α_（1A）-肾上腺素受体生物色谱的建立及应用
摘要
复杂体系中药物活性成分筛选是药学领域研究的热点科学问题,同时也是构建创新药物发现新模式的主要途径之一。已有的活性成分筛选方法包括：动物模型筛选法、溶液中细胞、酶和受体筛选法及经典的色谱筛选技术。上述技术的建立和应用对药物活性成分的筛选起到了积极的作用,但在特异性方面有待于进一步提高。本论文围绕α1A-肾上腺素受体(alphal A-adrenocepter,α1A-AR)生物色谱的构建及应用展开研究,旨在建立一种基于受体生物色谱的药物活性成分筛选新方法。全文分为三章,作者的主要贡献如下：
1.在现有药物活性成分筛选模型的基础上,利用体内受体识别药物配体的高特异性和色谱技术对复杂体系的高分离能力,提出了受体生物色谱药物活性成分筛选新模型。
2.采用细胞培养及亲和色谱等方法,获得了大量高纯度的α1A-AR.采用羰基二咪唑法将其固载在大孔硅胶表面,建立了αlA-AR生物色谱。用盐酸特拉唑嗪、去甲肾上腺素、坦索罗辛、间羟胺和乌拉地尔等工具药对色谱柱的保留特性进行了表征。在此基础上,应用该色谱方法对红花的活性成分进行了筛选。结果表明：盐酸特拉唑嗪等工具药在α1A-AR色谱柱上的容量因子分别为3.785、5.260、6.065、4.512、3.275,证明固载化受体具有特异性识别药物配体的能力；红花中与α1A-AR有特异性作用的活性成分为羟基红花黄色素A(Hydroxysafflor Yellow A,HSYA),证明受体生物色谱有望用于中药等复杂体系中药物活性成分的筛选。
3.分别采用前沿分析法和竞争置换法研究了固载化α1A-AR与盐酸哌唑嗪.盐酸特拉唑嗪作用的结合位点数、结合常数及其影响因素。结果表明：盐酸哌唑嗪与α1A-AR具有一类结合位点,结合位点数为5.8×10-6M,结合常数为1.6×105M-1,且其与受体作用过程的主要推动力为静电力；盐酸特拉唑嗪与α1A-AR具有两类结合位点,总结合位点数为6.1×10-6M,结合常数分别为1.1×105M-1,0.1×104M-1,其与受体的作用过程的主要推动力为疏水力。上述结果表明：受体生物色谱能用于在线研究受体与药物的相互作用。
Active components screening in complex system is a hot field of pharmacology, and is also an important way to find original new drugs. The active component screening methods include:animal model selection, solution-cells selection, enzymes and receptor model selection and classic chromatography model selection. The establishment and application of these models play a positive role for drug screening of active components, but these methods need to be further improved in the specific areas. This paper is mainly focus on the research of construction and application of alpha 1A-adrenocepter (α1A-AR) biochromatographic model, and aim to establish a new screening method for the active component. The paper has three chapters. The author's main contributions are as follows:
1. Based on existent methods of screening active component, the author make use of the high separate capacity of chromatography in complex system and the high specificity of adrenocptors to propose a new model of biochromatographic screening of drug active ingredients.
2. The author used the method of cell culturing and affinity chromatography to obtain alpha 1A-adrenocepters. Then, receptors were immobilized on the surface of macro porous silica gel by chemical coupling method, and the bonding sites and the bioactivity of the receptor on the surface of the stationary phase were characterized. Based on these, the biocharomatographic model was implicated to screening active components of Safflower. The result show that, capacity factors of terazosin, noradrenaline, tamsulosin, aramine and urapidil respectively are 3.785、5.260、6.065、4.512 and 3.275. Thus proves that the alpha 1A-adrenocepters biocharomatographic model can be used to screening active components of complex system.
3. The number of binding sites, association constant and influencing of immobilization alpha 1A-adrenocepters with prazosin and terazosin were determinated by the methods of frontal analysis and zonal elution. The result show that, the association constant of prazosin with alpha 1A-adrenocepters on the biochromatographic column was 1.6×105M-1, the binding sites was 5.8×10-6M. The main force on the binding process between prazosin and alpha 1A-adrenocepters was the electrostatic force. Terazosin and alpha 1 A-adrenocepters has two types of binding sites, the total binding sites was 6.1 x 10-6M, association constants were 1.1×105M-1,0.1 x 104M-1, The main force on binding process between prazosin and alpha 1A-adrenocepters was hydrophobic interaction. All the above results demonstrated that the receptor biocharomatographic model was a new technology for online investigating interaction between receptor and drug.
1.在现有药物活性成分筛选模型的基础上,利用体内受体识别药物配体的高特异性和色谱技术对复杂体系的高分离能力,提出了受体生物色谱药物活性成分筛选新模型。
2.采用细胞培养及亲和色谱等方法,获得了大量高纯度的α1A-AR.采用羰基二咪唑法将其固载在大孔硅胶表面,建立了αlA-AR生物色谱。用盐酸特拉唑嗪、去甲肾上腺素、坦索罗辛、间羟胺和乌拉地尔等工具药对色谱柱的保留特性进行了表征。在此基础上,应用该色谱方法对红花的活性成分进行了筛选。结果表明：盐酸特拉唑嗪等工具药在α1A-AR色谱柱上的容量因子分别为3.785、5.260、6.065、4.512、3.275,证明固载化受体具有特异性识别药物配体的能力；红花中与α1A-AR有特异性作用的活性成分为羟基红花黄色素A(Hydroxysafflor Yellow A,HSYA),证明受体生物色谱有望用于中药等复杂体系中药物活性成分的筛选。
3.分别采用前沿分析法和竞争置换法研究了固载化α1A-AR与盐酸哌唑嗪.盐酸特拉唑嗪作用的结合位点数、结合常数及其影响因素。结果表明：盐酸哌唑嗪与α1A-AR具有一类结合位点,结合位点数为5.8×10-6M,结合常数为1.6×105M-1,且其与受体作用过程的主要推动力为静电力；盐酸特拉唑嗪与α1A-AR具有两类结合位点,总结合位点数为6.1×10-6M,结合常数分别为1.1×105M-1,0.1×104M-1,其与受体的作用过程的主要推动力为疏水力。上述结果表明：受体生物色谱能用于在线研究受体与药物的相互作用。
Active components screening in complex system is a hot field of pharmacology, and is also an important way to find original new drugs. The active component screening methods include:animal model selection, solution-cells selection, enzymes and receptor model selection and classic chromatography model selection. The establishment and application of these models play a positive role for drug screening of active components, but these methods need to be further improved in the specific areas. This paper is mainly focus on the research of construction and application of alpha 1A-adrenocepter (α1A-AR) biochromatographic model, and aim to establish a new screening method for the active component. The paper has three chapters. The author's main contributions are as follows:
1. Based on existent methods of screening active component, the author make use of the high separate capacity of chromatography in complex system and the high specificity of adrenocptors to propose a new model of biochromatographic screening of drug active ingredients.
2. The author used the method of cell culturing and affinity chromatography to obtain alpha 1A-adrenocepters. Then, receptors were immobilized on the surface of macro porous silica gel by chemical coupling method, and the bonding sites and the bioactivity of the receptor on the surface of the stationary phase were characterized. Based on these, the biocharomatographic model was implicated to screening active components of Safflower. The result show that, capacity factors of terazosin, noradrenaline, tamsulosin, aramine and urapidil respectively are 3.785、5.260、6.065、4.512 and 3.275. Thus proves that the alpha 1A-adrenocepters biocharomatographic model can be used to screening active components of complex system.
3. The number of binding sites, association constant and influencing of immobilization alpha 1A-adrenocepters with prazosin and terazosin were determinated by the methods of frontal analysis and zonal elution. The result show that, the association constant of prazosin with alpha 1A-adrenocepters on the biochromatographic column was 1.6×105M-1, the binding sites was 5.8×10-6M. The main force on the binding process between prazosin and alpha 1A-adrenocepters was the electrostatic force. Terazosin and alpha 1 A-adrenocepters has two types of binding sites, the total binding sites was 6.1 x 10-6M, association constants were 1.1×105M-1,0.1 x 104M-1, The main force on binding process between prazosin and alpha 1A-adrenocepters was hydrophobic interaction. All the above results demonstrated that the receptor biocharomatographic model was a new technology for online investigating interaction between receptor and drug.
引文
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