CN105277684A - 一种基于荧光共振能量转移技术的黑素皮质素受体1药物筛选模型 - Google Patents
一种基于荧光共振能量转移技术的黑素皮质素受体1药物筛选模型 Download PDFInfo
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Abstract
本发明发现了一种筛选黑素皮质素受体1激动剂的药物筛选方法，本发明基于荧光共振能量转移原理，利用完整细胞生产的cAMP与反应体系中生物素标记的cAMP(b-cAMP)竞争性的和抗cAMP抗体结合，中断了荧光共振能量转移的过程，造成荧光信号的减弱，从而反映出化合物是否具有黑素皮质素受体1激动作用。包括以下步骤：(1)细胞培养；(2)标准曲线的测定；(3)模型验证：阳性药IC50与参考文献一致。方法简便快捷；荧光检测值灵敏度高，提高检测精确度；结果稳定可靠，重现性好。
Description
技术领域
本发明属于药理学领域，利用荧光检测技术，构建了一种黑素皮质素受体的药物筛选模型，用于待测样品对黑素皮质素受体1活性的药物筛选。
背景技术
MC1受体即黑素皮质素受体1(melanocortin1receptor，MC1R)，这一受体的基因是控制动物黑色素合成的重要基因，在黑色素细胞、肾上腺皮质细胞、神经系统和免疫系统中表现出不同的功能。通过控制真黑色素数量来决定动物的毛色，并影响动物的疾病发生。MC1R基因的突变与黑色素性状变异和皮肤癌等疾病有关。所以建立MC1R激动剂筛选模型对治疗皮肤癌等疾病具有重要意义。
LancecAMP方法基于荧光共振能量转移原理，通过检测荧光信号，反映出样品中环磷酸腺苷(cAMP)含量变化。实验中完整细胞生产的cAMP与反应体系中生物素标记的cAMP(b-cAMP)竞争性的和抗cAMP抗体结合，中断了荧光共振能量转移的过程，造成荧光信号的减弱，从而反映出化合物是否具有黑素皮质素受体1激动作用。
MC1R基因在哺乳动物中由extension位点编码，是G蛋白偶联受体(GPCR)家族成员之一，只有一个编码区，其编码的蛋白质有7个跨膜结构域，主要表达于动物的黑色素细胞。cAMP是其主要的信号分子，因此MC1R激动剂的筛选适用于LancecAMP方法。
目前，已有的黑素皮质素受体1激动剂/拮抗剂筛选方法，多利用荧光素酶报告基因法以及酶联免疫吸附法，但此两种方法费时费力，同时难以应用到高通量筛选。因此，建立方便快捷准确的检测方法，特别是体外的功能性检测在药物筛选中越来越受到重视。
发明内容
本发明的目的在于建立一种基于荧光的黑素皮质素受体1激动剂的药物筛选方法，具有信噪比高，使用安全，样品消耗量小的特点。
本发明的技术方案：采用荧光方法建立体外黑素皮质素受体1激动剂的药物筛选模型，初筛，复筛发现一类具有激动黑素皮质素受体1活性的候选化合物。具体步骤如下：
本发明利用荧光的方法建立了一种黑素皮质素受体1激动剂的药物筛选模型。
步骤一：细胞培养。
步骤二：标准曲线的测定。
步骤三：模型验证。
附图说明：
图1：以配制cAMP标准稀释液对标准曲线的测定。(n＝3，)
图2：Forskolin作用不同细胞数所产生的信号与标准曲线的对比。(n＝3，)
图3：(Nle4，D-Phe7)-α-MSH可刺激CHO稳转细胞株MC1受体产生cAMP且成量效依赖性。其IC50为1.03×10-11M。(n＝3，)
具体实施方式
以下结合附图说明本发明的具体实施方式：
细胞株：CHO-MC1
试剂：Ham’sF12，10％FBS，100IU/mlpenicillin，100μg/mlstreptomycin，400μg/mlG418，Hank平衡盐溶液(HBSS)，StimulationBufferA，StimulationBufferB。
仪器：二氧化碳培养箱；384孔板；Multidrop加样器；EnVision2014MultilabelReader。
一、细胞培养
(1)细胞的贴壁度要达到80％，达到对数生长期。
(2)用温和的PBS冲洗细胞一次。
(3)用0.05％trysin-lmMEDTA消化细胞。
(4)用1500rpm离心2分钟。
(5)停止后用完整培养基冲洗一次。
(6)对细胞计数，取细胞悬液到15ml培养基中，使细胞浓度达到7.5×105cells/ml，将此细胞悬液加入到T75培养瓶中，让细胞在37℃/5％CO2下过夜以进行培养。
二、标准曲线的测定
(1)配制cAMP标准稀释液：cAMP的标准溶液50μmol/L(试剂盒自带)，逐级稀释在Stimulatebuffer中。
(2)配制Alexa647-anticAMP抗体溶液：Alexa647-anticAMP溶液在StimulationBuffer中以1％的比例稀释(如将5μL的抗体液加入到495uL的StimulationBuffer中)，然后上下倒置混合均匀。
(3)配制DetectionMix
a.在cAMPDetectionBuffer中稀释标记了链霉素的LANCEEu-W8044(Eu-SA)：将5μL标记了链霉素的Eu-W8044(Eu-SA)加入到85μL的cAMPDetectionBuffer.
b.在cAMPDetectionBuffer中稀释Biotion-cAMP：将5μL的Biotion-cAMP(b-cAMP)加入到25μl的cAMPDetectionBuffer中。
c.根据以上两步的稀释物来进行混合检测：将615μl的DetectionBuffer加入到试管中；将5μl的Eu-W8044稀释物加入到试管中；轻轻地混合；将5μl的b-cAMP稀释物加入试管中；轻轻地混合；在室温下至少孵育15分钟。
(4)标准曲线检验步骤
实验方法在白色OptiPlate-384microplates上进行，总体积为20μL。
a.加5μL的cAMP标准稀释物.
b.加5μL的抗体溶液.
c.在室温下孵化30-60分钟.
d.加10μL的DetectionMix.
e.将板用TopSeal-A软片盖上并在室温下孵化60分钟.
f.在TRF检测仪上读取数据.在读取数据前要去掉TopSeal-A.
g.在检测的灵敏度范围内，阅读可在20小时以内进行。
三、模型验证
(1)配制StimulationbufferB：1×的HBSS中含5mMHEPES，0.1％BSA，IBMX以0.5mmol/L，pH7.4。
(2)用非胰酶的消化酶(0.2g/L的EDTA溶解于PBS中)消化细胞。以HBSS清洗细胞一次。
(3)在StimulationBuffer中重悬细胞使细胞浓度达到3x106cells/mL。将细胞稀释成11,500/5μL，5,750/5μL，2,880/5μLand960cells/5μL，以及没有细胞的空白孔。
(4)抗体溶液的制备：将5μL的抗体液加入到495μL的StimulationBuffer细胞悬液中，然后上下倒置混合均匀.
(5)将化合物溶于StimulationBuffer中制成4X浓度.如果有一种以上的化合物需要加入(如激动剂-forskolin)。
(6)LANCEcAMPAssay在optiplate384孔板中进行，其终体积为20μl：
a.向孔中加入2.5μl4X待测药物。
b.向孔中加入2.5μl的StimulationBuffer。
c.再加入5μl的细胞悬液(包含Alexa-labeledantibodies).
d.室温孵育细胞30-60分钟。
e.加入10μl的DetectionMix(配制方法如前).
f.用TopSeal-Afilm封板，在室温下孵育60minutes。
g.将封膜摘除，于TRF检测读取数据。
h.20小时以内可以进行再次的检测，不影响其灵敏度。
化合物稀释：
(1)将非GPCR依赖型激动cAMP化合物Forskolin使用DMSO配制成50mM母液，以StimulationBuffer逐级稀释为工作浓度的4倍。
(2)阳性药为黑素皮质素受体1激动剂(Nle4，D-Phe7)-α-MSH，使用无菌三蒸水配制成5mg/ml母液，以StimulationBuffer逐级稀释为4×药物浓度。
实验结果
确定激动稳转细胞株黑素皮质素受体1的药物筛选模型造模条件，每孔细胞数为192cells/μl，即为960cells/well(图2)，所得到forskolin刺激结果与标准曲线(图1)趋势最为一致。通过该模型验证了(Nle4，D-Phe7)-α-MSH对CHO稳转细胞株黑素皮质素受体1的激动作用，其IC50为1.03×10-11M(图3)。
Claims (7)
1.一种黑素皮质素受体1激动剂的药物筛选模型，其特征在于，包括步骤：
(1)细胞培养；
(2)标准曲线的测定；
(3)模型验证。
2.如权利要求1所述的方法，其特征在于，所述的黑素皮质素受体为黑素皮质素受体1。
3.如权利要求1所述的方法，其特征在于，步骤(1)中进行细胞培养，培养条件为：细胞悬液到15ml培养基中，使细胞浓度达到7.5×105cells/ml，将此细胞悬液加入到T75培养瓶中，让细胞在37℃/5％CO2下过夜以进行培养。
4.如权利要求1所述的方法，其特征在于，步骤(2)中，配制不同浓度的cAMP的标准溶液，每孔加入5μl，再每孔加入5μL的抗体溶液，在室温下孵化30-60分钟后，加10μL的DetectionMix，将板用TopSeal-A软片盖上并在室温下孵化60分钟。在TRF检测仪上读取数据。在读取数据前要去掉TopSeal-A，在检测的灵敏度范围内，阅读可在20小时以内进行。经数据统计，确定cAMP标准曲线。
5.如权利要求1所述的方法，其特征在于，步骤(3)配制不同浓度的非GPCR依赖型激动cAMP化合物Forskolin使用DMSO配制成50mM母液，以StimulationBuffer逐级稀释为工作浓度的4倍，每孔加入2.5μl，向每孔加入2.5μL的StimulationBuffer，再加入5μl的细胞悬液(包含Alexa-labeledantibodies)，在室温下孵化30-60分钟后，加10μL的DetectionMix，将板用TopSeal-A软片盖上并在室温下孵化60分钟。在TRF检测仪上读取数据。在读取数据前要去掉TopSeal-A，在检测的灵敏度范围内，阅读可在20小时以内进行。经数据统计，确定最佳每孔细胞数为960cells/well。
6.如权利要求1所述的方法，其特征在于，步骤(3)配制不同浓度的黑素皮质素受体l激动剂(Nle4，D-Phe7)-α-MSH，使用无菌三蒸水配制成5mg/ml母液，以StimulationBuffer逐级稀释为工作浓度的4倍。每孔加入2.5μl，向每孔加入2.5μL的StimulationBuffer，再加入5μl的细胞悬液(包含Alexa-labeledantibodies)，在室温下孵化30-60分钟后，加10μL的DetectionMix，将板用TopSeal-A软片盖上并在室温下孵化60分钟。在TRF检测仪上读取数据。在读取数据前要去掉TopSeal-A，在检测的灵敏度范围内，阅读可在20小时以内进行。经数据统计，(Nle4，D-Phe7)-α-MSH对CHO稳转细胞株黑素皮质素受体1具有激动作用，其IC50为1.03×10-11M，与文献报道一致。
7.权利要求1-6中所述的方法在筛选黑素皮质素受体1活性药物中的应用。
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| US6355473B1 (en) * | 1998-05-06 | 2002-03-12 | Cadus Pharmaceutical Corp. | Yeast cells having mutations in stp22 and uses therefor |
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| US20070105096A1 (en) * | 2002-04-16 | 2007-05-10 | Katsutoshi Sasaki | Endocrine cell lines and methods of using same |
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| US6355473B1 (en) * | 1998-05-06 | 2002-03-12 | Cadus Pharmaceutical Corp. | Yeast cells having mutations in stp22 and uses therefor |
| US20070105096A1 (en) * | 2002-04-16 | 2007-05-10 | Katsutoshi Sasaki | Endocrine cell lines and methods of using same |
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