/孔的密度种板;
步骤2,种板24h后,用水样作为染毒物质,稀释至浓度梯度:0、0.02%、0.2%、1%、2%,对细胞染毒24h;
步骤3,每孔加入10μL CCK-8试剂,孵育1~4h后,用酶标仪测定其450nm处OD值。
如图6所示,对比实施例4的检测结果显示,水样在0~0.2%浓度时均未引起显著的细胞活性抑制,1%浓度时则出现了显著的细胞活性抑制。
对比实施例5
细胞毒性试验-胞内活性氧水平ROS检测
步骤1,96孔板中,Hela细胞以10000个/孔的密度种板;
步骤2,种板24h后,用水样作为染毒物质,稀释至浓度梯度:0、0.02%、0.2%、1%、2%,对细胞进行染毒24h;
步骤3,吸去孔中培养基,用Hanks缓冲液清洗一遍,用Hanks缓冲液将DCF探针和Hoechst 33342探针分别稀释至10μM和2.5μg/mL,每孔加入100μL稀释后探针,孵育20min后,Hanks缓冲液清洗一遍,用酶标仪测定分别检测其488/530nm处和350/460nm处荧光值。
如图7所示,对比实施例5的检测结果显示,水样在0~0.02%浓度时均未引起显著的胞内ROS水平升高,0.2%浓度时则开始出现了显著的胞内ROS水平升高,表征氧化应激的产生。
对比实施例6
细胞毒性试验-线粒体膜电位JC-1检测
步骤1,96孔板中,Hela细胞以10000个/孔的密度种板;
步骤2,种板24h后,用水样作为染毒物质,稀释至浓度梯度:0、0.02%、0.2%、1%、2%,对细胞进行染毒24h;
步骤3,用ddH2O将10x孵育液稀释成1x孵育液,每500μL孵育液加入1μL JC-1,涡旋混匀配成JC-1工作液,10000rpm离心1min后取上清液;
步骤4,吸去孔中培养基,用Hanks缓冲液清洗一遍,每孔加入100μL JC-1工作液,孵育20min后,用酶标仪测定分别检测其488/530nm处和530/590nm处荧光值。
如图8所示,对比实施例6的检测结果表明,水样在0~0.02%浓度时均未引起显著的线粒体膜电位变化,0.2%浓度时则开始出现了显著的线粒体膜电位变化,表征早期细胞凋亡。
通过图1~图8可以说明,本发明报告基因细胞株能够表征细胞早期损伤,且具有低的毒性物或污染物浓度检测限值。
Claims (4)
1.一种基于内质网应激的报告基因细胞株构建方法,其特征在于:该方法选用与内质网应激相关的CHOP基因作为特异性诱导基因构建CHOP启动子,再将CHOP启动子与便于检测的SEAP基因连接,构建慢病毒CHOP-SEAP质粒载体;最后将CHOP-SEAP质粒转染至Hela细胞中得到所需的检测细胞株。
2.根据权利要求1所述的基于内质网应激的报告基因细胞株构建方法,其特征在于,具体包括如下步骤:
步骤1,构建慢病毒CHOP-SEAP质粒载体:将pHBLV-CMVIE-ZsGreen-Puro载体用EcoRI和XhoI酶切,载体酶切完成后进行胶回收;同时根据SEAP基因和CHOP基因序列设计引物并进行PCR扩增,将处理好的目的片段与载体进行连接反应,将连接反应后的片段转化至DH5a感受态细胞中,转化后进行平板挑菌,于37℃下250转/分钟摇菌14小时,用菌液进行PCR鉴定,得到阳性克隆菌液,将阳性克隆菌液进行测序;
步骤2,慢病毒CHOP-SEAP质粒载体抽提:从步骤1得到的阳性克隆菌液中抽提慢病毒CHOP-SEAP质粒载体,当慢病毒CHOP-SEAP质粒载体浓度大于1 μg/μL,A260/280在1.7~1.8之间时进入下一步的病毒包装;
步骤3,慢病毒CHOP-SEAP质粒载体包装:铺板 293T 细胞用于转染,操作完毕后置于37℃下、5 % CO2培养箱中培养,细胞汇合率至70~80 %时进行脂质体转染,每培养皿100mm的转染体系为:含有10μg pSPAX2、5μg pMD2G、10μg CHOP-SEAP质粒以及75μL LipofiterTM;转染后更换含10 %胎牛血清的新鲜完全培养基,转染后48 h和72 h分别两次收集病毒上清,将病毒上清于一定条件下离心处理去除细胞碎片;再进行超离心处理,最后将慢病毒超离液分装到灭菌处理的病毒管中,分装标记好后于-80℃冰箱保存;
步骤4,稳转细胞株构建:10cm大培养皿中待细胞长满后用胰酶消化稀释至细胞密度为3.0×105 /mL,接种于六孔板,每孔接种体积为2 mL,使得第二天细胞的融合率在60 %左右;感染时采用2 mL 5% FBS的DMEM+1 %双抗培养液,并加入适量的病毒悬液;感染24 h后,弃去病毒感染液,替换成新鲜完全培养基;病毒感染后待细胞的汇合率达到90%时将细胞转至10cm的大培养皿中;
步骤5,对稳转细胞株进行筛选:利用嘌呤霉素多次重复处理病毒感染后的细胞,从中筛选出成功感染病毒的细胞,得到所需的检测细胞株,随后对细胞进行扩大培养和冻存。
3.权利要求1或2所述基于内质网应激的报告基因细胞株构建方法构建的报告基因细胞株在表征毒害污染物毒性方面的应用。
4.权利要求1或2所述基于内质网应激的报告基因细胞株构建方法构建的报告基因细胞株在表征实际水体综合毒性方面的应用。
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