發(fā)布時(shí)間：2018-04-29 15:43

  本文選題：膽囊膽固醇沉著癥 + 過(guò)氧化物酶增殖體活化受體-γ��； 參考：《東南大學(xué)》2015年博士論文

【摘要】：目的：膽囊膽固醇沉著癥是因粘膜層及粘膜下層中過(guò)量的脂滴積聚所致。巨噬細(xì)胞吞噬脂滴后可以變成泡沫細(xì)胞,大量的泡沫細(xì)胞堆積于淋巴管,常導(dǎo)致淋巴管破壞,甚至影響局部的徽循環(huán),導(dǎo)致脂質(zhì)回流障礙。病理上,本病可分為兩個(gè)亞型：彌漫性膽固醇沉著癥和膽固醇息肉。其中,前者的粘膜下層脂質(zhì)回流通道尚存在,藥物干預(yù)能達(dá)到治療的目的；后者的粘膜下層脂質(zhì)回流通道受阻,只有手術(shù)切除息肉才能達(dá)到治療目的。膽囊膽固醇沉著癥脂滴中的主要成分是膽固醇酯,除去上皮細(xì)胞中的膽固醇酯對(duì)于維持細(xì)胞中膽固醇內(nèi)環(huán)境的穩(wěn)定和保護(hù)膽囊的生理功能具有重要意義。很多研究顯示過(guò)氧化物酶增殖體活化γ受體或者肝X受體α與細(xì)胞內(nèi)膽固醇內(nèi)環(huán)境的穩(wěn)態(tài)密切相關(guān)。22(R)-羥基膽固醇既是體內(nèi)固醇類物質(zhì)的中間代謝物,又可以上調(diào)肝X受體α的蛋白表達(dá)量；吡格列酮可以上調(diào)過(guò)氧化物酶增殖體活化γ受體的蛋白表達(dá)量。對(duì)于彌漫性膽固醇沉著癥,本實(shí)驗(yàn)的目的就是判定吡格列酮(過(guò)氧化物酶增殖體活化受體-γ的激動(dòng)劑)可否降低膽固醇沉著癥膽囊上皮細(xì)胞中膽固醇酯的含量及其作用機(jī)理,以及毗格列酮與細(xì)胞自身的調(diào)控因子肝X受體a能否協(xié)同降低膽固醇沉著癥膽囊上皮細(xì)胞中脂滴含量,進(jìn)而達(dá)到治療的目的。對(duì)于膽囊膽固醇息肉,既要達(dá)到切除息肉的目的,又要保留膽囊,以維持其生理功能才是最合理的手術(shù)方式。本實(shí)驗(yàn)同時(shí)建立了腹腔鏡內(nèi)鏡雙鏡聯(lián)合技術(shù)切除息肉保留膽囊的可行性,并進(jìn)行了保留膽囊后膽囊功能的短期隨訪。方法：病人來(lái)源于東南大學(xué)附屬中大醫(yī)院普外科,已通過(guò)倫理學(xué)驗(yàn)證。手術(shù)中取得的膽囊標(biāo)本,切取粘膜層的一半,行膽囊粘膜上皮細(xì)胞原代培養(yǎng),三天后經(jīng)毗格列酮(過(guò)氧化物酶增殖體活化γ受體激動(dòng)劑),22(R)-羥基膽固醇(肝X受體α激動(dòng)劑),或者過(guò)氧化物酶增殖體活化受體-γ小RNA的干預(yù)后,再使用Western蛋白質(zhì)印跡法檢測(cè)細(xì)胞中過(guò)氧化物酶增殖體活化受體-γ,肝X受體α, ATP-結(jié)合盒載體蛋白Al,中性膽固醇酯水解酶蛋白1蛋白含量的變化；并采用熒光定量的方法測(cè)量干預(yù)過(guò)程中細(xì)胞內(nèi)游離膽固醇外流量的變化,最后通過(guò)油紅O染色的方法直觀的觀察細(xì)胞中脂滴含量的變化。在臨床研究方面,選取中大醫(yī)院普外科的60例膽囊膽固醇息肉患者,術(shù)前常規(guī)B超檢查,采用全身麻醉,腹腔鏡下切開(kāi)膽囊底部,微波熱凝膽囊息肉的根部,活檢鉗取出體外。對(duì)所有的病人常規(guī)進(jìn)行隨訪,術(shù)后第1、3、6個(gè)月采用B超檢測(cè)保留膽囊的容積和收縮功能。結(jié)果：在膽囊上皮細(xì)胞培養(yǎng)中,1 μM的毗格列酮明顯的提高了中性膽固醇酯水解酶1和ATP-結(jié)合盒載體蛋白A1的表達(dá)量。在過(guò)氧化物酶增殖體活化受體-γ小RNA的作用下,膽囊上皮細(xì)胞內(nèi)中性膽固醇酯水解酶1和ATP-結(jié)合盒載體A1的蛋白含量分別下降到處理前的22.15%和23.62%；但是,10μM22(R)-羥基膽固醇加入到經(jīng)過(guò)氧化物酶增殖體活化受體-γ小RNA干擾后的膽囊上皮細(xì)胞后,ATP-結(jié)合盒載體Al蛋白含量增加了1.77倍,而中性膽固醇酯水解酶1的蛋白含量沒(méi)有明顯的變化。22(R)-羥基膽固醇可以少量的上調(diào)肝X受體α的蛋白表達(dá)量,同時(shí)增加ATP-結(jié)合盒載體Al約56%的蛋白表達(dá)量。在用吡格列酮處理過(guò)的膽囊上皮細(xì)胞,膽固醇外流量隨著毗格列酮濃度的增加或作用時(shí)間的延長(zhǎng)而外流量明顯增加。油紅O染色也證實(shí)1μM的吡格列酮可明顯的降低膽囊膽固醇沉著癥中脂滴的含量。為了判斷細(xì)胞自身膽固醇內(nèi)環(huán)境調(diào)節(jié)因子肝X受體α對(duì)吡格列酮的影響,我們采用22(R)-羥基膽固醇聯(lián)合毗格列酮干預(yù)的方式,發(fā)現(xiàn)二者共同上調(diào)3.64倍ATP-結(jié)合盒載體A1蛋白表達(dá)量,同時(shí)大幅度提高細(xì)胞外排游離膽固醇的能力。油紅O染色也顯示了二者聯(lián)合應(yīng)用可以更加明顯的降低膽固醇沉著癥患者膽囊上皮細(xì)胞中脂滴的含量。在臨床病例研究中,所有的手術(shù)過(guò)程是成功的,手術(shù)時(shí)間60～35分鐘。研究過(guò)程中發(fā)現(xiàn)雙鏡聯(lián)合技術(shù)更適用于肥胖病人,操作也更方便。所有病人短期隨訪無(wú)任何并發(fā)癥,無(wú)息肉復(fù)發(fā)；術(shù)后3個(gè)月,保留膽囊的容積和收縮功能基本上恢復(fù)到術(shù)前的水平,其后繼續(xù)隨訪發(fā)現(xiàn)膽囊的功能持續(xù)好轉(zhuǎn),并可以滿足生理功能。結(jié)論：(1)吡格列酮經(jīng)“過(guò)氧化物酶增殖體活化γ受體-肝X受體α -ATP-結(jié)合盒載體Al”通路提高ATP-結(jié)合盒載體A1的表達(dá)；而增加中性膽固醇酯水解酶1的含量和肝X受體a沒(méi)有明顯的關(guān)系。(2)吡格列酮通過(guò)增加膽囊上皮細(xì)胞中ATP-結(jié)合盒載體蛋白A1和中性膽固醇酯水解酶1的表達(dá)量,提高了膽固醇酯水解和游離膽固醇外流的能力,進(jìn)而達(dá)到降低膽固醇沉著癥膽囊上皮細(xì)胞內(nèi)脂滴含量的目的。(3)細(xì)胞自身的調(diào)節(jié)因子(肝X受體α)和吡格列酮,能夠協(xié)同作用于“過(guò)氧化物酶增殖體活化γ受體-肝X受體α -ATP-結(jié)合盒載體Al”通路,更大的增加細(xì)胞膜上ATP-結(jié)合盒載體Al的蛋白量,促進(jìn)膽固醇外排,減少細(xì)胞中脂滴沉著,進(jìn)而達(dá)到治療的目的。(4)對(duì)于膽囊膽固醇息肉,采用腹腔鏡內(nèi)鏡雙鏡聯(lián)合技術(shù)切除息肉安全、可靠,更加適合于肥胖的病人。
[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.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R657.4

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