胆囊Cajal间质细胞损害在胆囊胆固醇结石形成中的作用及其机制的研究

发布时间：2018-06-06 04:36

本文选题：胆囊结石 + 胆囊动力　；参考：《南京医科大学》2014年博士论文

【摘要】：[研究背景和目的]胆囊结石是消化系统常见病,其发病率逐年上升。胆囊结石分为胆固醇结石(cholesterol gallstone, CG)及胆色素结石,其中70%为CG。胆囊动力障碍、胆汁淤积是胆囊结石特别是CG的重要成因,也是保胆治疗术后结石复发的重要原因。Cajal间质细胞(interstitial cells of Cajal, ICC)是胃肠起搏细胞,其数量结构或功能异常与许多胃肠疾病密切相关；ICC的特异标志为酪氨酸激酶受体c-Kit,被广泛用于鉴定ICC及其功能研究。近年来,随着人胆囊及胆道ICC的发现,ICC在胆囊及胆道中所起的作用倍受关注。2013年,有研究报道,胆囊结石患者胆囊壁ICC显著减少,提示ICC损害可能参与了胆囊结石的发生过程。但胆囊壁ICC损害是否介导了胆囊动力障碍而参与胆囊结石形成?其机制鲜有报道。胆囊收缩素(cholecystokinin, CCK)是胆囊收缩的主要激素,血清CCK浓度降低、胆囊CCK1受体(Type 1 cholecystokinin receptor, CCK1)表达减少与胆囊动力障碍、结石形成密切相关。近年研究表明,ICC可能是CCK的重要靶细胞：有研究报道,大鼠胃幽门ICC及豚鼠的胆囊ICC均表达CCK1。因此本研究通过：①临床胆囊标本：探讨胆囊ICC损害与胆囊动力障碍有无相关性,同时研究ICC减少是否可引起CCK1减少；②动物模型：探讨胆囊ICC损害在胆囊动力障碍、结石形成中的作用,及长期高胆固醇饮食对胆囊ICC的影响；③细胞实验：高浓度胆固醇对ICC生长的作用,以期阐明CG患者胆囊ICC损害的机制。[方法]1、临床标本：CG患者胆囊ICC损害与胆囊动力障碍的相关性实验组：CG胆囊切除患者,对照组：早期胰头癌或肝右叶肝癌未累积胆囊而行胆囊切除患者。胆囊切除后,取部分胆囊体部组织及胆汁,进行以下实验：(1)免疫荧光双标,鉴定人胆囊ICC上是否存在CCK1；(2)制备离体胆囊平滑肌肌条、记录胆囊肌条的收缩功能；(3)免疫组化标记c-Kit+细胞,免疫荧光双标技术标记ICC,计算c-Kit+细胞及ICC密度；将c-Kit+细胞密度、ICC密度分别与胆囊肌条收缩功能进行相关性分析；(4)免疫荧光双标,标记CCK1及c-Kit,观察CCK1表达与c-Kit表达间的关系；(5)HE染色,测量胆囊粘膜层+肌层厚度；Western检测c-Kit蛋白表达；(6)检测胆汁胆固醇浓度。2、动物模型：胆囊ICC损害在胆囊动力障碍、CG形成中的作用(1)按照文献,建立CG豚鼠模型：4周龄豚鼠40只,随机分配至“普通饲料组(Regular diet, RD)"及“致石饲料组(lithogenic diet, LD)";(2)B超监测成石情况,喂养6周处死豚鼠,观察胆囊大体标本情况；(3)检测豚鼠胆囊肌条的收缩功能;(4) real-time PCR检测c-Kit、CCK1 mRNA表达；对胆囊肌条收缩功能及c-Kit mRNA表达水平进行相关性分析。3、细胞实验： CG胆囊ICC损害的机制——高浓度胆固醇对ICC生长的影响(1)酶解法分离小鼠胆囊细胞,标记以下抗体：PE-Cy7(PC7)标记的抗c-Kit、PC5标记的抗CD45、PC5标记的抗F4/80、PC5标记的抗CD11b、PE标记的抗CD34抗体,流式细胞仪分选出c-Kit+CD45-F4/80-CD11b-CD34-的细胞(即具有成熟ICC标志的细胞)；将分选出的细胞接种至鼠尾胶原包被的培养皿中、含高浓度血清(15%胎牛血清)的M199培养基进行培养；(2) Real-time PCR鉴定分选效率、免疫荧光鉴定ICC；(3)细胞分组：对照组、实验组(给予不同浓度的水溶性胆固醇(water-soluble cholesterol,WSC));培养72h,real-time PCR检测c-Kit mRNA水平。[结果]1、临床标本研究：CG胆囊ICC损害与胆囊动力障碍的相关性(1)对照组(n=10例),CG组(n=30例)。两组患者平均年龄与男女性别构成比均无统计学差异(2)人胆囊ICC上存在CCK1表达(3)CG组离体胆囊肌条收缩能力较对照组明显减弱(4)CG组胆囊壁c-Kit+细胞密度及ICC密度均较对照组显著降低；c-Kit+密度/ICC密度与胆囊肌条收缩能力呈正相关(5)CCK1表达与c-Kit表达变化一致(6)CG组胆囊壁厚度较对照组增厚,胆囊c-Kit蛋白表达较对照组降低(7)CG组胆汁胆固醇浓度较对照组升高2.动物模型研究：胆囊ICC损害在胆囊动力障碍、CG形成中的作用(1)RD组豚鼠均未形成胆囊结石,LD组豚鼠成石率为60%(2)LD组(形成结石及未形成结石的)其离体胆囊肌条收缩能力均较RD组减弱(3)LD组豚鼠胆囊c-Kit、CCK1 mRNA表达较RD组明显降低(4)胆囊c-Kit mRNA表达水平与胆囊肌条收缩能力呈正相关3、细胞实验：CG胆囊壁ICC损害机制——高浓度胆固醇对ICC生长的影响(1)流式细胞术分选ICC：具有成熟ICC标志的细胞占全部酶解法分离出的小鼠胆囊细胞的10.32±2.21%(2)ICC的培养与鉴定：培养12小时后,可见细胞贴壁；c-Kit及ANO1 (ICC另一特异标志)免疫荧光染色显示：细胞c-Kit、ANO1表达均为阳性,c-Kit标记时细胞胞质着色,ANO1标记时胞质及胞核均着色。收集分选前及分选后的ICC进行real-time PCR检测分选效率,结果提示：分选后的c-Kit mRNA与GAPDH mRNA的比率较分选前提高102±3.18倍(3)外源性WSC干预：加入10mM、20mmM、40mM的WSC作用72h后,与对照组相比,ICC数量显著减少,c-Kit mRNA表达水平显著降低(P0.05)[结论]1、临床标本(CG患者及对照组胆囊、胆汁标本)的研究提示：①人胆囊ICC存在CCK1表达,CCK通过作用于ICC表面的CCK1使胆囊收缩；②胆囊壁ICC损害可致CCK1表达减少,CCK对胆囊收缩作用减弱,进而引起胆囊动力障碍；③CG患者胆汁中胆固醇浓度较对照组增高。2.动物实验(胆囊胆固醇结石豚鼠模型)的研究提示：长期高胆固醇摄入可致胆囊ICC减少、胆囊动力障碍、进而促进胆固醇结石的形成；在结石形成前,即发生胆囊动力减弱。3.细胞研究提示：高浓度胆固醇可显著抑制ICC的生长,CG患者胆囊的ICC损害可能由于长期的高浓度胆固醇胆汁所致。一、研究背景和目的ICC是胃肠起搏细胞,产生慢波并传导电兴奋,ICC起搏功能的产生与胞内Ca2+振荡有关。CCK是一种双重分布在胃肠道及中枢神经系统中的脑肠肽,通过其受体对胃肠运动、胆囊收缩和胰酶分泌等起重要调节作用。既往研究发现大鼠胃幽门ICC上存在CCK1,提示CCK可能直接作用于ICC。本研究首先通过小鼠胃窦组织全层铺片及切片的c-Kit与CCK1免疫荧光共同标记,来证明小鼠胃窦ICC存在CCK1；其次通过分离并培养小鼠胃窦ICC,观察CCK对ICC胞内钙振荡的影响,并探讨具体的分子机制。二、方法1、对小鼠胃窦组织全层铺片、切片及体外培养ICC进行c-Kit与CCK1免疫荧光双标2、CCK对ICC胞内钙离子振荡影响的检测：(1)酶法分离、流式分选、培养并鉴定小鼠胃窦ICC;(2) Fluo-3/AM负载ICC,激光共聚焦显微镜下观察CCK、CCK1受体拮抗剂氯戊米特、内质网钙泵拮抗剂毒胡萝卜素、1,4,5-三磷酸肌醇受体(1,4,5-triphosphate, InsP3R)拮抗剂Xestospongin C及L-型Ca2+通道阻滞剂硝苯地平等对ICC胞内钙振荡的影响。三、结果1、小鼠胃窦组织、体外培养ICC存在c-Kit与CCK1共定位,免疫荧光鉴定培养的细胞为ICC;2、CCK对ICC胞内钙离子振荡影响的检测：CCK可显著升高ICC胞内钙离子浓度(intracellular calcium concentration,[Ca2+]i),该效应可分别被氯戊米特、毒胡萝卜素、Xestospongin C所拮抗,给予硝苯地平也可稍减弱CCK的作用。四、结论CCK通过作用于ICC表面上CCK1受体促进其内质网上InsP3R介导的内钙释放,导致ICC胞内[Ca2+]i显著升高。
[Abstract]:Cholecystolithiasis is a common disease of digestive system. The incidence of cholecystolithiasis is rising year by year. Gallstone is divided into cholesterol gallstone (CG) and cholelithiasis. 70% of them are CG. gallbladder motility disorder. Cholestasis is the important cause of gallstone, especially CG, and it is also important for the recurrence of gallstones. .Cajal interstitial cells of Cajal (ICC) is a gastrointestinal pacing cell whose quantitative structure or dysfunction is closely related to many gastrointestinal diseases. The specific marker of ICC is a tyrosine kinase receptor c-Kit, which is widely used to identify ICC and its functional study. In recent years, with the discovery of human gallbladder and biliary ICC, ICC is in the gallbladder and The role of the biliary tract has attracted much attention for.2013 years. It has been reported that the gallbladder wall of the patients with gallbladder stones decreased significantly, suggesting that ICC damage may be involved in the process of gallstone. But whether the ICC damage of the gallbladder wall is mediated by gallbladder motility disorder and the formation of gallbladder stones? The mechanism is rarely reported. Cholecystokinin (cholecystokinin, CCK) is the main hormone of gallbladder contraction and the decrease of serum CCK concentration. The decrease of CCK1 receptor (Type 1 cholecystokinin receptor, CCK1) is closely related to gallbladder motility disorder and stone formation. In recent years, the study shows that ICC may be an important target cell of CCK: there are research reports, rat gastric pylorus ICC and guinea pig gallbladder ICC all express CCK1. This study was carried out: (1) clinical gallbladder specimens: To investigate whether there is a correlation between gallbladder ICC damage and gallbladder motility disorder, and whether the reduction of ICC can cause CCK1 reduction or not; 2. Animal models: To explore the effect of gallbladder ICC damage on gallbladder motility, the formation of gallstones, and the effect of long-term high cholesterol diet on gallbladder ICC; Experiment: the effect of high concentration of cholesterol on ICC growth in order to clarify the mechanism of ICC damage to the gallbladder in CG patients. [methods]1, clinical specimens: the experimental group on the correlation between ICC damage and gallbladder motility disorders in CG patients: CG cholecystectomy patients, control group: early pancreatic head cancer or liver right lobe liver cancer without gallbladder and cholecystectomy patients. Gallbladder. After excision, taking part of the body tissue and bile of the gallbladder, the following experiments were carried out: (1) whether there was CCK1 on the gall bladder ICC by double immunofluorescence; (2) the contractile function of the isolated gallbladder smooth muscle muscle was prepared and the contractile function of the gallbladder muscle strips was recorded; (3) the immunohistochemical labeling c-Kit+ fine cell, the immunofluorescence double labeling technique marked ICC, and the calculation of c-Kit+ cell and ICC density Degree; correlation analysis of c-Kit+ cell density, ICC density and contractile function of gallbladder muscle strips; (4) double immunofluorescence labeling, labeling CCK1 and c-Kit, and observing the relationship between CCK1 expression and c-Kit expression; (5) HE staining, measuring the thickness of gallbladder mucosa and muscularis layer; Western detected the expression of c-Kit protein; (6) detection of cholesterol concentration.2, movement of bile. Object model: the function of gallbladder ICC damage in the gallbladder dyskinesia and the formation of CG (1) the CG guinea pig model was established according to the literature: 40 guinea pigs of 4 weeks old were randomly assigned to "Regular diet, RD" and "lithogenic diet, LD"); (2) B ultrasonic monitoring of stone formation and feeding the guinea pigs for 6 weeks to observe the gross specimen of the gallbladder (3) detect the contractile function of the gallbladder muscle strips in guinea pigs; (4) real-time PCR detection of c-Kit, CCK1 mRNA expression, the correlation analysis of the contractile function of the gallbladder muscle strips and the level of c-Kit mRNA expression,.3, cell experiment: the mechanism of CG gallbladder ICC damage: the effect of high concentration cholesterol on ICC growth (1) the isolation of mouse gallbladder cells by enzymatic method. The following antibodies: PE-Cy7 (PC7) labeled anti c-Kit, PC5 labeled anti CD45, PC5 labeled anti F4/80, PC5 labeled anti CD11b, PE labeled anti CD34 antibody, flow cytometry was used to select the c-Kit+CD45-F4/80-CD11b-CD34- cells (that is, the cells with mature markers), and the selected cells were inoculated into the culture dish of the rat tail collagen envelope, M199 medium was cultured with high concentration serum (15% fetal bovine serum); (2) Real-time PCR identification efficiency, immunofluorescence identification of ICC; (3) cell grouping: control group, experimental group (given different concentrations of water-soluble cholesterol, WSC); culture 72h, real-time PCR detection c-Kit mRNA level. [results, clinical Specimen study: the correlation between CG gallbladder ICC damage and gallbladder motility disorder (1) control group (n=10), CG group (n=30). The average age of the two groups was not statistically significant (2) there was a CCK1 expression on the ICC of the gallbladder (3) the contractile ability of the gallbladder muscle in the CG group was significantly lower than that in the control group (4) CG group of the gallbladder wall c-Kit+ cell density The density of /ICC and the density of ICC were significantly lower than that of the control group. The density of c-Kit+ density and the contractile ability of gallbladder muscle strips were positively correlated (5) the expression of CCK1 was in accordance with the changes of c-Kit expression (6) the thickness of gallbladder wall in the group CG was thicker than that of the control group, and the expression of c-Kit protein in the gallbladder was lower than that of the control group (7) the cholesterol concentration in the CG group was increased by 2. animal models compared with the control group. The effect of gallbladder ICC damage on gallbladder motility disorder and CG formation (1) the guinea pigs in group RD did not form gallbladder stones, and the rate of stone formation in group LD was 60% (2) LD (formation of stones and no stones), the contractile ability of the isolated gallbladder muscle in the isolated group was lower than that of the RD group (3) the LD group of guinea pig gallbladder c-Kit, and the CCK1 mRNA expression was significantly lower than that in the RD group (4) the cholecystoc-Kit table. There was a positive correlation between the level and the contractile ability of the gallbladder muscle strips. Cell test: the ICC damage mechanism of CG gallbladder wall - the effect of high concentration of cholesterol on the growth of ICC (1) flow cytometry: ICC: the cells with mature ICC markers accounted for 10.32 + 2.21% (2) ICC of the gallbladder cells isolated from the whole enzyme solution and culture and identification: 12 small culture. C-Kit and ANO1 (another specific marker of ICC) immunofluorescence staining showed that the cell c-Kit, ANO1 expression were all positive, the cytoplasm of the cells were stained with c-Kit markers, and the cytoplasm and nucleus were coloured by ANO1 markers. The sorting efficiency of real-time PCR was collected before and after the sorting, and the results showed: c-Ki after sorting. The ratio of t mRNA and GAPDH mRNA increased by 102 + 3.18 times (3) exogenous WSC intervention: after adding 10mM, 20mmM, 40mM's WSC action 72h, the ICC quantity decreased significantly compared with the control group. The expression of CCK1 in C, CCK through the action of CCK1 on the ICC surface to constrict the gallbladder; (2) the ICC damage of the gallbladder wall may reduce the expression of CCK1, and the contraction of the gallbladder decreases with CCK, and then causes the gallbladder motility disorder; (3) the study of cholesterol concentration in the bile of CG patients with.2. animal experiment (the guinea pig model of gallbladder cholesterol stone) suggests that the cholesterol concentration in the bile is higher than that in the control group. Long term high cholesterol intake may lead to gallbladder ICC reduction and gallbladder motility disorder, which further promote the formation of cholesterol gallstones. Before the formation of the gallstones, the occurrence of gallbladder motility weakened.3. cell research suggests that high concentration of cholesterol can significantly inhibit the growth of ICC, and the ICC damage of the gallbladder in CG patients may be caused by long-term high concentration of cholesterol bile. First, research background and objective ICC is a gastrointestinal pacing cell, which produces slow wave and conduction excitation. The production of ICC pacing function is related to the intracellular Ca2+ oscillation..CCK is a double distribution of the brain gut peptide in the gastrointestinal tract and the central nervous system. Through its receptor, it plays an important role in the gastrointestinal motility, gallbladder contraction and the secretion of pancreatin. It was found that there was CCK1 on the ICC of the gastric pylorus in rats, suggesting that CCK may directly affect the ICC. in this study. First, the c-Kit and CCK1 immunofluorescence of the gastric antrum of mice were first labeled with the c-Kit and CCK1 immunofluorescence, to prove that the ICC existed CCK1 in the gastric antrum of mice. Secondly, the effect of CCK on the ICC intracellular calcium oscillation was observed and the effect of CCK on the ICC intracellular calcium oscillation was observed. To discuss the specific molecular mechanism. Two, method 1, the whole layer of the gastric antrum of mice was paved, the c-Kit and CCK1 immunofluorescence double standard 2, the effect of CCK on the intracellular calcium ion oscillation of ICC were detected: (1) enzyme separation, flow sorting, culture and identification of mouse gastric antrum ICC; (2) Fluo-3/AM loaded ICC, and laser confocal microscope to observe CC K, CCK1 receptor antagonist chloropamyl, endoplasmic reticulum calcium pump antagonist venom carotene, 1,4,5- three phosphoric acid inositol receptor (1,4,5-triphosphate, InsP3R) antagonist Xestospongin C and L- type Ca2+ channel blocker nifedipine, and other effects on the intracellular calcium oscillation in ICC. Three. Results 1, mouse gastric antrum tissue, in vitro culture ICC c-Kit and co localization, The cells cultured by immunofluorescence are ICC; 2, CCK can detect the effect of ICC intracellular calcium ion oscillation: CCK can significantly increase the intracellular calcium concentration of ICC (intracellular calcium concentration, [Ca2+]i). This effect can be antagonized by chloropamyl, carotene, Xestospongin C, and nifedipine can also slightly weaken the action of CCK. Four, conclusion CCK can promote the InsP3R mediated calcium release in the endoplasmic reticulum through the action of CCK1 receptors on the ICC surface, resulting in a significant increase in [Ca2+]i in ICC cells.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.62

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