CN114891750A - 筛选经cyp3a4介导代谢毒性外源化合物的细胞模型及其构建方法、应用 - Google Patents
筛选经cyp3a4介导代谢毒性外源化合物的细胞模型及其构建方法、应用 Download PDFInfo
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Abstract
本发明公开了一种筛选经CYP3A4介导代谢毒性外源化合物的细胞模型及其构建方法、应用。所述的筛选经CYP3A4介导代谢毒性外源化合物的细胞模型,采用Flp‑InTM‑CHO作为目的细胞,将含有CYP3A4和POR的编码DNA序列分别转染入目的细胞,得到所述筛选成人代谢毒性外源化合物的细胞模型。本发明所建立的模型新颖、可靠、简便,可极大提高经CYP3A4介导的外源化合物代谢毒性筛选细胞模型的灵敏性。
Description
技术领域
本发明涉及药物毒理学技术领域,具体涉及一种筛选经CYP3A4介导代谢毒性外源化合物的细胞模型及其构建方法、应用。
背景技术
外源化合物代谢毒性是指外源化合物本身没有毒性或毒性较低,经过机体的代谢会产生毒性更强的的中间代谢物或副产物对机体造成损害,称之为代谢毒性(或代谢损伤),如黄曲霉素、苯并芘、对乙酰氨基酚等外源化合物均通过细胞色素P450(CytochromeP450,CYP450)超家族代谢生成致毒甚至致癌的代谢物发挥毒性作用。CYP是介导外源化合物代谢毒性的主要代谢酶之一,CYP3A4是成人肝脏表达量最高也是参与外源化合物代谢最多的CYP亚型。目前对代谢毒性的评价手段大多是将整体动物脏器损伤结果和基因/蛋白-代谢物结合物作为外源化合物发育毒性的衡量指标,但存在动物用量大、检测方法要求高、灵敏度低等缺点,而已有的外源化合物代谢毒性的体外评价体系,如HepaRG或肝瘤细胞系,前者价格昂贵且不能传代,后者因代谢酶基础表达低需转染随机插入CYP的DNA序列,影响不同时期不同实验室的检测重复性。综上,外源化合物代谢毒性体外评价体系存在较大局限,灵敏度高、成本低、重复性好的外源化合物代谢毒性体外评价体系尚待开发。
Flp-InTM-CHO细胞系来源于赛默飞世尔科技公司(货号:R75807),在转录活性基因组基因座处包含一个稳定整合的FRT位点。构建含有CYP3A4编码DNA序列的Flp-InTM表达载体和Flp-In重组酶载体pOG44共转染Flp-InTM-CHO,可以在每个细胞中将CYP3A4表达载体靶向整合到同一个位点中,确保不同时期或不同实验室都能得到均一CYP3A4表达水平的细胞模型。文献报道的其它类似代谢毒性评价细胞模型体系是否表达POR也并不清楚。
发明内容
本发明目的之一在于提供一种筛选经CYP3A4介导代谢毒性的外源化合物的细胞模型,所用细胞为Flp-InTM-CHO细胞系来源于赛默飞世尔科技公司(货号:R75807),来自于中国仓鼠卵巢,不涉及复杂伦理问题并可多次传代,转录活性基因组基因座处包含一个稳定整合的FRT位点,可以在每个细胞中将CYP3A4表达载体靶向整合到同一个位点中。共转染POR后可极大提高对经CYP3A4介导代谢毒性的外源化合物的敏感性。所述模型以细胞活力为检测指标,是一个方法简单、成本低、敏感、快速的检测模型。
本发明目的之二在于提供一种筛选经CYP3A4介导代谢毒性的外源化合物的细胞模型的构建方法,所述造模方法简单,可重复性强。
本发明目的之三在于提供一种筛选经CYP3A4介导代谢毒性的外源化合物的细胞模型在检测具有代谢毒性的外源化合物中的应用,所述模型检测过程快速,应用范围广,可用于高通量筛选。
本发明实现目的之一采用以下技术方案:
一种筛选经CYP3A4介导代谢毒性外源化合物的细胞模型,其特征在于:采用Flp-InTM-CHO作为目的细胞,将含有CYP3A4和POR的编码DNA序列分别转染入目的细胞,得到所述筛选成人代谢毒性外源化合物的细胞模型。
作为优选方案,所述筛选经CYP3A4介导代谢毒性外源化合物的细胞模型具有两个指标,细胞活力和半数抑制浓度(IC50)。
本发明实现目的之二采用以下技术方案:
一种如上所述的筛选经CYP3A4介导代谢毒性外源化合物的细胞模型构建方法,其特征在于:包括如下步骤:
S1:在基础培养基中复苏Flp-InTM-CHO细胞,所述基础培养基含10%胎牛血清和1%青霉素和链霉素的F12;
S2:构建含CYP3A4编码DNA序列的pcDNA5质粒;
S3:构建含POR编码DNA序列的pCMV质粒;
S4:将步骤S2构建的含CYP3A4编码DNA序列的pcDNA5质粒稳定转染入步骤S1传代后的Flp-InTM-CHO中,在筛选DMEM/F12-1培养基中培养,所述筛选DMEM/F12-1培养基含10%胎牛血清和500μg/ml潮霉素的F12;
S5:将步骤S3构建的含POR编码DNA序列的pCMV质粒稳定转染入步骤S4得到的Flp-InTM-CHO-CYP3A4细胞中,在筛选DMEM/F12-2培养基中培养,所述筛选DMEM/F12-2培养基含10%胎牛血清、500μg/ml潮霉素和50μg/ml嘌呤霉素的F12,得到所述筛选经CYP3A4介导代谢毒性外源化合物的细胞模型。
本发明实现目的之三采用以下技术方案:
一种如上述筛选经CYP3A4介导代谢毒性外源化合物的细胞模型在筛选具有代谢毒性的外源化合物中的应用。
作为优选方案,当细胞活力降低时,则提示所述待筛选外源化合物具有经CYP3A4介导的代谢毒性。
进一步地,根据不同待筛选外源化合物的IC50值能对不同外源化合物代谢毒性进行比较。
与现有的技术相比,本发明具有以下优点及有益效果:
本发明首次发现Flp-InTM-CHO缺乏人细胞色素P450氧化还原酶(EC 1.6.2.4;NADPH-Cytochrome P450 Oxidoreductase;POR)表达,它是唯一的传递电子给CYP酶系以帮助完成CYP氧化功能的辅酶。本发明提出,将POR稳定转染入表达CYP3A4的Flp-InTM-CHO中,可极大提高经CYP3A4介导的外源化合物代谢毒性筛选细胞模型的灵敏性。具体优点如下:
1、本发明提供的筛选经CYP3A4介导代谢毒性外源化合物的细胞模型与其他代谢毒性体外筛选系统相比,所用Flp-In-CHO-3A4-POR细胞具有以下优势:①细胞来源于中国仓鼠卵巢,不涉及复杂伦理问题;②无CYP3A4基础表达和诱导表达,需重组表达CYP3A4,无细胞本身CYP3A4表达对筛选结果的干扰;③在每个细胞中将CYP3A4表达载体靶向整合到同一个位点中,得到CYP3A4表达均一的细胞模型,重复性好;④共转染POR极大提高了筛选系统的灵敏性;⑤在潮霉素和嘌呤霉素作用下可稳定保持CYP3A4和POR表达性状,对经CYP3A4介导代谢毒性的化合物反应敏感,可用于高通量筛选。
2、本发明提供的筛选经CYP3A4介导代谢毒性外源化合物的细胞模型以细胞活力和IC50值作为检测指标,当细胞活力降低时,提示所述待筛选外源化合物具有经CYP3A4介导的代谢毒性,简易、灵敏度高、应用范围广。根据不同待筛选外源化合物的IC50值可以对不同外源化合物代谢毒性进行比较,对于快速筛选经CYP3A4介导代谢毒性的外源化合物具有重要意义。
附图说明
图1为本发明中构建含CYP3A4编码和POR编码的DNA序列Flp-InTM-CHO模型的方法示意图。
图2为本发明实施例1中构建含CYP3A4编码DNA序列所用工具载体基本骨架图。
图3为本发明实施例1中构建含POR编码DNA序列所用工具载体基本骨架图。
图4为本发明实施例1中蛋白鉴定重组克隆的免疫印迹图;
图4中:vector为表达空载体(vector)的CHO细胞提取的蛋白,POR为转染了人POR基因组的CHO细胞提取的蛋白,3A4为转染了人CYP3A4基因组的CHO细胞提取的蛋白,POR-3A4为转染了人POR和CYP3A4基因组的CHO细胞提取的蛋白。
图5为本发明实施例1中倒千里光碱(retrorsine,RTS)的MTS检测细胞活力图;
图5中:给予药物RTS后,相比较于CHO细胞和CHO-CYP3A4细胞而言,对于CHO-POR-CYP3A4细胞的抑制性更强。如图所示,药物RTS的IC50在CHO-POR-CYP3A4细胞中最低。
图6为本发明实施例1中野百合碱(monocrotaline,MCT)的MTS检测细胞活力图;
图6中:给予药物MCT后,相比较于CHO细胞和CHO-CYP3A4细胞而言,对于CHO-POR-CYP3A4细胞的抑制性更强。如图所示,药物MCT的IC50在CHO-POR-CYP3A4细胞中最低。
图7为本发明实施例2中倒千里光碱(retrorsine,RTS)暴露后雌性成年大鼠肝苏木精-伊红染色病理图。
图8为本发明实施例2中倒千里光碱(retrorsine,RTS)暴露后雌性成年大鼠肝功能相关指标图;
图8中:为RTS暴露后大鼠血清中ALT活性。
图9为本发明实施例2中野百合碱(monocrotaline,MCT)暴露后雌性成年大鼠肝苏木精-伊红染色病理图。
图10为本发明实施例2中野百合碱(monocrotaline,MCT)暴露后雌性成年大鼠肝功能相关指标图;
图10中:为MCT暴露后大鼠血清中ALT活性。
具体实施方式
以下结合附图及具体实施例对本发明的技术方案作进一步地详细阐述。
实施例1:构建体外成人药物毒性筛选的CHO-POR-CYP3A4的细胞模型
1.实验细胞
本实验所用的Flp-InTM CHO细胞购自赛默飞世尔科技公司。
2.实验步骤
2.1将构建好的含有人CYP3A4全长cDNA的pcDNA5重组质粒与辅助质粒pOG44一起共转染Flp-InTM CHO细胞。具体转染步骤如下:
1)CHO细胞复苏后在含10%FBS和1%青链霉素的F12(Ham’s)培养液中维持培养,并且传3代。
2)转染前24小时,将CHO细胞接种于六孔板中,使得转染当天CHO细胞长至80~90%融合。
3)在250μL无血清培养基中加入0.3μg的POR-pcDNA5质粒(同时以不含CYP3A4基因cDNA的pcDNA5空载体平行操作,作为阴性对照)及5μg的辅助质粒pOG44并与10μL的P3000转染试剂混和,室温放置5min。
4)在250μL无血清培养基中加入7.5μL的Lipo3000转染试剂混合均匀后,室温放置5min。
5)将S2)和S3)的试剂混合均匀,室温放置10-15min后,与以1mL的无血清Ham’sF12培养基培养的Flp-InTM CHO细胞共孵育24小时,之后用含10%胎牛血清的完全Ham’sF12培养基2mL换液以终止反应。
6)以完全培养基恢复培养12小时后,将转染细胞从6孔板中转种到100mm直径的培养皿中。
7)转种12小时以后,向培养基中加入潮霉素B(Hygromycin B)至终浓度为500μg/mL以筛选阳性克隆。
8)待筛选到阳性克隆长出后,挑选单克隆并继续以终浓度为500μg/mL的潮霉素B(Hygromycin B)维持培养。
2.2将包装好的含有人POR全长cDNA的慢病毒感染CHO-CYP3A4细胞。
具体感染步骤如下:
1)感染前24小时,将CHO-CYP3A4细胞接种于六孔板中,使得感染当天细胞长至80~90%融合。
2)将500uL包装有人POR全长cDNA的病毒液(同时以不含POR全长cDNA的病毒空载平行操作,作为阴性对照)与2ml的Ham’s F12培养基混合,并随后加入聚凝胺polybrene(8mg/mL),使其终浓度为8ug/mL(2mL+0.5mL=2.5mL,即加2.5uL聚凝胺)。混匀后,常温,3000rpm离心1.5h(离心时做好保护,防污染)。
3)感染24h后,再感染一次。
4)感染48h后,将六孔板每孔的细胞分别传部分到100mm直径的培养皿中,剩下的细胞提蛋白做western验证是否转染成功。待12h贴壁后,向培养基中加入嘌呤霉素(puromycin)至终浓度为50μg/mL以筛选阳性克隆。
5)待培养皿中的细胞长成单克隆(吸走大部分培养基后可以用肉眼看到小白点)后,可选用滤纸或者小枪头挑单克隆,到24孔板中,并继续以终浓度为50μg/mL的嘌呤霉素(puromycin)维持培养。
6)待24孔板中的细胞长起来后,将细胞转移到6孔板中,6孔板的细胞长起来后,收部分细胞检测POR蛋白的表达,并进行活性检测。将表达量高且活性高的细胞冻存。
3.Western blot检测CHO-POR-CYP3A4细胞的蛋白表达
1)构建的CHO-POR-CYP3A4细胞蛋白提取:
a)用预冷的PBS清洗六孔板中的细胞两次,以除去残留的培养液;每孔加入100μL的细胞裂解液(含1%的PMSF),用细胞刮将细胞刮下后将细胞转移到1.5mL的EP管中,用移液器吹打5次以充分裂解细胞,冰上放置5分钟。
b)于4℃,12000rpm离心5分钟。
c)将离心后的上清转移至新的0.5mL的Eppendorf管中,-80℃保存备用。
2)BCA法测定样品蛋白浓度:
a)将0.5mg/mL的牛血清白蛋白(BSA)标准品分别取0、1、2、4、8、12、16和20μL至96孔板中,加双蒸水补足至20μL;
b)加200μL的BCA工作液,37℃放置30分钟;
c)用紫外分光光度计测定波长为570nm的吸光度(A)值,根据标准曲线计算出蛋白浓度。
3)蛋白质的聚丙酰胺凝胶电泳。
4.RTS和MCT对表达CYP3A4以及POR和CYP3A4的Flp-In CHO细胞系的毒性作用
将筛选成功的CHO-CYP3A4和CHO-POR-CYP3A4细胞扩大培养并且冻存保种,然后将CHO&CHO-CYP3A4&CHO-POR-CYP3A4按5-9×103/孔的密度种96孔板(n=4),24h后分别给予0、0.000001、0.00001、0.0001、0.001、0.01和0.1mM的RTS或MCT处理72h,处理结束时每孔加入20μL的MTS,37℃孵育60min至颜色变为黄棕色,在酶标仪中OD 490nm处检测吸光度值。正式实验完成后,根据样品OD值及对照孔OD值在prism软件中进行计算和作图,得出RTS和MCT的药物抑制曲线。
5.实验结果
5.1构建的CHO-POR-CYP3A4细胞的蛋白表达
构建的CHO-POR-CYP3A4细胞的POR和CYP3A蛋白表达如图4所示。与表达空载体(vector)的CHO细胞相比,转染了人POR基因组检测到POR蛋白的表达,转染了人CYP3A4基因组检测到CYP3A4蛋白的表达,转染了人POR和CYP3A4基因组检测到POR和CYP3A4蛋白的表达。实验结果说明,人的POR和CYP3A4基因已经稳定转染到CHO细胞中并成功表达。
5.2吡咯里西啶生物碱(pyrrolizidine alkaloids,PAs)对CHO-POR-CYP3A4细胞的毒性
吡咯里西啶生物碱(pyrrolizidine alkaloids,PAs),主要是倒千里光碱(retrorsine,RTS)和野百合碱(Monocrotaline,MCT)对CHO&CHO-CYP3A4细胞的毒性如图5和6所示,本发明发现两种药物RTS和MCT,相比较于CHO细胞和CHO-CYP3A4细胞而言,对于CHO-POR-CYP3A4细胞活力的抑制性更强。如图5和6所示两种药物RTS和MCT的IC50在CHO-POR-CYP3A4细胞中最低。上述数据说明筛选经CYP3A4代谢增毒的药物时,使用CHO-POR-CYP3A4细胞较CHO-CYP3A4细胞更灵敏。
本实施例结果提示,采用在Flp-In CHO细胞中分别稳定转染人POR和CYP3A4基因,获得稳定表达人POR的CHO-CYP3A4的细胞株的方法,本发明证明了在体外研究成人药物毒性筛选是可能的。建立CHO-POR-CYP3A4细胞模型具有多种应用,包括临床药物和环境外源物的毒性筛选相关研究。
实施例2:基于实施例1中毒性外源化合物RTS和MCT的在体毒性验证
1.实验动物
SPF级健康成年雌性Wistar大鼠(体重:200±20g)购自湖北省疾病预防控制中心。许可证号:SCXK(鄂)2020-0006。
2.动物处理
Wistar大鼠于25±2℃,12小时光照循环的环境中,适应性喂养1w后,将雌性大鼠随机分为5组:对照组、RTS5和RTS20组、MCT5和MCT20组,每组3-4只。RTS组:雌性大鼠第一天开始灌胃给予5或20mg/kg RTS,每天一次;MCT组:雌性大鼠第一天开始灌胃给予5或20mg/kg MCT,每天一次;对照组:母鼠给予等容量的溶媒,连续给药21天。每组随机选3只母肝置于4%中性多聚甲醛固定,用于HE染色。血清与剩余肝脏组织标本保存于-80℃。
3.雌性大鼠肝脏形态学检测
雌性大鼠肝脏组织固定48小时后脱水浸蜡、包埋和切片,然后用苏木精-伊红染液染色,最后在显微镜下观察和拍照。
4.实验结果
4.1 RTS和MCT暴露对雌性大鼠肝脏形态学的影响
如图7所示,对照组肝组织中央静脉轮廓和肝细胞间界限清晰,肝索沿着中央静脉呈放射状排列,肝板排列规则有序,肝窦结构正常,未见脂滴。而给药组RTS20肝细胞排列紊乱,界限不清晰,肝索结构模糊,肝细胞内出现明显的脂肪空泡变性。如图9所示与对照组相比,给药组MCT20肝细胞内出现明显的脂肪空泡变性。
4.2 RTS和MCT暴露对雌性大鼠肝脏的损伤
如图8所示,与对照组相比,RTS暴露可致雌性大鼠血清中ALT活性升高。如图10所示与对照组相比,MCT暴露亦可致雌性大鼠血清中ALT活性升高,产生肝损伤。
综上,本发明基于Flp-InTM-CHO细胞系将含有CYP3A4和POR的编码DNA序列分别转染入目的细胞,得到所述筛选成人代谢毒性外源化合物的细胞模型。通过检测两种外源性化合物RTS和MCT在CHO-POR-CYP3A4模型上的毒性,提示该细胞模型相较于以前更加稳定,敏感和便捷。之后本发明进一步在整体动物模型上,给予雌性大鼠RTS和MCT暴露,证实了两种毒物对大鼠肝脏功能的不良影响,与本发明构建的体外成人药物毒性筛选的CHO-POR-CYP3A4的细胞模型结果一致。本发明所建立的外源物筛选系统具有高敏感性、高稳定性等特点,可用于经CYP3A4代谢增毒的外源化合物筛选。
Claims (6)
1.一种筛选经CYP3A4介导代谢毒性外源化合物的细胞模型,其特征在于:采用Flp-InTM-CHO作为目的细胞,将含有CYP3A4和POR的编码DNA序列分别转染入目的细胞,得到所述筛选成人代谢毒性外源化合物的细胞模型。
2.如权利要求1所述的筛选经CYP3A4介导代谢毒性外源化合物的细胞模型,其特征在于:所述筛选经CYP3A4介导代谢毒性外源化合物的细胞模型具有两个指标,细胞活力和半数抑制浓度(IC50)。
3.一种如权利要求1所述的筛选经CYP3A4介导代谢毒性外源化合物的细胞模型构建方法,其特征在于:包括如下步骤:
S1:在基础培养基中复苏Flp-InTM-CHO细胞,所述基础培养基含10%胎牛血清和1%青霉素和链霉素的F12;
S2:构建含CYP3A4编码DNA序列的pcDNA5质粒;
S3:构建含POR编码DNA序列的pCMV质粒;
S4:将步骤S2构建的含CYP3A4编码DNA序列的pcDNA5质粒稳定转染入步骤S1传代后的Flp-InTM-CHO中,在筛选DMEM/F12-1培养基中培养,所述筛选DMEM/F12-1培养基含10%胎牛血清和500μg/ml潮霉素的F12;
S5:将步骤S3构建的含POR编码DNA序列的pCMV质粒稳定转染入步骤S4得到的Flp-InTM-CHO-CYP3A4细胞中,在筛选DMEM/F12-2培养基中培养,所述筛选DMEM/F12-2培养基含10%胎牛血清、500μg/ml潮霉素和50μg/ml嘌呤霉素的F12,得到所述筛选经CYP3A4介导代谢毒性外源化合物的细胞模型。
4.一种如权利要求1所述筛选经CYP3A4介导代谢毒性外源化合物的细胞模型在筛选具有代谢毒性的外源化合物中的应用。
5.如权利要求4所述的筛选经CYP3A4介导代谢毒性外源化合物的细胞模型在筛选具有代谢毒性的外源化合物中的应用,其特征在于:当细胞活力降低时,则提示所述待筛选外源化合物具有经CYP3A4介导的代谢毒性。
6.如权利要求4所述的筛选经CYP3A4介导代谢毒性外源化合物的细胞模型在筛选具有代谢毒性的外源化合物中的应用,其特征在于:根据不同待筛选外源化合物的IC50值能对不同外源化合物代谢毒性进行比较。
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