胆固醇结石的体外模拟研究

发布时间：2018-11-17 09:34

【摘要】：人体胆汁中主要包含胆盐、卵磷脂和胆固醇这三种物质，其中胆固醇在胆汁中的溶解、成核与生长主要受其他两种物质含量变化的影响。当前对胆固醇结石的研究主要集中于在体外进行模拟，即采用类似于人体胆汁的模拟胆汁，通过实验获得胆固醇结石的产生机制。本文的研究主要是从胆固醇在模拟胆汁中的溶解、成核发生机制和成核时间、以及胆汁中可能影响胆固醇成核的因素这几个方面着手。首先，通过制备四元体系（胆盐-胆固醇-卵磷脂-水）的模拟胆汁，测定胆固醇在该体系中的溶解度（胆汁能够溶解胆固醇的量），获得了胆汁中卵磷脂、胆盐的含量变化以及模拟胆汁总脂度（胆汁中胆盐、胆固醇与卵磷脂总量之和）对胆固醇溶解度的影响情况。其中，胆汁中各组分的含量通过高效液相色谱法（HPLC）测定。结果表明，当胆盐含量越高，胆固醇在模拟胆汁中的溶解度越大；胆盐/卵磷脂摩尔比越大，胆固醇在模拟胆汁中溶解度也变大，摩尔比在0.5-1.77之间时，溶解度达到最大，当摩尔比继续增大时，胆固醇的溶解度则逐渐减小；此外，随着胆汁总脂度的增大，胆汁对胆固醇的溶解能力逐渐增强。最后，绘制了胆固醇在三元相图中的饱和溶解度曲线。其次，通过显微镜法、光散射法考察了不同组成的胆汁中各分散相（包括胶束和囊泡）的粒度分布情况。实验发现，对于含有复杂体系的胆汁，其组分含量的不同，标志着胆汁中存在着不同的相态。其中，模拟胆汁中囊泡的平均粒径主要在455.5nm左右，胶束的平均粒径主要在5.7nm左右。此外，通过实验观察模拟胆汁中胆固醇晶核的形成过程发现，胆汁中囊泡出现到囊泡发生聚集，胆固醇嵌入到囊泡中成核，最终形成液态晶体或固态晶体。最后，由于人体胆汁中的成分比较复杂，不仅含有以上三种主要物质，还包括钙盐、蛋白质、胆红素、细菌以及其他常量元素和微量元素等，所以本文还研究了钙盐和蛋白质对胆固醇成核的影响情况。对于钙盐的影响，本文以四元体系所构成的模拟胆汁作为参照胆汁，添加Ca2+（氯化钙）后得到的五元体系模拟胆汁（胆盐-胆固醇-卵磷脂-水-氯化钙），其成核时间相对变短，胆汁中胆固醇的溶解度减小。在不同总脂度下，Ca2+的浓度越大，溶解度愈小、成核时间愈短，而且，随着总脂度的增大，成核也越来越快。另外，含钙的模拟胆汁相图中其单相区域变小。因而，基于胆固醇成核时间和溶解度的测定结果，我们认为，Ca2+在胆固醇成核的过程中以促成核因子存在。通过粒度分布分析得到，含钙的模拟胆汁的囊泡平均粒径较大。对于蛋白质的影响，本文选择了牛血清白蛋白（BSA）作为胆汁蛋白质，将BSA替换钙盐，添加到四元体系的模拟胆汁中，构成另一五元体系。溶解度和成核时间测定结果表明，，BSA通过静电和疏水作用与卵磷脂结合，从而影响了卵磷脂形成的囊泡的稳定性，胆固醇的溶解度因而随着BSA浓度的升高而减小。但添加BSA后，胆汁中胆固醇的成核时间并没有明显的变化，即BSA的存在，仅对胆固醇的溶解度存在一定的影响，但对胆固醇的成核过程影响很小。
[Abstract]:The bile of human body mainly contains three substances of bile salt, lecithin and cholesterol, wherein the dissolution, nucleation and growth of cholesterol in the bile are mainly influenced by the change of other two substances. The present study on the cholesterol gallstone is mainly focused on the in vitro simulation, that is to use the simulated bile similar to the human bile, and the generation mechanism of the cholesterol stone is obtained through the experiment. The study of this paper is mainly about the dissolution of cholesterol in the simulated bile, the mechanism of nucleation and the time of nucleation, and the factors that may affect the nucleation of cholesterol in the bile. In the first place, the solubility of cholesterol in the system (the amount of bile capable of dissolving cholesterol) was determined by the preparation of the simulated bile of the quaternary system (bile salt-cholesterol-lecithin-water), and the bladder was obtained. The changes in the content of lecithin and bile salt in the juice and the total lipid of the bile (the sum of the total amount of bile salt, cholesterol and lecithin) in the bile to the solubility of cholesterol The content of each component in the bile is determined by high performance liquid chromatography (HP The results showed that the higher the bile salt content, the higher the solubility of cholesterol in the simulated bile, the higher the bile salt/ lecithin molar ratio, the higher the solubility of the cholesterol in the simulated bile and the molar ratio of 0. 5-1.77, when the molar ratio When continued to increase, the solubility of cholesterol is gradually reduced; in addition, with the increase of the total lipid of the bile, the dissolution of the bile to the cholesterol The force is gradually enhanced. Finally, the saturation of cholesterol in the ternary phase diagram is plotted. Solubility curve. Secondly, the disperse phase (including micelles and vesicles) in the bile of different composition was investigated by the method of microscope and light scattering. The results showed that in the bile with complex system, the content of the components of the bile is different, and the contents of the bile are marked in the bile. The average particle size of the vesicles in the simulated bile is about 455. 5nm, and the average particle size of the micelles is mainly about 455. 5nm. In addition, the formation of the cholesterol crystal nucleus in the simulated bile was observed by the experiment. The formation of the cholesterol crystal nucleus in the bile was observed. The formation of the vesicles in the bile and the formation of the cholesterol in the vesicles and the formation of the liquid were found. finally, because the composition of human bile is more complex, not only contains the above three main substances, but also contains calcium salt, protein, bilirubin, bacteria and other constant elements and trace elements, so the calcium salt and the protein are also studied in this paper. The effects of calcium salt on cholesterol nucleation were studied. The five-membered system of bile (bile salt-cholesterol-lecithin-water-water-water-water-water-water-water-water-water-water-water-water-water-water-water-water-water-water-water-water-water) was prepared by using the simulated bile composed of the four-element system as the reference bile and the addition of Ca2 + (calcium chloride). Calcium chloride), and the nucleation time of the calcium chloride is relatively variable. The higher the concentration of Ca2 +, the smaller the solubility, the shorter the nucleation time, and the shorter the nucleation time. The increase of the total lipid and the faster the nucleation. In addition, the calcium-containing The single-phase region of the simulated bile phase diagram is small. Thus, based on the determination of the time and solubility of cholesterol nucleation, we believe that Ca2 + is in cholesterol in the process of nucleation to contribute to the presence of a nuclear factor, obtained by particle size distribution analysis, In this paper, bovine serum albumin (BSA) was selected as the bile protein, and the BSA was replaced with calcium salt and added to the quaternion. In the simulated bile of the system, another five-membered system was constructed. The results of the determination of the solubility and the nucleation time showed that the BSA was combined with the lecithin by static and hydrophobic interactions, thus affecting the stability of the vesicles formed by the lecithin and the dissolution of cholesterol. The degree of solution is thus reduced with the increase of the concentration of BSA. However, after the addition of BSA, there is no significant change in the nucleation time of the cholesterol in the bile, i.e., the presence of BSA, and there is only a certain degree of solubility in the solubility of the cholesterol.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.62

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