本文關(guān)鍵詞： 膽囊結(jié)石 Cajal間質(zhì)細胞 膽囊收縮功能 凋亡 微創(chuàng)保膽取石術(shù)　出處：《東南大學》2015年博士論文　論文類型：學位論文

【摘要】：目的：1、膽囊結(jié)石是一種常見的消化系統(tǒng)疾病。膽囊結(jié)石的成因復雜,膽囊運動功能異常是一個關(guān)鍵的因素。以往的研究多集中在膽囊對膽囊收縮素(Cholecystokinin, CCK)敏感性下降、CCK受體功能失調(diào)、膽囊平滑肌功能減退以及肝外膽道系統(tǒng)調(diào)節(jié)功能紊亂等方面。隨著對消化道Cajal間質(zhì)細胞(Interstitial ICC of Cajal,ICC)的深入研究,發(fā)現(xiàn)ICC異常與多種消化道動力紊亂性疾病關(guān)系密切。最新的研究表明ICC存在于動物和人類膽囊,其對膽囊動力的調(diào)控以及與膽囊結(jié)石發(fā)病的關(guān)系成為了研究熱點。我們在構(gòu)建兔膽囊膽固醇結(jié)石模型的基礎(chǔ)上,通過一系列體內(nèi)、外實驗,對膽囊結(jié)石形成過程中膽囊收縮功能與ICC的變化關(guān)系以及相關(guān)原因和機制進行探討,為膽囊結(jié)石的病因研究和預(yù)防提供新的理論依據(jù)。2、膽囊切除被認為是治療膽囊結(jié)石的“金標準”,但是術(shù)后多種并發(fā)癥以及膽囊切除后對人體生理機能的影響,使得一些專家對此進行了反思,提出了“新式的微創(chuàng)保膽取石手術(shù)”。但經(jīng)過多年的臨床實踐,專家們發(fā)現(xiàn)該術(shù)式在適應(yīng)癥及技術(shù)操作標準等方面仍有一些細節(jié)值得改進和規(guī)范。我們根據(jù)ICC在膽囊的分布特點,對保膽取石手術(shù)的方法學進行探索研究,取得了初步結(jié)果。方法：(1)52只新西蘭大白兔隨機分為對照組20只,實驗組32只,分別予以普通飼料和致石飼料喂養(yǎng),6周后行B超檢查成石情況。核素掃描評估膽囊收縮功能；多導生理記錄儀檢測在體膽囊基本電節(jié)律和膽囊肌條對CCK反應(yīng)張力的改變。免疫熒光全層鋪片計數(shù)膽囊ICC變化；電鏡觀察ICC超微結(jié)構(gòu)改變。Western Blot檢測膽囊c-kit蛋白表達。(2)全自動生化儀檢測兩組膽汁膽固醇飽和指數(shù)、膽囊組織氧化應(yīng)激指標,TUNEL染色觀察兩組膽囊ICC凋亡的變化。應(yīng)用膠原酶II消化法分離培養(yǎng)兔膽囊原代ICC,c-kit免疫熒光鑒定,通過梯度離心、顯微機械刮除技術(shù)對其進行純化。用不同濃度的膽固醇(0,25,50,100mg/L)對ICC進行干預(yù),通過細胞膜片鉗、流式細胞儀等檢測ICC起搏電位、氧化應(yīng)激和凋亡的變化。隨后在含100mg/L膽固醇的培養(yǎng)基加入不同濃度的膽固醇清除劑甲基-p-環(huán)糊精(0,2,4,6mM)對ICC進行干預(yù)后,檢測ICC起搏電位、氧化應(yīng)激和凋亡的變化。(3)65名符合保膽取石術(shù)指征并自愿接受手術(shù)的膽囊結(jié)石患者,根據(jù)ICC在膽囊的分布特點設(shè)計不同膽囊切口位置,隨機分為膽囊底部切口組(35名),膽囊體部切口組(30名),對比兩組患者術(shù)后膽囊功能恢復的情況；同時就手術(shù)的適應(yīng)癥、禁忌癥進行補充和完善；并定期隨訪保膽術(shù)后患者的療效及結(jié)石復發(fā)情況。結(jié)果：(1)B超檢測實驗組兔膽囊成石率為74.2%(23/31)。核素掃描結(jié)果顯示,實驗組膽囊收縮功能較對照組明顯下降(P0.05)。實驗組膽囊基本電節(jié)律的頻率和振幅和膽囊肌條對CCK反應(yīng)張力明顯低于對照組(P0.05)。膽囊全層鋪片免疫熒光檢測示實驗組ICC數(shù)目明顯少于對照組(P0.05)。結(jié)石膽囊ICC超微結(jié)構(gòu)發(fā)生線粒體腫脹、自噬小體形成、細胞核內(nèi)陷、染色質(zhì)邊集等病理改變。Westen Blot顯示結(jié)石膽囊組織c-kit蛋白表達低于正常膽囊(P0.05)。(2)成石膽汁膽固醇飽和指數(shù)明顯高于對照組(P0.05)。結(jié)石膽囊組織氧化應(yīng)激指標顯著高于正常膽囊(P0.05)。全層鋪片CD117/TUNEL免疫熒光雙染顯示正常膽囊組織有極少ICC凋亡,而結(jié)石組ICC凋亡明顯增多(P0.05)。體外試驗中,成功對膽囊原代ICC進行了分離、培養(yǎng)和純化,純化率約為85%。隨著培養(yǎng)液中膽固醇濃度的逐漸增加(0,25,50, 100mg/L),ICC的起搏電流的振幅和頻率逐漸減小、氧化應(yīng)激反應(yīng)逐漸增強、ICC凋亡率逐漸升高,呈劑量依耐性變化,各組間比較差異均有顯著性(P0.05)。隨著甲基-p-環(huán)糊精濃度的增加,對細胞結(jié)合膽固醇清除的增多,ICC起搏電流的振幅和頻率逐漸恢復、氧化應(yīng)激反應(yīng)逐漸減弱；ICC凋亡率逐漸降低,呈劑量依耐性變化,各組間比較差異均有顯著性(P0.05)。(3)65名膽囊結(jié)石患者接受保膽取石手術(shù),中轉(zhuǎn)腹腔鏡膽囊切除4例,保膽取石手術(shù)61例,均無手術(shù)并發(fā)癥。術(shù)后隨訪18(6-24)月,結(jié)石復發(fā)3例,復發(fā)率為4.92%。術(shù)前存在的消化道癥狀,術(shù)后基本消失。觀察膽囊不同切口部位對術(shù)后膽囊收縮功能恢復的影響,發(fā)現(xiàn)術(shù)后1月底部切口和體部切口兩組膽囊射膽分數(shù)(Gallbladder EjectionFraction, GEF)較術(shù)前均有減弱,且兩者無明顯差異(P0.05)；術(shù)后3月及6月的GEF檢測顯示,底部切口術(shù)后膽囊收縮功能的恢復明顯優(yōu)于體部切口,兩者差異有顯著性(P0.05)。結(jié)論：(1)飲食誘導兔膽囊膽固醇結(jié)石形成過程中,膽囊ICC明顯減少,膽囊運動功能下降。(2)成石膽汁中膽固醇過飽和誘發(fā)膽囊組織氧化應(yīng)激反應(yīng),導致膽囊ICC凋亡。(3)高膽固醇影響膽囊ICC電生理活動,使ICC起搏電位減弱,妨礙了膽囊平滑肌基本電節(jié)律的產(chǎn)生,介導了膽囊收縮功能減弱。(4)保膽取石術(shù)中選擇膽囊底部切口,更有利于術(shù)后膽囊收縮功能的恢復。(5)保膽取石手術(shù)有嚴格的適應(yīng)癥和禁忌癥。其遠期療效尚需通過前瞻性、大樣本、多中心的隨機對照研究進行臨床評估。
[Abstract]:Objective: 1, gallstone is a common disease of digestive system. The causes of gallstone complicated gallbladder motor dysfunction is one of the key factors. Previous studies have focused on the gallbladder of cholecystokinin (Cholecystokinin, CCK) decreased CCK receptor dysfunction, gallbladder smooth muscle dysfunction and extrahepatic biliary system regulation disorders. With the digestive tract interstitial cells of Cajal (Interstitial ICC of Cajal, ICC) of the in-depth study, found that abnormal ICC and a variety of gastrointestinal motility disorders related closely. The latest research shows that ICC exists in animal and human gallbladder, regulating the gallbladder motility and relationship with the pathogenesis of gallstone. The US has become a research hotspot. In the foundation of rabbit cholesterol gallstone model, through a series of experiments, the formation of a body, in the process of the contraction of gallbladder gallstone Discusses the relationship between the function and the change of ICC and related causes and mechanism,.2 provides a new theoretical basis for the study on etiology and prevention of gallstone, gallbladder resection is considered the treatment of gallbladder stones "gold standard", but the postoperative complications after cholecystectomy and to the human body physiological effects, makes some experts a reflection, put forward "new cholecystolithotomy operation". But after years of clinical practice, the experts found that the operation there are still some details to improve and regulate the indications and technical standards. According to the distribution of ICC in gallbladder stone surgery, method for preserving the common bile exploration and research, has achieved initial results. Methods: (1) 52 New Zealand white rabbits were randomly divided into control group 20, experimental group 32, were given ordinary feed and lithogenic diet. After 6 weeks B Super check stone. Radionuclide scan evaluation function of gallbladder; polygraph testing in gallbladder basic electrical rhythm and the change of gallbladder muscle strips in response to CCK tension. Immunofluorescence wholemount preparation count gallbladder ICC changes; electron microscopy ultrastructural changes of ICC.Western Blot detected the expression of c-kit protein in gallbladder (2). Automatic biochemical detection of two groups of bile cholesterol saturation index, the index of oxidative stress in tissues of gallbladder, gallbladder of two groups were observed by TUNEL staining. The apoptosis of ICC cultured rabbit gallbladder primary ICC using collagenase II digestion method, c-kit immunofluorescence identification, by gradient centrifugation, micro mechanical scraping technology to carry on the purification of different. The concentration of cholesterol (0,25,50100mg/L) intervention on ICC, through cell patch clamp, flow cytometry detection of ICC pacemaker potential changes, oxidative stress and apoptosis. Then in 100mg/L containing cholesterol The cultivation of cholesterol scavenger methyl -p- cyclodextrin radicals with different concentrations of ICC (0,2,4,6mM) intervention, detection of ICC pacemaker potential changes, oxidative stress and apoptosis. (3) 65 with lrcl refers to patients with gallstone syndrome and voluntarily accept surgery, according to the ICC design of different incision position in gallbladder the distribution characteristics of gallbladder, were randomly divided into the gallbladder incision group (35 patients), the gallbladder body incision group (30), gallbladder function recovery were compared between two groups after operation; at the same time, operation indications, contraindications to supplement and perfect; and regular follow-up surgery curative effect and preservation of gallbladder stone recurrence after the patients. Results: (1) the stone rate of ultrasound in experimental group rabbit gallbladder was 74.2% (23/31). Radionuclide scanning showed that the experimental group gallbladder contraction function decreased significantly compared with the control group (P0.05). The experimental group of gallbladder basic electrical rhythm, frequency and amplitude and bile Sac muscle strips in response to CCK tension was significantly lower than the control group (P0.05). The gallbladder whole mount immunofluorescence assay shows the number of ICC in the experimental group was significantly less than the control group (P0.05). The ultrastructural changes of gallbladder ICC mitochondrial swelling, autophagosome formation, nucleus invagination, chromatin margination and other pathological changes of.Westen Blot c-kit protein expression was lower than that of normal gallbladder gallbladder stones (P0.05). (2) the lithogenic bile cholesterol saturation index was significantly higher than the control group (P0.05). The indexes of oxidative stress in gallbladder tissue was significantly higher than that of normal gallbladder stones (P0.05). All layers of CD117/TUNEL double immunofluorescence staining showed normal gallbladder tissue have rarely ICC apoptosis, and stone group. ICC apoptosis was significantly increased (P0.05). In vitro, the success of the gallbladder primary ICC were isolated, cultured and purified, the purification rate is about 85%. with the cholesterol concentration in the culture medium gradually increased (0,25,50, 100mg/L ), amplitude and frequency of the pacemaker current ICC decreased gradually, oxidative stress gradually increased, the apoptosis rate of ICC gradually increased, in a dose dependent change, the difference was significant (P0.05). With the increase of -p- methyl cyclodextrin concentration, the cells with cholesterol removal increased, amplitude and frequency ICC pacemaker current gradually restored, oxidative stress gradually decreased; the apoptosis rate of ICC decreased in a dose dependent change, the difference was significant (P0.05). (3) of 65 patients with gallstone receiving Paul gall stone surgery, converted to laparoscopic cholecystectomy in 4 cases, gallbladder stone surgery in 61 cases, there were no operative complications. Postoperative follow-up of 18 months (6-24), 3 cases of recurrence, the recurrence rate was 4.92%. preoperative gastrointestinal symptoms disappeared after operation. To observe the different effect of gallbladder incision reduction on function recovery of postoperative gallbladder operation After the end of January two incision and body incision gallbladder bile fraction (Gallbladder EjectionFraction, at GEF) than before were weakened, and had no significant difference (P0.05); March and June GEF detection after operation, the contraction function of gallbladder bottom incision postoperative recovery was significantly better than the body cut, there was the difference between them (P0.05). Conclusion: (1) the process of the formation of cholesterol gallstone in rabbits induced by diet, gallbladder ICC significantly reduced gallbladder motor function decline. (2) lithogenic bile cholesterol saturation response to oxidative stress induced by gallbladder tissue, resulting in gallbladder ICC apoptosis. (3) high cholesterol gallbladder ICC electrophysiological effect the activities of ICC pacing potential reduced hinders gallbladder smooth muscle of basic electric rhythm, mediates the gallbladder contraction. (4) choose the gallbladder incision lrcl, more conducive to the gallbladder contraction function of postoperative recovery. (5) gallbladder The operation of stone removal has strict indications and contraindications. The long-term effect of the surgery needs to be evaluated through a prospective, large sample, multicenter randomized controlled study.

【學位授予單位】：東南大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R657.42

【參考文獻】

相關(guān)期刊論文 前10條

1 姜皓;施維錦;;保膽取石術(shù)的現(xiàn)狀分析[J];中國實用外科雜志;2014年S1期

2 Yuan Cheng;Ze-Sheng Jiang;Xiao-Ping Xu;Zhi Zhang;Ting-Cheng Xu;Chen-Jie Zhou;Jia-Sheng Qin;Guo-lin He;Yi Gao;Ming-Xin Pan;;Laparoendoscopic single-site cholecystectomy vs three-port laparoscopic cholecystectomy: A large-scale retrospective study[J];World Journal of Gastroenterology;2013年26期

3 Jie Chen;Lei Du;Yong-Tao Xiao;Wei Cai;;Disruption of interstitial cells of Cajal networks after massive small bowel resection[J];World Journal of Gastroenterology;2013年22期

4 Vasiliy Ivanovich Reshetnyak;;Concept of the pathogenesis and treatment of cholelithiasis[J];World Journal of Hepatology;2012年02期

5 劉京山;榮萬水;鄧勇;劉衍民;喬鐵;周望先;胡乃海;李志東;賀憲;胡海;孫文生;楊玉龍;張陽德;張寶善;;膽石癥術(shù)后不良反應(yīng)多中心聯(lián)合調(diào)查分析[J];中國內(nèi)鏡雜志;2011年10期

6 李斌輝;董軍亞;;膽囊壁內(nèi)結(jié)石研究進展[J];武警醫(yī)學院學報;2011年09期

7 徐龍;余保平;陳靈丹;呂農(nóng)華;朱萱;陳幼祥;;Cajal細胞調(diào)節(jié)豚鼠膽囊動力的實驗研究[J];第二軍醫(yī)大學學報;2010年07期

8 Randa M Mostafa;Yasser M Moustafa;Hosam Hamdy;;Interstitial cells of Cajal, the Maestro in health and disease[J];World Journal of Gastroenterology;2010年26期

9 王惠群;傅賢波;;我國內(nèi)鏡微創(chuàng)保膽取石術(shù)的發(fā)展現(xiàn)狀分析[J];中國微創(chuàng)外科雜志;2010年06期

10 喬鐵;張陽德;張寶善;馮禹陽;王興強;王小鋒;黃萬潮;黃海儀;馬瑞紅;;硬質(zhì)超聲膽囊內(nèi)鏡系統(tǒng)在保膽取石手術(shù)中的應(yīng)用價值[J];中國內(nèi)鏡雜志;2010年02期

相關(guān)博士學位論文 前4條

1 劉W，

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