CN111349607A - 一种无标记蛙皮素受体bb3的细胞筛选模型 - Google Patents
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Abstract
本发明提供了一种无标记蛙皮素受体BB3的细胞筛选模型。本发明基于无标记细胞整合药理学技术,利用BB3稳定表达的细胞系,建立了筛选BB3受体的激动剂和拮抗剂的方法。此方法还可以用于研究影响BB3受体下游通路的调节剂。本发明构建的BB3细胞筛选模型不需要荧光标记且检测过程无需额外添加指示剂,具有靶点‑通路整合响应、对细胞无损伤、检测结果可靠、灵敏度高、筛选通量高及操作简便等特点。其用于从天然产物库、代谢产物库及组合化学库中寻找BB3受体的激动剂、拮抗剂和通路调节剂,及BB3受体参与的能量代谢、神经内分泌、细胞生长和增殖相关疾病的药物筛选。
Description
技术领域
本发明涉及细胞筛选领域,具体涉及一种无标记蛙皮素受体BB3的细胞筛选模型。
背景技术
G蛋白偶联受体(G-protein-coupled receptor,GPCR)是细胞信号传导中最重要的一类膜受体,也是小分子药物开发中最受关注的药物靶点之一,约34%的现代药物直接靶向该受体家族。蛙皮素受体BB3属于GPCR家族的一员,于1992年通过同源筛选方法被发现,由399个氨基酸组成,此序列与胃泌素释放肽受体(GRP-R)有51%的同源性,与神经介素B受体(NMB-R)有47%的同源性,因此BB3被视为另一种蛙皮素受体。BB3受体主要表达于下丘脑、脑干、肺、乳腺、睾丸、妊娠子宫等器官,在发育的肺组织和一些肿瘤细胞也有较高表达,广泛表达于肺癌细胞;研究发现,BB3基因敲除小鼠可引起轻度肥胖、糖代谢紊乱和高血压,表明BB3受体在调节神经内分泌功能和能量代谢方面具有重要作用;此外,合成的BB3激动剂引起交感神经张力,表明BB3在调节交感神经系统中发挥作用。目前已发现BB3受体在肺和某些癌细胞的发育过程中过表达,表明该受体与细胞生长和增殖也有关系;而BB3受体的天然配体至今尚未发现,仍被视为一种孤儿受体。因此,构建BB3受体的细胞筛选模型,以发现BB3受体的内源性配体、高活性激动剂、拮抗剂及通路调节剂,对揭示BB3的生物学功能及药理学特征具有重大意义。
目前受体的高通量筛选方法主要有传统的放射性配体受体结合实验法、GTPγS结合实验法、环磷酸腺苷(cAMP)分析法、钙流检测法、报告基因检测法、受体的内吞检测法及β-arrestin的招募检测法等。这些方法都有一定的局限性,如传统的放射性配体受体结合实验法需要洗涤和过滤,实验周期长及通量低等不足,此技术还不能区分受体的激动剂和拮抗剂;其余的GPCR检测方法主要针对某条信号通路的激活,往往不考虑多条通路的激活,常常需要荧光蛋白标记或者额外加入指示剂,使操作变得繁琐,而且这些指示剂的加入对细胞也会产生一定的损伤,影响筛选结果的可靠性。
发明内容
本发明的目的是针对现有技术中存在的问题,借助于新型无标记细胞整合药理学技术,提供一种无标记蛙皮素受体BB3的细胞筛选模型,以高通量筛选BB3受体激动剂、拮抗剂和通路调节剂,及BB3受体参与的能量代谢、神经内分泌、细胞生长和增殖相关疾病的药物筛选应用。
本发明的技术方案为:
基于无标记细胞整合药理学技术,利用稳定表达BB3的细胞系HEK-293-BB3,借助于已知的激动剂和拮抗剂,建立BB3受体的细胞筛选模型。根据待测样品的DMR信号谱与已知激动剂和拮抗剂的DMR特征信号谱的相似性,判断待测样品的激动活性、拮抗活性或者下游通路的调节影响。
所述的无标记细胞整合药理学技术为利用共振波导光栅(RWG)生物传感器将药物导致的细胞内成分的动态再分布现象转化为整体的、动态的波长位移响应信号,此信号为波长变化的响应值(pm),通过Epic光学生物传感器384微孔板实现。
一种无标记蛙皮素受体BB3的细胞筛选模型的建立过程为:
1)在细胞兼容的具有光学生物传感功能的384微孔板中接种HEK-293-BB3细胞,接种的细胞密度为1.0~4.5×104个/孔,细胞培养液体积为40μL/孔,接种后细胞培养时间为18~24h。
2)将溶解在含0.1%BSA的HBSS缓冲盐中的BA 1激动剂以浓度为10~10000nM加入到接种HEK-293-BB3细胞的384微孔板中,检测其DMR特征信号谱;
3)将溶解在含0.1%BSA的HBSS缓冲盐中的BANTAG-1拮抗剂以浓度为0.1~1000nM加入到接种HEK-293-BB3细胞的384微孔板中,检测其DMR特征信号谱;
4)获得的所有DMR特征信号谱具有浓度-响应依赖关系且具有灵敏性、饱和性及特异性。
进一步的,待测样品具有激动活性的筛选步骤如下:
1)将溶解在含0.1%BSA的HBSS缓冲盐中的BA 1激动剂以浓度为10~10000nM加入到接种HEK-293-BB3细胞的384微孔板中,检测其DMR特征信号谱;
2)将待测样品以0.01nM~100μM加入到接种HEK-293-BB3细胞的微孔板中,检测其DMR信号谱;
3)关联分析步骤1)和步骤2)中的DMR信号谱,若步骤2)的DMR信号谱与1)中的DMR特征谱没有相似性,则样品没有激动活性;若具有轮廓相似性,则进行下一步骤;
4)将BB3拮抗剂BANTAG-1以浓度0.1~1000nM加入到接种HEK-293-BB3细胞的微孔板中,预处理5~60min,加入与步骤2)中相同浓度的待测样品,检测其DMR信号,若此DMR信号强度低于步骤2)中的DMR信号强度,则判断此样品为BB3受体的激动剂。
进一步的,待测样品具有拮抗活性的筛选步骤如下:
1)将待测样品和BA 1分别加入到接种HEK-293-BB3细胞的微孔板中,待测样品浓度为0.01nM~100μM,BA 1浓度为10~10000nM,检测DMR信号谱;
2)若步骤1)中待测样品不引起DMR信号谱,再向步骤1)中加入了待测样品的细胞板中继续加入与步骤1)中相同浓度的BA 1,检测DMR信号谱;若此DMR信号比步骤1)中BA 1的信号弱,可判断待测样品是BB3受体的拮抗剂。
进一步的,待测样品对BB3通路有调节活性的步骤如下:
1)将待测样品和BA 1分别加入到接种HEK-293-BB3细胞的微孔板中,待测样品浓度为0.01nM~100μM,BA 1浓度为10~10000nM,检测DMR信号谱;
2)再向步骤1)中加入了待测样品的细胞板中继续加入与步骤1)中相同浓度的BA1,检测DMR信号谱,检测时间为1~60min;若此DMR信号比步骤1)中BA 1的信号在上升期(1~10min)、平台期(10~20min)和延滞期(20~60min)某一个阶段不同;
3)将BB3拮抗剂BANTAG-1以浓度0.1~1000nM加入到接种HEK-293-BB3细胞的微孔板中,预处理5~60min,加入与步骤1)中相同浓度的待测样品,检测其DMR信号,若此DMR信号谱与步骤1)中的样品的DMR信号谱一致,可判断待测样品是BB3受体下游信号通路的调节剂。
本发明采用的新型无标记细胞整合药理学技术是基于无标记的共振波导光栅(RWG)生物传感器将药物导致的细胞内成分的动态再分布过程转化为整体的、动态的波长位移响应信号,称为动态质量重置(DMR)信号,具有无损伤、高时空分辨、高灵敏度、高通量、能靶点-通路整合研究及操作简单、实验周期短等特点,检测过程无需标记及额外指示剂的添加,更真实的响应药物在活细胞整体水平的作用。因此,采用无标记细胞整合药理学技术构建BB3无标记高通量筛选模型可大大提高BB3的激动剂、拮抗剂及通路调节剂的发现效率,对阐述BB3的药理学和生理学功能具有重大意义,同时为BB3受体参与的能量代谢、神经内分泌、细胞生长和增殖相关疾病的药物筛选提供指导。
附图说明
图1(A)不同浓度的BA 1在HEK-293-BB3细胞上的DMR特征信号谱;(B)不同浓度的BA 1在HEK-293-BB3细胞上的浓度-响应依赖曲线;其中BA 1的浓度单位为nM。
图2BANTAG-1在HEK-293-BB3细胞上的DMR特征信号谱;其中BANTAG-1的浓度单位为nM。
图3(A)不同浓度的BA 1预处理HEK-293-BB3细胞1h后,固定浓度BA 1的DMR信号谱;(B)不同浓度的BA 1预处理HEK-293-BB3细胞1h后,固定浓度BA 1的DMR信号谱对应的浓度-响应依赖曲线;其中BA 1的浓度单位为nM。
图4(A)不同浓度的BANTAG-1预处理HEK-293-BB3细胞1h后,固定浓度BA 1的DMR信号谱;(B)不同浓度的BANTAG-1预处理HEK-293-BB3细胞1h后,固定浓度BA 1的DMR信号谱对应的浓度-响应依赖曲线;其中BA 1和BANTAG-1的浓度单位为nM。
具体实施方式
现结合实例,对本发明做进一步说明。实例仅限于说明本发明,而非对本发明的限定。
实施例1:激动剂BA 1在HEK-293-BB3细胞上的DMR特征信号谱
人胚肾细胞HEK-293-BB3细胞来源于实验室自主构建细胞库,倒置显微镜购于OLYMPUS,BA 1购于Tocris公司,BANTAG-1来自加州大学欧文分校。细胞培养板为Epic光学生物传感384微孔板,购于康宁公司,检测平台为康宁第三代成像仪,检测的信号为细胞动态质量重置(DMR)引起的波长位移。
将处于对数生长期的HEK-293-BB3细胞,接种于细胞兼容的384微孔板中,所用培养基为DMEM(#SH30022.01B,Thermo),每孔的接种体积为40μL,每孔接种的细胞数目为2.0×104个,将接种好的细胞板置于细胞培养箱中培养20~22h,至细胞融合度达95%左右,进行活性实验。将在微孔板中的细胞培养液换成Hank's平衡盐溶液(含10mM的HEPES),每孔加入体积为30μL,加入之后,放置于成像仪上平衡1h;重新扫描2min的基线,将BA 1加入微孔板中,每孔加入体积为10μL,浓度为5000nM、2500nM、1250nM、625nM、312.5nM、156.25nM、78.125nM、39.06nM、19.53nM、9.76nM、4.88nM和2.44nM,平行3次,置于Epic仪器上实时监测DMR信号1h,基于细胞经BA 1作用的40min内DMR最大响应值处计算BA 1的EC50值,结果见图1。研究表明BA 1呈剂量依赖的激动BB3受体,剂量响应曲线呈单相“S”型且都达到饱和响应,最高的DMR响应值达500pm,对应的EC50值为0.27±0.01μM。
实施例2:拮抗剂BT-1在HEK-293-BB3细胞上的DMR特征信号谱
将处于对数生长期的HEK-293-BB3细胞,接种于细胞兼容的384微孔板中,所用培养基为DMEM(#SH30022.01B,Thermo),每孔的接种体积为40μL,每孔接种的细胞数目为2.0×104个,将接种好的细胞板置于细胞培养箱中培养20~22h,至细胞融合度达95%左右,进行活性实验。将在微孔板中的细胞培养液换成Hank's平衡盐溶液(含10mM的HEPES),每孔加入体积为30μL,加入之后,放置于成像仪上平衡1h;重新扫描2min的基线,将不同浓度的BANTAG-1加入微孔板中,每孔加入体积为10μL,浓度为200nM、100nM、50nM、25nM、12.5nM、6.25nM、3.13nM、1.56nM、0.78nM、0.39nM、0.20nM、0.10nM、0.05nM和0.02nM,平行3次,置于Epic仪器上实时监测DMR信号1h,结果见图2。研究表明不同浓度的BANTAG-1的DMR响应信号接近于零。
实施例3:HEK-293-BB3细胞的脱敏DMR特征信号谱
将处于对数生长期的HEK-293-BB3细胞,接种于细胞兼容的384微孔板中,所用培养基为DMEM(#SH30022.01B,Thermo),每孔的接种体积为40μL,每孔接种的细胞数目为2.0×104个,将接种好的细胞板置于细胞培养箱中培养20~22h,至细胞融合度达95%左右,进行活性实验。将在微孔板中的细胞培养液换成Hank's平衡盐溶液(含10mM的HEPES),每孔加入体积为30μL,加入之后,放置于成像仪上平衡1h;将不同浓度的BA1加入微孔板中预处理HEK-293-BB3细胞1h,每孔加入体积为10μL,浓度为10000nM、5000nM、2500nM、1250nM、625nM、312.5nM、156.25nM、78.125nM、39.06nM、19.53nM、9.76nM、4.88nM和2.44nM,平行3次;重新扫描2min的基线,将固定浓度的BA1加入微孔板中,每孔加入体积为10μL,浓度为3000nM,平行3次,置于Epic仪器上实时监测DMR信号1h,基于细胞经BA 1作用的40min内DMR最大响应值处计算IC50值,结果见图3。研究表明BA 1呈剂量依赖的脱敏BB3受体,剂量响应曲线呈单相“S”型且都达到饱和响应,对应的IC50值为1.43±0.13μM。
实施例4:HEK-293-BB3细胞的拮抗DMR特征信号谱
将处于对数生长期的HEK-293-BB3细胞,接种于细胞兼容的384微孔板中,所用培养基为DMEM(#SH30022.01B,Thermo),每孔的接种体积为40μL,每孔接种的细胞数目为2.0×104个,将接种好的细胞板置于细胞培养箱中培养20~22h,至细胞融合度达95%左右,进行活性实验。将在微孔板中的细胞培养液换成Hank's平衡盐溶液(含10mM的HEPES),每孔加入体积为30μL,加入之后,放置于成像仪上平衡1h;将不同浓度的BANTAG-1加入微孔板中预处理细胞1h,每孔加入体积为10μL,浓度为200nM、100nM、50nM、25nM、12.5nM、6.25nM、3.13nM、1.56nM、0.78nM、0.39nM、0.20nM、0.10nM、0.05nM和0.02nM,平行3次;重新扫描2min的基线,将固定浓度的BA 1加入微孔板中,每孔加入体积为10μL,浓度为3000nM,平行3次,置于Epic仪器上实时监测DMR信号1h,基于细胞经BA 1作用的40min内DMR最大响应值处计算IC50值,结果见图4。研究表明BANTAG-1呈剂量依赖的拮抗BB3受体,剂量响应曲线呈单相“S”型且都达到饱和响应,对应的IC50值为0.023±0.007μM。
本发明基于无标记细胞整合药理学技术,建立了BB3无标记筛选模型,此模型具有不需要荧光标记且检测过程无需额外添加指示剂的优势,高效可靠的筛选商品化的小分子库、自主制备的天然产物提取物、组分或化合物库及化学修饰物,以获得BB3受体的激动剂、拮抗剂和通路调节剂及BB3受体调控的能量代谢、神经内分泌、细胞生长和增殖相关疾病的药物。
Claims (5)
1.一种无标记蛙皮素受体BB3的细胞筛选模型,其特征在于,建立过程为:
1)在细胞兼容的具有光学生物传感功能的384微孔板中接种HEK-293-BB3细胞,接种的细胞密度为1.0 ~ 4.5 × 104个/孔,细胞培养液体积为40 µL/孔,接种后细胞培养时间为18 ~ 24 h;
2)将溶解在含0.1%BSA的HBSS缓冲盐中的BA 1激动剂以浓度为10 ~ 10000 nM加入到接种HEK-293-BB3细胞的384微孔板中,检测其DMR特征信号谱;
3)将溶解在含0.1%BSA的HBSS缓冲盐中的BANTAG-1拮抗剂以浓度为0.1 ~ 1000 nM加入到接种HEK-293-BB3细胞的384微孔板中,检测其DMR特征信号谱;
4)获得的所有DMR特征信号谱具有浓度-响应依赖关系。
2.根据权利要求1所述一种无标记蛙皮素受体BB3的细胞筛选模型,其特征在于,待测样品具有激动活性的筛选步骤如下:
1)将溶解在含0.1%BSA的HBSS缓冲盐中的BA 1激动剂以浓度为10~ 10000 nM加入到接种HEK-293-BB3细胞的384微孔板中,检测其DMR特征信号谱;
2)将待测样品以0.01 nM ~ 100 µM加入到接种HEK-293-BB3细胞的微孔板中,检测其DMR信号谱;
3)关联分析步骤1)和步骤2)中的DMR信号谱,若步骤2)的DMR信号谱与1)中的DMR特征谱没有相似性,则样品没有激动活性;若具有轮廓相似性,则进行下一步骤;
4)将BB3拮抗剂BANTAG-1以浓度0.1 ~ 1000 nM加入到接种HEK-293-BB3细胞的微孔板中,预处理5 ~ 60 min,加入与步骤2)中相同浓度的待测样品,检测其DMR信号,若此DMR信号强度低于步骤2)中的DMR信号强度,则判断此样品为BB3受体的激动剂。
3.根据权利要求1所述一种无标记蛙皮素受体BB3的细胞筛选模型,其特征在于,待测样品具有拮抗活性的筛选步骤如下:
1)将待测样品和BA 1分别加入到接种HEK-293-BB3细胞的微孔板中,待测样品浓度为0.01 nM ~ 100 µM,BA 1浓度为10 ~ 10000 nM,检测DMR信号谱;
2)若步骤1)中待测样品不引起DMR信号谱,再向步骤1)中加入了待测样品的细胞板中继续加入与步骤1)中相同浓度的BA 1,检测DMR信号谱;若此DMR信号比步骤1)中BA 1的信号弱,可判断待测样品是BB3受体的拮抗剂。
4.根据权利要求1所述一种无标记蛙皮素受体BB3的细胞筛选模型,其特征在于,待测样品对BB3通路有调节活性的步骤如下:
1)将待测样品和BA 1分别加入到接种HEK-293-BB3细胞的微孔板中,待测样品浓度为0.01 nM ~ 100 µM,BA 1浓度为10 ~ 10000 nM,检测DMR信号谱;
2)再向步骤1)中加入了待测样品的细胞板中继续加入与步骤1)中相同浓度的BA 1,检测DMR信号谱,检测时间为1 ~ 60 min;若此DMR信号比步骤1)中BA 1的信号在上升期、平台期和延滞期某一个阶段不同;
3)将BB3拮抗剂BANTAG-1以浓度0.1 ~ 1000 nM加入到接种HEK-293-BB3细胞的微孔板中,预处理5 ~ 60 min,加入与步骤1)中相同浓度的待测样品,检测其DMR信号,若此DMR信号谱与步骤1)中的样品的DMR信号谱一致,可判断待测样品是BB3受体下游信号通路的调节剂。
5.根据权利要求4所述一种无标记蛙皮素受体BB3的细胞筛选模型,其特征在于,所述上升期为1 ~ 10 min、平台期为10 ~ 20 min和延滞期为20 ~ 60 min。
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