Cajal间质细胞在胆囊结石形成中的作用以及保胆取石术方法学研究

发布时间：2018-01-22 17:02

本文关键词： 胆囊结石 Cajal间质细胞胆囊收缩功能凋亡微创保胆取石术　出处：《东南大学》2015年博士论文　论文类型：学位论文

【摘要】：目的：1、胆囊结石是一种常见的消化系统疾病。胆囊结石的成因复杂,胆囊运动功能异常是一个关键的因素。以往的研究多集中在胆囊对胆囊收缩素(Cholecystokinin, CCK)敏感性下降、CCK受体功能失调、胆囊平滑肌功能减退以及肝外胆道系统调节功能紊乱等方面。随着对消化道Cajal间质细胞(Interstitial ICC of Cajal,ICC)的深入研究,发现ICC异常与多种消化道动力紊乱性疾病关系密切。最新的研究表明ICC存在于动物和人类胆囊,其对胆囊动力的调控以及与胆囊结石发病的关系成为了研究热点。我们在构建兔胆囊胆固醇结石模型的基础上,通过一系列体内、外实验,对胆囊结石形成过程中胆囊收缩功能与ICC的变化关系以及相关原因和机制进行探讨,为胆囊结石的病因研究和预防提供新的理论依据。2、胆囊切除被认为是治疗胆囊结石的“金标准”,但是术后多种并发症以及胆囊切除后对人体生理机能的影响,使得一些专家对此进行了反思,提出了“新式的微创保胆取石手术”。但经过多年的临床实践,专家们发现该术式在适应症及技术操作标准等方面仍有一些细节值得改进和规范。我们根据ICC在胆囊的分布特点,对保胆取石手术的方法学进行探索研究,取得了初步结果。方法：(1)52只新西兰大白兔随机分为对照组20只,实验组32只,分别予以普通饲料和致石饲料喂养,6周后行B超检查成石情况。核素扫描评估胆囊收缩功能；多导生理记录仪检测在体胆囊基本电节律和胆囊肌条对CCK反应张力的改变。免疫荧光全层铺片计数胆囊ICC变化；电镜观察ICC超微结构改变。Western Blot检测胆囊c-kit蛋白表达。(2)全自动生化仪检测两组胆汁胆固醇饱和指数、胆囊组织氧化应激指标,TUNEL染色观察两组胆囊ICC凋亡的变化。应用胶原酶II消化法分离培养兔胆囊原代ICC,c-kit免疫荧光鉴定,通过梯度离心、显微机械刮除技术对其进行纯化。用不同浓度的胆固醇(0,25,50,100mg/L)对ICC进行干预,通过细胞膜片钳、流式细胞仪等检测ICC起搏电位、氧化应激和凋亡的变化。随后在含100mg/L胆固醇的培养基加入不同浓度的胆固醇清除剂甲基-p-环糊精(0,2,4,6mM)对ICC进行干预后,检测ICC起搏电位、氧化应激和凋亡的变化。(3)65名符合保胆取石术指征并自愿接受手术的胆囊结石患者,根据ICC在胆囊的分布特点设计不同胆囊切口位置,随机分为胆囊底部切口组(35名),胆囊体部切口组(30名),对比两组患者术后胆囊功能恢复的情况；同时就手术的适应症、禁忌症进行补充和完善；并定期随访保胆术后患者的疗效及结石复发情况。结果：(1)B超检测实验组兔胆囊成石率为74.2%(23/31)。核素扫描结果显示,实验组胆囊收缩功能较对照组明显下降(P0.05)。实验组胆囊基本电节律的频率和振幅和胆囊肌条对CCK反应张力明显低于对照组(P0.05)。胆囊全层铺片免疫荧光检测示实验组ICC数目明显少于对照组(P0.05)。结石胆囊ICC超微结构发生线粒体肿胀、自噬小体形成、细胞核内陷、染色质边集等病理改变。Westen Blot显示结石胆囊组织c-kit蛋白表达低于正常胆囊(P0.05)。(2)成石胆汁胆固醇饱和指数明显高于对照组(P0.05)。结石胆囊组织氧化应激指标显著高于正常胆囊(P0.05)。全层铺片CD117/TUNEL免疫荧光双染显示正常胆囊组织有极少ICC凋亡,而结石组ICC凋亡明显增多(P0.05)。体外试验中,成功对胆囊原代ICC进行了分离、培养和纯化,纯化率约为85%。随着培养液中胆固醇浓度的逐渐增加(0,25,50, 100mg/L),ICC的起搏电流的振幅和频率逐渐减小、氧化应激反应逐渐增强、ICC凋亡率逐渐升高,呈剂量依耐性变化,各组间比较差异均有显著性(P0.05)。随着甲基-p-环糊精浓度的增加,对细胞结合胆固醇清除的增多,ICC起搏电流的振幅和频率逐渐恢复、氧化应激反应逐渐减弱；ICC凋亡率逐渐降低,呈剂量依耐性变化,各组间比较差异均有显著性(P0.05)。(3)65名胆囊结石患者接受保胆取石手术,中转腹腔镜胆囊切除4例,保胆取石手术61例,均无手术并发症。术后随访18(6-24)月,结石复发3例,复发率为4.92%。术前存在的消化道症状,术后基本消失。观察胆囊不同切口部位对术后胆囊收缩功能恢复的影响,发现术后1月底部切口和体部切口两组胆囊射胆分数(Gallbladder EjectionFraction, GEF)较术前均有减弱,且两者无明显差异(P0.05)；术后3月及6月的GEF检测显示,底部切口术后胆囊收缩功能的恢复明显优于体部切口,两者差异有显著性(P0.05)。结论：(1)饮食诱导兔胆囊胆固醇结石形成过程中,胆囊ICC明显减少,胆囊运动功能下降。(2)成石胆汁中胆固醇过饱和诱发胆囊组织氧化应激反应,导致胆囊ICC凋亡。(3)高胆固醇影响胆囊ICC电生理活动,使ICC起搏电位减弱,妨碍了胆囊平滑肌基本电节律的产生,介导了胆囊收缩功能减弱。(4)保胆取石术中选择胆囊底部切口,更有利于术后胆囊收缩功能的恢复。(5)保胆取石手术有严格的适应症和禁忌症。其远期疗效尚需通过前瞻性、大样本、多中心的随机对照研究进行临床评估。
[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.

【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】：R657.42

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