药物与蛋白和细胞相互作用的毛细管电泳研究
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摘要
前人在毛细管电泳分子相互作用分析领域已做了大量工作，并取得了丰硕成果。在研究生物大分子或细胞与药物之间的相互作用时，常会遇到生物大分子在毛细管上吸附、紫外检测背景高等问题。为解决这些难题，人们对研究方法进行了不断改进和发展，提出了区段灌注技术。
血清白蛋白是血液中重要的转运蛋白，研究血清白蛋白与药物的相互作用，对了解药物在体内的转运、吸收有重要意义，也可为新药的筛选和开发提供一定的依据。细胞是生物体形态结构和生命活动的基本单位，药物与细胞的相互作用研究无疑对药理研究还是新药开发都具有重要的意义。本文包括两个部分，第一部分是药物与血清白蛋白的相互作用分析，第二部分是药物与细胞的相互作用研究。
第一部分，在区段灌注技术基础上，建立了区段灌注-自标记的毛细管电泳分析方法，并将该方法应用于药物与蛋白相互作用的毛细管电泳分析。本部分对药物蛋白相互作用的两个体系进行了研究，并求得了相应的结合常数。这两个体系分别是：人血清白蛋白(HSA)-烟碱相互作用体系，其结合常数为1.66×103mol-1L；牛血清白蛋白(BSA)-麻黄碱(EPH)，咖啡因(CAF)和扑热息痛(ACEP)相互作用体系，它们的结合常数分别为KBSA-EPH=3.38 ×104mol-1L，KBSA-CAF=4.79×104 mol-1L和KBSA-ACEP= 6.13 ×104 mol-1L。
第二部分，采用第一部分建立的区段灌注-自标记技术，对麻黄碱、咖啡因、肾上腺素与红细胞的相互作用进行了研究，并采用了在形式上与分子间相互作用结合常数相似的常数——结合参数，来表达药物与细胞间的结合情况。这样，利用结合参数，我们就可对药物与细胞的结合情况进行判断。通过对不同实验所得的麻黄碱与细胞的结合参数比较，我们认为药物与细胞的结合参数重现性不高，因而对药物间结合参数的比较要在同一次实验中完成。本研究还对麻黄碱、咖啡因与细胞的结合参数进行了比较，结果表明咖啡因与细胞的结合能力比麻黄碱的强。本文认为这种差别是由麻黄碱和咖啡因在细胞膜上不同的分配行为所致。另外，本研究在对麻黄碱、肾上腺素与红细胞间的结合参数的比较中发现，肾上腺素与细胞的结合比麻黄碱强，并从理论上分析认为，这种差异源于麻黄碱和肾上腺素与红细胞膜上相应受体的不同结合能力。
结果表明，区段灌注-自标记技术是毛细管电泳相互作用分析的一种有效方法。该方法不仅有效地消除了蛋白背景吸收所带来的干扰问题，成功地克服了蛋白吸附对研究的影响，而且对峰漂移模型也作了进一步发展。使用区段灌注-自标记技术，结合常数的计算比采用Scatchard方程计算更为精确，适用范围更广。该技术将在毛细
管电泳药物与蛋白相互作用分析中发挥积极的作用，为非涂层毛细管药物蛋白分析提供了一条有效途径。本文还借助该技术，将分子相互作用的毛细管电泳研究的思路引入细胞和药物相互作用分析中，成功地建立了细胞与药物相互作用的毛细管电泳分析方法。
A amount of work has been done in the field of molecular interaction analysis by capillary electrophoresis, and a great deal of achievement has been obtained. But in the study of interaction on the drug molecules with the bio-molecules or cells, the adsorption of proteins on the capillary wall and the background induced by the biological particles are serious. To solve the problems, the interaction analytical methods have been improved and developed in this thesis.
The study of interaction between serum albumen and drugs is important in the investigation of the drug transfer and absorption in body which can be acted as assistant in the drug screening. The interaction analysis between cell and drug is important in the drug development and pharmacology. The paper is made up of two correlative parts. In the first part, the interaction between serum albumen and drugs were involved; in the second part, the interactions between red blood cell and drugs were studied.
The studies on interaction between drugs and serum albumen were consisted of Two interaction systems with the partial filling-self marker technique. One was the study on the interaction between human serum albumen (HSA) and nicotine and the binding constant of them was 1.66×103mol-1L. Another system is the interaction between bovine serum albumen(BSA) and ephedrine, caffeine and acetaminophen, and the binding constants between BSA to them were got, to be 3.38 ×104 mol-1L, 4.79×104mol-1L and 6.13 ×104mol-1L, respectively. In the study, the partial filling with the fore plug self marker technique for CE was built, which successfully reduced the interference from the absorption of the protein buffer.
In the second part, the partial filling -self marker technique was employed to study the interaction between the red blood cell (RBC) and ephedrine, caffeine and epinephrine and the interaction was evaluated by a parameter in the form of binding constant named as binding parameter. By the parameter, the interaction between cells and drugs can be described. The experiment fidelity was proved to be poor by the study of the ephedrine in different experiment. The comparing between the interaction between different drugs
and BRC was advised to be done in a same experiment. The binding parameters between ephedrine and caffeine to RBC was compared and the difference between was explained. The binding parameters between ephedrine and epinephrine to RBC was compared and the difference between was explained in principle.
The thesis indicated that the partial filling fore plug drug self marker technique is an effective method in the CE interaction study which has developed the peak-shifting model refined Scatchard plot and enlarged its scope. A way of uncovered capillary studying the interaction between protein and drugs was developed, which can not only be successfully used in the interaction study on of protein to drugs, but be used in the study of interaction between cells and drugs and a way to study the interaction between cells and drugs by CE was exploited.
血清白蛋白是血液中重要的转运蛋白，研究血清白蛋白与药物的相互作用，对了解药物在体内的转运、吸收有重要意义，也可为新药的筛选和开发提供一定的依据。细胞是生物体形态结构和生命活动的基本单位，药物与细胞的相互作用研究无疑对药理研究还是新药开发都具有重要的意义。本文包括两个部分，第一部分是药物与血清白蛋白的相互作用分析，第二部分是药物与细胞的相互作用研究。
第一部分，在区段灌注技术基础上，建立了区段灌注-自标记的毛细管电泳分析方法，并将该方法应用于药物与蛋白相互作用的毛细管电泳分析。本部分对药物蛋白相互作用的两个体系进行了研究，并求得了相应的结合常数。这两个体系分别是：人血清白蛋白(HSA)-烟碱相互作用体系，其结合常数为1.66×103mol-1L；牛血清白蛋白(BSA)-麻黄碱(EPH)，咖啡因(CAF)和扑热息痛(ACEP)相互作用体系，它们的结合常数分别为KBSA-EPH=3.38 ×104mol-1L，KBSA-CAF=4.79×104 mol-1L和KBSA-ACEP= 6.13 ×104 mol-1L。
第二部分，采用第一部分建立的区段灌注-自标记技术，对麻黄碱、咖啡因、肾上腺素与红细胞的相互作用进行了研究，并采用了在形式上与分子间相互作用结合常数相似的常数——结合参数，来表达药物与细胞间的结合情况。这样，利用结合参数，我们就可对药物与细胞的结合情况进行判断。通过对不同实验所得的麻黄碱与细胞的结合参数比较，我们认为药物与细胞的结合参数重现性不高，因而对药物间结合参数的比较要在同一次实验中完成。本研究还对麻黄碱、咖啡因与细胞的结合参数进行了比较，结果表明咖啡因与细胞的结合能力比麻黄碱的强。本文认为这种差别是由麻黄碱和咖啡因在细胞膜上不同的分配行为所致。另外，本研究在对麻黄碱、肾上腺素与红细胞间的结合参数的比较中发现，肾上腺素与细胞的结合比麻黄碱强，并从理论上分析认为，这种差异源于麻黄碱和肾上腺素与红细胞膜上相应受体的不同结合能力。
结果表明，区段灌注-自标记技术是毛细管电泳相互作用分析的一种有效方法。该方法不仅有效地消除了蛋白背景吸收所带来的干扰问题，成功地克服了蛋白吸附对研究的影响，而且对峰漂移模型也作了进一步发展。使用区段灌注-自标记技术，结合常数的计算比采用Scatchard方程计算更为精确，适用范围更广。该技术将在毛细
管电泳药物与蛋白相互作用分析中发挥积极的作用，为非涂层毛细管药物蛋白分析提供了一条有效途径。本文还借助该技术，将分子相互作用的毛细管电泳研究的思路引入细胞和药物相互作用分析中，成功地建立了细胞与药物相互作用的毛细管电泳分析方法。
A amount of work has been done in the field of molecular interaction analysis by capillary electrophoresis, and a great deal of achievement has been obtained. But in the study of interaction on the drug molecules with the bio-molecules or cells, the adsorption of proteins on the capillary wall and the background induced by the biological particles are serious. To solve the problems, the interaction analytical methods have been improved and developed in this thesis.
The study of interaction between serum albumen and drugs is important in the investigation of the drug transfer and absorption in body which can be acted as assistant in the drug screening. The interaction analysis between cell and drug is important in the drug development and pharmacology. The paper is made up of two correlative parts. In the first part, the interaction between serum albumen and drugs were involved; in the second part, the interactions between red blood cell and drugs were studied.
The studies on interaction between drugs and serum albumen were consisted of Two interaction systems with the partial filling-self marker technique. One was the study on the interaction between human serum albumen (HSA) and nicotine and the binding constant of them was 1.66×103mol-1L. Another system is the interaction between bovine serum albumen(BSA) and ephedrine, caffeine and acetaminophen, and the binding constants between BSA to them were got, to be 3.38 ×104 mol-1L, 4.79×104mol-1L and 6.13 ×104mol-1L, respectively. In the study, the partial filling with the fore plug self marker technique for CE was built, which successfully reduced the interference from the absorption of the protein buffer.
In the second part, the partial filling -self marker technique was employed to study the interaction between the red blood cell (RBC) and ephedrine, caffeine and epinephrine and the interaction was evaluated by a parameter in the form of binding constant named as binding parameter. By the parameter, the interaction between cells and drugs can be described. The experiment fidelity was proved to be poor by the study of the ephedrine in different experiment. The comparing between the interaction between different drugs
and BRC was advised to be done in a same experiment. The binding parameters between ephedrine and caffeine to RBC was compared and the difference between was explained. The binding parameters between ephedrine and epinephrine to RBC was compared and the difference between was explained in principle.
The thesis indicated that the partial filling fore plug drug self marker technique is an effective method in the CE interaction study which has developed the peak-shifting model refined Scatchard plot and enlarged its scope. A way of uncovered capillary studying the interaction between protein and drugs was developed, which can not only be successfully used in the interaction study on of protein to drugs, but be used in the study of interaction between cells and drugs and a way to study the interaction between cells and drugs by CE was exploited.
引文
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