胆囊胆固醇沉着症的发病机制及相关干预研究

发布时间：2018-04-29 15:43

本文选题：胆囊胆固醇沉着症 + 过氧化物酶增殖体活化受体-γ　；参考：《东南大学》2015年博士论文

【摘要】：目的：胆囊胆固醇沉着症是因粘膜层及粘膜下层中过量的脂滴积聚所致。巨噬细胞吞噬脂滴后可以变成泡沫细胞,大量的泡沫细胞堆积于淋巴管,常导致淋巴管破坏,甚至影响局部的徽循环,导致脂质回流障碍。病理上,本病可分为两个亚型：弥漫性胆固醇沉着症和胆固醇息肉。其中,前者的粘膜下层脂质回流通道尚存在,药物干预能达到治疗的目的；后者的粘膜下层脂质回流通道受阻,只有手术切除息肉才能达到治疗目的。胆囊胆固醇沉着症脂滴中的主要成分是胆固醇酯,除去上皮细胞中的胆固醇酯对于维持细胞中胆固醇内环境的稳定和保护胆囊的生理功能具有重要意义。很多研究显示过氧化物酶增殖体活化γ受体或者肝X受体α与细胞内胆固醇内环境的稳态密切相关。22(R)-羟基胆固醇既是体内固醇类物质的中间代谢物,又可以上调肝X受体α的蛋白表达量；吡格列酮可以上调过氧化物酶增殖体活化γ受体的蛋白表达量。对于弥漫性胆固醇沉着症,本实验的目的就是判定吡格列酮(过氧化物酶增殖体活化受体-γ的激动剂)可否降低胆固醇沉着症胆囊上皮细胞中胆固醇酯的含量及其作用机理,以及毗格列酮与细胞自身的调控因子肝X受体a能否协同降低胆固醇沉着症胆囊上皮细胞中脂滴含量,进而达到治疗的目的。对于胆囊胆固醇息肉,既要达到切除息肉的目的,又要保留胆囊,以维持其生理功能才是最合理的手术方式。本实验同时建立了腹腔镜内镜双镜联合技术切除息肉保留胆囊的可行性,并进行了保留胆囊后胆囊功能的短期随访。方法：病人来源于东南大学附属中大医院普外科,已通过伦理学验证。手术中取得的胆囊标本,切取粘膜层的一半,行胆囊粘膜上皮细胞原代培养,三天后经毗格列酮(过氧化物酶增殖体活化γ受体激动剂),22(R)-羟基胆固醇(肝X受体α激动剂),或者过氧化物酶增殖体活化受体-γ小RNA的干预后,再使用Western蛋白质印迹法检测细胞中过氧化物酶增殖体活化受体-γ,肝X受体α, ATP-结合盒载体蛋白Al,中性胆固醇酯水解酶蛋白1蛋白含量的变化；并采用荧光定量的方法测量干预过程中细胞内游离胆固醇外流量的变化,最后通过油红O染色的方法直观的观察细胞中脂滴含量的变化。在临床研究方面,选取中大医院普外科的60例胆囊胆固醇息肉患者,术前常规B超检查,采用全身麻醉,腹腔镜下切开胆囊底部,微波热凝胆囊息肉的根部,活检钳取出体外。对所有的病人常规进行随访,术后第1、3、6个月采用B超检测保留胆囊的容积和收缩功能。结果：在胆囊上皮细胞培养中,1 μM的毗格列酮明显的提高了中性胆固醇酯水解酶1和ATP-结合盒载体蛋白A1的表达量。在过氧化物酶增殖体活化受体-γ小RNA的作用下,胆囊上皮细胞内中性胆固醇酯水解酶1和ATP-结合盒载体A1的蛋白含量分别下降到处理前的22.15%和23.62%；但是,10μM22(R)-羟基胆固醇加入到经过氧化物酶增殖体活化受体-γ小RNA干扰后的胆囊上皮细胞后,ATP-结合盒载体Al蛋白含量增加了1.77倍,而中性胆固醇酯水解酶1的蛋白含量没有明显的变化。22(R)-羟基胆固醇可以少量的上调肝X受体α的蛋白表达量,同时增加ATP-结合盒载体Al约56%的蛋白表达量。在用吡格列酮处理过的胆囊上皮细胞,胆固醇外流量随着毗格列酮浓度的增加或作用时间的延长而外流量明显增加。油红O染色也证实1μM的吡格列酮可明显的降低胆囊胆固醇沉着症中脂滴的含量。为了判断细胞自身胆固醇内环境调节因子肝X受体α对吡格列酮的影响,我们采用22(R)-羟基胆固醇联合毗格列酮干预的方式,发现二者共同上调3.64倍ATP-结合盒载体A1蛋白表达量,同时大幅度提高细胞外排游离胆固醇的能力。油红O染色也显示了二者联合应用可以更加明显的降低胆固醇沉着症患者胆囊上皮细胞中脂滴的含量。在临床病例研究中,所有的手术过程是成功的,手术时间60～35分钟。研究过程中发现双镜联合技术更适用于肥胖病人,操作也更方便。所有病人短期随访无任何并发症,无息肉复发；术后3个月,保留胆囊的容积和收缩功能基本上恢复到术前的水平,其后继续随访发现胆囊的功能持续好转,并可以满足生理功能。结论：(1)吡格列酮经“过氧化物酶增殖体活化γ受体-肝X受体α -ATP-结合盒载体Al”通路提高ATP-结合盒载体A1的表达；而增加中性胆固醇酯水解酶1的含量和肝X受体a没有明显的关系。(2)吡格列酮通过增加胆囊上皮细胞中ATP-结合盒载体蛋白A1和中性胆固醇酯水解酶1的表达量,提高了胆固醇酯水解和游离胆固醇外流的能力,进而达到降低胆固醇沉着症胆囊上皮细胞内脂滴含量的目的。(3)细胞自身的调节因子(肝X受体α)和吡格列酮,能够协同作用于“过氧化物酶增殖体活化γ受体-肝X受体α -ATP-结合盒载体Al”通路,更大的增加细胞膜上ATP-结合盒载体Al的蛋白量,促进胆固醇外排,减少细胞中脂滴沉着,进而达到治疗的目的。(4)对于胆囊胆固醇息肉,采用腹腔镜内镜双镜联合技术切除息肉安全、可靠,更加适合于肥胖的病人。
[Abstract]:Objective: cholecystocaosis is caused by excessive accumulation of lipid droplets in the mucous layer and submucosa. Macrophages can become foam cells after phagocytosis of lipid droplets. A large number of foam cells accumulate in the lymphatic vessels, often causing lymphatic destruction, even local emblem circulation, leading to lipid reflux disorder. Pathological, this disease can be divided into two diseases. Subtype: diffuse cholesterosis and cholesterol polyp. Among them, the former is still present in the submucosal lipid reflux channel, and drug intervention can achieve the purpose of treatment; the latter is hindered by the lipid recirculation channel of the submucosa, and only surgical excision of polyps can achieve the purpose of treatment. The main ingredient in the cholesterol drop of gallbladder cholesterol is the bile. Sterol esters, removing cholesterol esters in epithelial cells, are important for maintaining the stability of the intracellular cholesterol and protecting the physiological functions of the gallbladder. Many studies have shown that the peroxidase proliferator activated gamma receptor or the liver X receptor alpha is closely related to the homeostasis of intracellular cholesterol (.22 (R) - hydroxycholesterol as a body. The intermediate metabolites of the steroids can also increase the protein expression of the liver X receptor alpha, which can up regulate the protein expression of the peroxidase proliferator activated gamma receptor. For the diffuse cholesterosis, the aim of this experiment is to determine the activity of pioglitazone (the activator of the peroxisome activation receptor - gamma). Whether the content of cholesteryl ester in the gallbladder epithelial cells and its mechanism of action, and whether the liver X receptor A of vishlone and cell itself can be used to reduce the lipid droplet content in the gallbladder epithelial cells of cholesterosis, and then to achieve the purpose of treatment. For gallbladder cholesterol polyps, it is necessary to achieve the removal of polyps Objective to maintain the gallbladder to maintain its physiological function is the most reasonable mode of operation. This experiment also established the feasibility of the laparoscopic endoscopic double mirror combined technique for the resection of the gallbladder, and the short-term follow-up of the gallbladder function after the retention of the gallbladder. Method: the patients were derived from the Department of general surgery in Zhongda Hospital Affiliated to Southeast University. It was verified by ethics. The gallbladder specimens obtained during the operation were cut in half of the mucosa, primary culture of the epithelial cells of the gallbladder mucosa, three days after the peroxidase (peroxidase proliferator activated gamma receptor agonist), 22 (R) - hydroxycholesterol (liver X receptor alpha agonist), or the stem of the peroxidase proliferator activated receptor - gamma small RNA Prognosis, Western Western blot was used to detect the changes in the content of pod proliferator activated receptor - gamma, liver X receptor alpha, ATP- binding cassette carrier protein Al and neutral cholesteryl ester hydrolase protein 1 protein, and the fluorescence quantitative method was used to measure the changes of intracellular free cholesterol flow in the intervention process. In the clinical study, 60 patients with gallbladder cholesterol polyp were selected in the Department of general surgery of Zhongda Hospital. In the clinical study, 60 cases of gallbladder cholesterol polyps in Department of general surgery were selected, general B ultrasound examination, general anesthesia, laparoscope incision of the gallbladder bottom, microwave coagulation of the root of cholecystis polyps, biopsy forceps removed in vitro. The patients were followed up and the volume and contractile function of the gallbladder were retained after 1,3,6 months after the operation. Results: in the culture of the gallbladder epithelial cells, 1 u M vishlone significantly increased the expression of the neutral cholesteryl ester hydrolase 1 and the ATP- binding cassette carrier protein A1. Under the action, the protein content of the neutral cholesteryl ester hydrolase 1 and ATP- binding cassette carrier A1 in the epithelial cells of the gallbladder decreased to 22.15% and 23.62% before the treatment, but 10 mu M22 (R) hydroxycholesterol was added to the epithelial cells of the gallbladder after the activated receptor gamma small RNA interference by the oxide enzyme proliferator, and the ATP- binding cassette carrier Al protein contained The amount increased by 1.77 times, while the protein content of the neutral cholesteryl ester hydrolase 1 did not change significantly..22 (R) - hydroxycholesterol could increase the protein expression of liver X receptor alpha in a small amount and increase the protein expression of about 56% of the ATP- binding cassette carrier Al. The increase in the concentration of ketone or the prolongation of the action time increased significantly. The oil red O staining also confirmed that 1 M of pioglitazone significantly reduced the lipid droplets in the gallbladder cholesterol deposit. In order to determine the effect of the liver X receptor alpha on the pioglitazone, we used 22 (R) - hydroxy cholesterol. Combined with vishlone intervention, it was found that the two together up up 3.64 times the expression of ATP- binding cassette carrier A1 protein, and greatly improving the ability of extracellular free cholesterol. The oil red O staining also showed that the combination of the two groups could significantly reduce the content of lipid droplets in the gallbladder epithelial cells of the patients with cholesterosis. In the case study, all surgical procedures were successful and the operation time was 60~35 minutes. The study found that the double mirror combined technique was more suitable for obese patients and was more convenient to operate. All patients were followed up without any complications and no polyp recurrence; 3 months after the operation, the volume and contractile function of the retained gallbladder was basically restored before the operation. The function of gallbladder continues to improve and can meet the physiological functions. Conclusion: (1) the expression of pioglitazone through the "peroxidase proliferator activated gamma receptor X receptor alpha -ATP- binding cassette carrier Al" pathway improves the expression of ATP- binding cassette carrier A1, and increases the content of neutral cholesteryl ester hydrolase 1 and the X receptor A of the liver. There is no obvious relationship. (2) pioglitazone improves the ability of cholesteryl ester hydrolysis and free cholesterol Exodus by increasing the expression of ATP- binding cassette carrier protein A1 and neutral cholesteryl ester hydrolase 1 in gallbladder epithelial cells, thereby reducing the lipid droplet content in the upper gallbladder cells of the cholesterosis. (3) cell itself The regulatory factor (liver X receptor alpha) and pioglitazone can cooperate with the "peroxidase proliferator activated gamma receptor - liver X receptor alpha -ATP- binding cassette carrier Al" pathway to increase the protein amount of the ATP- binding cassette carrier Al on the cell membrane, promote the cholesterol efflux, reduce the lipid droplet in the cell, and then achieve the purpose of treatment. (4) (4) Laparoscopic cholecystectomy combined with polypectomy is safe, reliable and suitable for obese patients.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R657.4

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