肝内胆管结石病人胆汁糖蛋白、钙离子测定的意义

发布时间：2018-06-24 10:57

本文选题：肝内胆管结石 + 糖蛋白　；参考：《山东大学》2015年博士论文

【摘要】：前言肝内胆管结石是发生在左右肝管汇合部以上各分枝胆管内的结石,在西方国家很少见,中国的肝内胆管结石病人在所有的这类病人中占据着非常大的比例。肝内胆管结石是一种发病率高、病情严重复杂、治疗困难、容易复发的疾病。肝内胆管结石的发生与环境因素、饮食结构、感染、胆汁淤积、寄生虫、遗传等因素密切相关。结石形成的基础是胆固醇在胆汁中呈饱和甚至过饱和状态或者是非结合胆红素在胆汁中相对增多,胆汁中的糖蛋白、金属离子等均参与结石的形成并起着非常重要的作用。肝内胆管结石可引起反复发作的胆管炎、败血症、门脉高压、肝功能损害甚至衰竭等严重并发症,还可导致肝癌,严重威胁了病人的健康和生命。但是,肝内胆管结石不是恶性肿瘤,是一类良性疾病,应该是可以防治的。第一部分胆汁糖蛋白含量测定及其意义目的通过测定胆汁中糖蛋白含量,对比肝内胆管结石病人与肝外胆管结石病人胆汁中糖蛋白含量的不同,获得更有说服力的研究结果,探讨肝内胆管结石多发、复发的相关因素,为指导病人在生活工作中如何注意相关事项以预防其发生,指导医生如何选择恰当的治疗方法以取得更好的疗效提供重要依据。材料和方法1.病人资料：选择2012年12月-2014年02月间因胆石症入院的病人38例,肝内胆管结石组病人17例,肝外胆管结石组病人21例,同期住院的非胆石症病人18例为对照组,共56例。2.标本采集：胆石症病人胆汁标本于术后(或EST后)3天通过T型引流管或鼻胆管取20ml,放置在无菌试管内进行密封保存,并放入冰箱内,设定温度为零下20摄氏度。对非胆石症病人均于术中细针穿刺胆囊取胆汁20ml,存放置在无菌试管内进行密封保存,并放入冰箱内,设定温度为零下20摄氏度。3.方法：胆汁标本解冻后,经过离心、沉淀、除盐等过程后,通过SDS-PAGE电泳跑出蛋白条带,将分子量在200KD以下的蛋白条带转移至硝酸纤维素膜上,再以糖蛋白测定试剂盒,采用过碘酸一席夫液试剂染色法(PAS法)进行糖蛋白定性试验。糖蛋白的定量按Lowry法,以血清白蛋白为标准,计算分子量在200KD以下的糖蛋白含量值。4.统计学处理：分别计算各组病人的胆汁糖蛋白含量,所得数据用X±S表示,以SPSS17.0软件进行统计学处理。采用独立样本实验的均值比较,方差齐则取对应方差齐的P值,方差不齐则取对应方差不齐的P值,P0.05为差异有统计学意义。结果1.关于胆汁糖蛋白含量：肝内胆管结石组病人高于肝外胆管结石组病人,肝外胆管结石组病人高于对照组病人。2.关于均值比较统计学处理：肝内胆管结石组病人与肝外胆管结石组病人两组数据间的差异具有显著性；肝内胆管结石组病人、肝外胆管结石组病人分别与对照组病人数据比较的差异均具有显著性。讨论1.研究肝内胆管结石发病机制的重要性：肝内胆管结石因其治疗困难及并发症严重可能会导致病人死亡,但是它不是恶性肿瘤,而是一类良性疾病,应该是可以预防和治愈的。因此,探索其病因及发病规律,为指导病人在生活工作中如何注意相关事项以及指导医生如何选择恰当的治疗方法提供依据就显得非常重要。2.糖蛋白在肝内胆管结石形成中的作用机制：研究发现糖蛋白的凝聚作用是胆石形成的主要因素。许多实验结果表明：一定分子量范围内的糖蛋白在胆石形成过程中起促进作用。糖蛋白或糖蛋白胆色素复合物可以作为核心,使胆固醇结晶或胆色素钙盐不断沉积而形成结石。糖蛋白分子之间互相交联在胆管粘膜表面形成胶体网状结构中,微泡的运动受到限制,微泡脱水融合,形成胆固醇结晶。糖蛋白形成的凝胶体粘性很高,胆色素分子及钙离子被粘附在凝胶体中,相互结合形成胆色素钙复合物。在糖蛋白凝胶体中,胆固醇结晶或胆色素钙盐在糖蛋白复合物或糖蛋白胆色素复合物周围不断沉积、粘附、结合而形成结石。3.糖蛋白的相关研究：很多学者通过各自的研究一致认为,糖蛋白在胆石形成过程中起着重要的作用。4本研究的特点：我们以肝内胆管结石病人为研究对象,直接用胆汁为标本测定糖蛋白含量,从深层次探讨与肝内胆管结石形成及复发相关的因素。采用SDS-PAGE电泳分离后PAS法染色,测定胆汁糖蛋白含量,这种方法是目前比较先进的测定糖蛋白的方法,经济,可靠,数据容易得出。促进胆管结石形成的糖蛋白分子量都在200KD左右以下。结论本研究结果表明胆汁糖蛋白含量与胆管结石的形成相关,而胆汁糖蛋白含量与肝内胆结石的形成关系更为密切,测定胆汁糖蛋白含量对肝内胆管结石病人具有重要的意义。第二部分胆汁钙离子含量测定及意义目的本研究通过测定胆汁钙离子含量,对比肝内胆管结石病人与肝外胆管结石病人胆汁钙离子含量的不同,获得更有说服力的研究结果,探讨肝内胆管结石多发、复发的相关因素,为指导病人在生活工作中如何注意相关事项以预防其发生,指导医生如何选择恰当的治疗方法以取得更好的疗效提供重要依据。材料和方法1.病人资料及标本采集同第一部分。2.方法：胆汁标本冻融后,在酸性铬蓝K缓冲溶液中加入胆汁标本,胆汁中的钙与酸性铬蓝K间形成一种络合物,此络合物的最大吸收波长为496 n m,用分光光度法在波长496 n m处测量酸性铬蓝K溶液吸光度减少值,能测定的钙离子含量范围为0～60mol/L。3.统计学处理：分别计算各组病人的胆汁钙离子含量,所得数据用X±S表示,以SPSS17.0软件行统计学处理。采用独立样本实验的均值比较,方差齐则取对应方差齐的P值,方差不齐则取对应方差不齐的P值,P0.05为差异有统计学意义。结果1.关于胆汁钙离子含量：肝内胆管结石组病人高于肝外胆管结石组病人,肝外胆管结石组病人高于对照组病人。2.关于均值比较统计学处理：肝内胆管结石组病人与肝外胆管结石组病人两组数据间的差异具有显著性；肝内胆管结石组病人、肝外胆管结石组病人分别与对照组病人数据比较的差异均具有显著性。讨论1肝内胆管结石的特点：肝内胆管结石在我国的发病率比较高,其发生与环境、饮食、感染等许多因素有关,其形成涉及到游离胆红素所占比例增高；结石核心的形成；糖蛋白的凝聚作用以及金属离子的参与等。主要是胆色素结石,常伴有胆汁淤积及胆道感染。临床特点为间歇性、反复性发作。其治疗比较困难,疗效不够满意。2病因机制的特殊性：肝内胆管结石主要成分是胆色素和钙。由于胆道感染等原因导致胆汁中游离胆红素所占的比例增高,可与胆汁中的钙结合,形成不溶性的胆红素钙而析出,进而形成肝内胆管结石3钙离子在肝内胆管结石形成中的作用机制研究：游离胆红素和其他物质与金属离子集结是肝内胆管结石形成的一个主要过程,并且钙离子是所有离子中的首要作用物质。钙离子与胆红素、糖蛋白分子结构中的羟基很容易结合形成不能分解的聚合物。钙离子在胆石网状结构的形成中起着离子桥的作用。4本研究的特点：我们以肝内胆管结石病人为研究对象,直接用胆汁为标本测定胆汁中钙离子含量,这是本研究的与以往相关研究的不同之处,希望从深层次探讨与肝内胆管结石形成及复发相关的因素。钙离子测定选用酸性铬蓝K分光光度法,此方法能测定胆汁中微量钙,误差很小,比较准确,方法简单易行。结论本研究结果表明胆汁钙离子含量与胆管结石的形成相关,尤其是与肝内胆结石形成关系更为密切。测定胆汁糖蛋白含量对肝内胆管结石病人具有重要的意义。小结1.肝内胆管结石介绍：肝内胆管结石在西方国家较为少见,我国的这类病人相对比较多。可引起胆管梗阻、感染,病情加重可引起肝脓肿、重症胆管炎、感染性休克。长期不愈可能导致肝纤维化、肝硬化甚至肝癌。肝内胆管结石病人的治疗方法有肝切除术、内镜治疗、肝移植等等。肝内胆管结石的特点是发病率高、病情复杂、治疗困难、容易复发,严重影响了病人的健康甚至生命。但是,它是一类良性疾病,应该是可以预防和治愈的。2.本研究结果：肝内胆管结石是一种特殊类型的胆管结石,在结石形成机理方面既具有一般胆管结石形成的基本规律,又具有其自身的特点。3.本研究对预防的意义：对于具有肝内胆管结石高发因素同时胆汁糖蛋白、钙离子含量增高的病人,在生活工作中应特别注意习惯,饮食,环境等事项或采取一定的预防措施如应用疏肝利胆的一些药物等,尽可能减少或避免该病的发生。4.在治疗中的意义：对于胆汁糖蛋白、钙离子含量明显增高的肝内胆管结石病人,应采取更积极、确实的治疗方法；要为后续治疗预留途径；采用综合性治疗措施；做好随访。5.与相关研究的比较：很多相关研究与本研究的结果是一致的,说明了结石形成机理的一般规律。个别结果不一致,可能与地区、种族、测定方法及计算统计方法不完全一致有,但不是本质的、规律的不同,不影响本研究的结果。6.不足之处：本研究的所有过程都严格按照课题及实验设计的要求进行的,但毕竟样本量有限,实验结果尚需要多中心大样本的随机对照实验来进一步证实。
[Abstract]:Intrahepatic choledocholithiasis, which occurs in the bile duct of the left and right hepatic duct, is rare in the western countries. In China, the patients with intrahepatic cholangiolithiasis occupy a very large proportion in all these patients. Intrahepatic bile duct stones are a high incidence, serious and complicated disease, difficult treatment, and easy to relapse. The occurrence of intrahepatic bile duct stones is closely related to environmental factors, dietary structure, infection, cholestasis, parasite, heredity and other factors. The basis of the formation of the stones is that the cholesterol is saturated or even supersaturated in the bile, or the unbound bilirubin is relatively increased in the bile, and the glycoproteins and metal ions in the bile are all involved in the shape of the stones. Hepatolithiasis can cause severe complications such as recurrent cholangitis, septicemia, portal hypertension, liver function damage and even failure. It can also cause liver cancer and seriously threaten the health and life of the patients. However, the hepatolithiasis is not a malignant tumor. It is a kind of benign disease and should be prevented. The determination of the content of bile glycoprotein in part 1 and its purpose by measuring the content of glycoprotein in bile and comparing the difference of the glycoprotein content in the bile of patients with intrahepatic bile duct stones and extrahepatic bile duct stones, to obtain more convincing results, to explore the related factors of the recurrence of intrahepatic bile duct stones, and to guide the patients. In the life work, how to pay attention to the related matters in order to prevent its occurrence and how to guide the doctor to choose the appropriate treatment to achieve better curative effect provides an important basis. Material and methods 1. patients: 38 cases of cholelithiasis, 17 cases of intrahepatic bile duct stones, and extrahepatic bile duct stones in 02 months of -2014 in December 2012. Group 21 cases, 18 cases of non cholelithiasis patients in the same period as control group, 56 cases of.2. specimens were collected: bile specimens of cholelithiasis patients were sealed and preserved in aseptic test tubes for 3 days after operation (or after EST) by T type drainage tube or nasal bile duct, and placed in the refrigerator to set the temperature of 20 degrees below zero. The bile 20ml was taken from the gallbladder in the intraoperative fine needle puncture, stored in a sterile test tube to be sealed and preserved, and placed in the refrigerator to set a.3. method of 20 degrees Celsius under zero temperature. After the bile samples were defrosting, after the centrifugation, precipitation, and desalination, the protein strip was run out by SDS-PAGE electrophoresis, and the protein strips with molecular weight below 200KD were transferred. It was moved to the nitrocellulose membrane, then the kit was measured with glycoprotein, and the qualitative test of glycoprotein was carried out by the PAS method. The quantitative Lowry method of glycoprotein and the serum albumin as the standard were used to calculate the content of glycoprotein content below 200KD by.4. statistics: the bile of each group of patients was calculated respectively. The content of glycoprotein was expressed with X + S, and the statistical treatment was performed with SPSS17.0 software. The mean comparison of the independent sample experiment was used to take the P value of the corresponding variance homogeneous, and the P value of the corresponding variance was not homogeneous, and the difference was statistically significant. Results 1. about the bile glycoprotein content: the intrahepatic bile duct stone group disease. The patients were higher than the extrahepatic bile duct stone group, and the patients with extrahepatic bile duct stone group were higher than those of the control group. The mean value of.2. was statistically significant. The difference between the two groups of the patients in the hepatolithiasis group and the extrahepatic bile duct stone group was significant; the patients in the hepatolithiasis group and the extrahepatic bile duct stone group were respectively with the control group. The difference in human data comparison is significant. Discuss the importance of the 1. study on the pathogenesis of intrahepatic bile duct stones: intrahepatic bile duct stones may lead to death because of their treatment difficulties and serious complications, but it is not a malignant tumor, but a kind of benign disease, which should be prevented and cured. Therefore, explore its cause and hair. The rule of disease, in order to guide the patient to pay attention to the related matters in the life work and to guide the doctor how to choose the appropriate treatment method, it is very important that the mechanism of.2. glycoprotein in the formation of intrahepatic bile duct stone formation: the study found that the coagulation of glycoprotein is the main factor of the formation of bile stones. The glycoproteins in a certain molecular weight range play a role in the formation of cholelithiasis. Glycoprotein or glycoprotein cholangio complex can be used as the core to make the cholesterol crystals or calcium salts of calcium salts to form stones. The cross linking of glycoproteins to the surface of the bile duct mucosa forms a colloidal network, and the microbubbles are transported. The gelatin molecules and calcium ions are adhered to the gel to form a calcium complex of calcium pigments. In the glycoprotein gel, the cholesterol crystallization or the calcium salt of the cholesterol is in the glycoprotein complex or the glycoprotein cholesterin complex. The related research on the formation of.3. glycoprotein by continuous deposition, adhesion and binding: many scholars agree that glycoproteins play an important role in the formation of cholelithiasis through their respective studies. We take the hepatolithiasis patients as the research object and determine the glycoprotein content directly by bile. The factors related to the formation and recurrence of intrahepatic bile duct stones were deeply discussed. The content of bile glycoprotein was determined by PAS staining after SDS-PAGE electrophoresis separation. This method is a more advanced method for the determination of glycoprotein at present. It is economical, reliable, and the data are easy to be obtained. The molecular weight of glycoprotein in the formation of bile duct stones is below 200KD Conclusion the results of this study show that the content of bile glycoprotein is related to the formation of bile duct stones, and the content of bile glycoprotein is more closely related to the formation of intrahepatic gallstones. The determination of the content of bile glycoprotein is of great significance for the patients with intrahepatic bile duct stones. The determination of the content and significance of the second parts of the bile cacalate is the purpose of the study. The content of calcium ion of fixed bile and the difference of calcium content of bile in patients with hepatolithiasis and extrahepatic bile duct stone were compared. The results of more persuasive research were obtained, and the related factors of recurrent intrahepatic bile duct stones were discussed. Material and methods 1. patient data and samples were collected with the first part.2. method: bile specimens were added to the acid chrome blue K buffer solution after the bile specimens were frozen and thawing, and the calcium in the bile was formed into a complex between the acid chrome blue K and the maximum absorption wave of the complex. With a length of 496 n m, the absorbency reduction value of acid chrome blue K solution was measured at the wavelength of 496 n m by spectrophotometric method. The content of calcium ion content was measured by 0 to 60mol/L.3.. The calcium content of the bile was calculated in each group of patients respectively. The obtained data were expressed in X + S, using SPSS17.0 software. The independent sample experiment was used. The average variance was compared with the P value of the homogeneity of variance, and the P value of the variance was not homogeneous, and the difference of P0.05 was statistically significant. Results 1. of the bile calcium ion content: the patients in the intrahepatic bile duct stone group were higher than those in the extrahepatic bile duct stone group, and the patients in the extrahepatic bile duct stone group were higher than those of the control group with the mean comparison of the mean value of.2.. Statistical treatment: the difference of data between the two groups of patients with hepatolithiasis group and extrahepatic bile duct stone group was significant. The difference of the data of the patients in the intrahepatic bile duct stone group and the extrahepatic bile duct stone group were significantly different from those of the control group. The characteristics of the 1 intrahepatic bile duct stones were discussed: the intrahepatic bile duct stones were in me. The incidence of the disease is high in the country, which is related to many factors such as environment, diet, infection and so on. Its formation involves the increase in the proportion of free bilirubin, the formation of stone core, the coagulation of glycoprotein and the involvement of metal ions, mainly the cholestasis, often accompanied by cholestasis and biliary tract infection. A recurrent attack. The treatment is difficult and the curative effect is not satisfied with the specificity of the.2 etiological mechanism: the main components of the intrahepatic bile duct stones are bile pigment and calcium. The proportion of free bilirubin in bile increases as a result of biliary tract infection, which can combine with calcium in the bile to form insoluble bilirubin calcium and form the liver. The mechanism of 3 calcium ion in the formation of intrahepatic choledocholithiasis: the aggregation of free bilirubin and other substances and metal ions is a major process in the formation of intrahepatic bile duct stones, and calcium ions are the primary substances in all ions. Calcium ions and bilirubin, the hydroxyl groups in the molecular structure of glycoprotein are easy to knot. The formation of an incapable polymer. Calcium ions play the role of ion bridge in the formation of the cholelithiasis reticular structure.4: we take the intrahepatic cholelithiasis patients as the research object and determine the calcium content in the bile by direct bile. This is the difference between this study and the related research. Study on the factors associated with the formation and recurrence of intrahepatic bile duct stones. Calcium ion determination is used to determine the trace calcium K in the bile. This method can be used to determine trace calcium in bile. The error is very small and accurate. Conclusion the results of this study show that the calcium content of bile is related to the formation of bile duct stones, especially in the liver gallstones. The determination of the content of bile glycoprotein is of great significance for the patients with intrahepatic bile duct stones. The introduction of 1. intrahepatic bile duct stones: intrahepatic bile duct stones are relatively rare in western countries. There are relatively many cases of these patients in our country. It can cause bile duct obstruction, infection, and aggravation of the disease can cause liver abscess, severe cholangitis and feeling. In the long run, it may lead to liver fibrosis, liver cirrhosis and even liver cancer. The treatment of hepatolithiasis patients include hepatectomy, endoscopic treatment, liver transplantation and so on. The characteristics of hepatolithiasis are high incidence, complicated condition, difficult treatment, easy recurrence, severe weight affecting the health and even life of the patients. However, it is one of the patients. Benign diseases of the class should be the results of the.2. study that can be prevented and cured: intrahepatic bile duct stones are a special type of bile duct stones. The mechanism of stone formation not only has the basic law of the formation of common bile duct stones, but also has its own characteristics: the significance of this Study on prevention: the high incidence of intrahepatic bile duct stones. Patients with higher levels of bile glycoprotein and calcium ions should pay special attention to habits, diet, environment, etc. in life work, or take some preventive measures, such as some drugs, such as the use of Liver Soothing and gallbladder, to reduce or avoid the significance of.4. in the treatment of the disease: for bile glycoprotein and calcium content, The patients with elevated intrahepatic bile duct stones should take more active and true treatment methods; to reserve the way for follow-up treatment; adopt comprehensive treatment measures; do a good job of follow-up.5. and related research: many related studies are consistent with the results of this study, indicating the general rule of the formation mechanism of the formation of stone. It may not be entirely consistent with area, race, determination method and calculation and statistical method, but it is not essential, and the law is different. It does not affect the.6. deficiency of the results of this study. All the processes of this study are strictly based on the requirements of the subject and experimental design, but the sample size is limited and the experimental results still need many central samples. This randomized controlled trial is further confirmed.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R657.42

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