【作者】 张广军；

【导师】 刘宝华；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 慢传输性便秘(slow transit constipation, STC)是较常见的一类功能性顽固性便秘,发病机制不清,严重者需要手术切除全(次全)结肠。流行病学调查表明,STC多发于女性,男女比例为1:9,提示性激素可能在STC的发生及发展过程中起着重要的作用。雌激素作为最重要的性激素之一,除对生殖系统的重要作用外,对消化系统、心血管系统、泌尿系统、中枢神经系统、免疫系统等都发挥重要的调控作用。雌激素是与其特异性受体-雌激素受体(estrogen receptor,ER)结合而发挥生理作用的。ER包括ERα及ERβ两种亚型,每种亚型又包括多种同分异构体。研究发现ERβ是结肠内主要表达的雌激素受体,其表达水平决定肠道对雌激素及其拮抗剂的反应。我们前期免疫组化研究发现,女性STC患者乙状结肠粘膜层ERβ表达较对照组明显减少,提示ERβ表达降低可能与STC的发病有关。但ERβ在结肠壁其它部位的分布及表达如何?STC患者结肠存在哪几种ERβ同分异构体,其表达如何?ERβ在STC发病中的作用如何?国内外尚未见报道。近年来的研究证实Cajal间质细胞(interstitial cells of Cajal, ICC)是介于肠神经系统和平滑肌细胞之间的一类特殊的间质细胞,是胃肠道慢波的起搏细胞,对肠神经信号传递到平滑肌起重要的调控作用,探索ICC的病理变化可望成为STC发病机制研究的突破点。c-kit是ICC的特异性标志物,属酪氨酸激酶受体家族第三亚类,与其配体干细胞因子(stem cell factor, SCF)结合后所启动的信号通路,对ICC的发育、分化及表型维持至关重要。中和抗体阻断c-kit可导致ICC数目减少,肠道慢波消失。目前研究发现STC患者结肠ICC显著减少,并存在形态异常,同时发现结肠c-kit表达也显著降低,提示STC患者结肠ICC数量和形态异常变化与c-kit基因表达的异常有关,但这种异常是否与ERβ有关联?其具体机制如何?国内外尚未见报道。近年来研究发现,阻断ER可以导致c-kit mRNA几乎不表达,因此,ER有可能参与调控c-kit信号通路。为此,我们将进一步从转录和翻译水平研究STC患者及模型大鼠结肠ERβ表达,以及阻断ERβ信号通路后结肠c-kit及ICC的变化,观察ERβ对ICC的影响,探讨ERβ影响结肠动力的机制,为今后深入探讨STC的发病机制及其激素治疗提供新思路。目的:1.研究STC患者血清性激素变化及ERβ在乙状结肠的分布,从转录和翻译水平探讨STC患者乙状结肠ERβ基因表达的变化。2.建立STC大鼠模型,研究STC大鼠血清性激素及结肠ERβ表达变化。3.阻断ERβ信号通路,检测大鼠结肠肌电的变化,观察大鼠结肠ICC数量及c-kit表达的变化,探讨ERβ在ICC异常变化中的作用。方法:1.采用电化学发光法检测STC患者血清性激素的变化;采用免疫组织化学方法观察STC患者乙状结肠ERβ的分布和表达变化;采用RT-PCR及Western blot技术检测STC患者乙状结肠ERβ同分异构体mRNA及ERβ蛋白的表达变化。2.建立STC大鼠模型,采用电化学发光法检测STC大鼠血清性激素的变化;采用电生理方法检测STC大鼠结肠离体肌条慢波频率及振幅;采用RT-PCR及Western blot技术检测STC大鼠结肠ERβ同分异构体mRNA及ERβ蛋白的表达变化。3.采用他莫昔芬(tamoxifen,TAM)阻断ERβ信号通路,通过电生理方法检测大鼠在体及离体结肠肌电慢波频率及振幅的变化;采用RT-PCR技术检测大鼠结肠c-kit mRNA的表达变化;采用Western blot技术检测大鼠结肠c-kit蛋白的表达变化;采用免疫组织化学及免疫荧光染色方法观察大鼠结肠ICC的分布、形态及ICC的数量变化。结果:1.电化学发光法检测显示,STC患者血清促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E2)、泌乳素(PRL)、孕酮(P)及睾酮(Testo)与对照组相比均无显著性差异(P>0.05)。2.免疫组化结果显示,ERβ在STC患者及对照组乙状结肠粘膜层、肌间神经丛及粘膜下神经丛都有表达,环肌层及纵肌层未见表达。与对照组相比,STC患者乙状结肠粘膜层、肌间神经丛及粘膜下神经丛ERβ表达均明显减少(p<0.01)。3.RT-PCR结果显示,STC患者及对照组乙状结肠均表达ERβ1、ERβ2、ERβ5 mRNA,而ERβ3、ERβ4 mRNA未见表达。与对照组比较,STC组ERβ1、ERβ2、ERβ5 mRNA表达显著降低(p<0.01)。Western blot结果显示,STC患者乙状结肠ERβ蛋白表达明显低于对照组(p<0.01)。4.成功建立STC大鼠模型,电生理方法检测显示,STC大鼠结肠离体肌条慢波频率及振幅明显低于对照组(p<0.05)。5.电化学发光法检测显示,STC大鼠血清促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E2)、孕酮(P)及睾酮(Testo)与对照组相比均无显著性差异(P>0.05),泌乳素(PRL)检测量太少,无意义。6.RT-PCR结果显示,STC大鼠及对照组结肠均表达rERβ1、rERβ2 mRNA,而rERβ1δ3、rERβ2δ3及rERβ1δ4 mRNA均未见表达。与对照组相比,STC大鼠结肠rERβ1、rERβ2 mRNA表达均显著降低(p<0.01)。Western blot结果显示,STC大鼠结肠ERβ蛋白表达明显低于对照组(p<0.01)。7.阻断ERβ信号通路,电生理检测发现大鼠在体及离体结肠肌电慢波频率及振幅均显著降低(p<0.05)。RT-PCR结果显示,实验组大鼠结肠c-kit mRNA表达显著降低(p<0.01)。Western blot结果显示,实验组大鼠结肠c-kit蛋白表达显著降低(p<0.01)。免疫组化结果显示,结肠ICC主要包括四个部分,即粘膜下环肌层表面ICC、环肌层ICC、肌间丛ICC和纵肌层ICC,实验组大鼠结肠各部分ICC均显著减少,残存的ICC突起变短、变钝。免疫荧光染色分析结果显示,实验组大鼠结肠粘膜下环肌层表面ICC、环肌层ICC、肌间丛ICC和纵肌层ICC阳性面积均显著减少,结肠环肌层和纵肌层ICC几乎消失。结论:1.STC患者及模型大鼠血清性激素与对照组均无明显差异。提示血清性激素与STC的发病无直接联系。2.STC患者乙状结肠肌间及粘膜下神经丛ERβ表达较对照组均明显降低。提示肌间及粘膜下神经丛ERβ表达降低可能和STC发病有关。3.STC患者及模型大鼠结肠ERβ蛋白及ERβ同分异构体mRNA表达较对照组均显著降低。提示STC发病与ERβ基因在转录和(或)翻译水平的异常表达有关。4.ERβ表达异常可能通过影响c-kit信号通路进而影响ICC数量和形态,导致STC发病。更多还原

【Abstract】 Background:Slow transit constipation (STC) is a colonic motor disorder, which is characterized by measurably delayed movement of materials through the colon. Although abnormalities in the neuronal networks of the colon have been demonstrated in patients with STC, the aetiology of it remains unclear. The severity of its symptoms and the failure of the conservative therapy ultimately led to colectomy for some patients with STC.Health surveys revealed that chronic constipation caused by slow transit is common in women with an F/M ratio of 9:1. The cause and mechanism responsible for this syndrome are unknown. Among the female hormones, estrogen is one of the most important female homones,it can have an indispensable effect on body with estrogen receptor (ER), ER have two kinds of recptors, ie, ERαand ERβ. Studies reported ERβis the major ER in colon.That is to say, ERβcan determine the effect of estrogen and its antagonist on colon. Previous studies in our laboratories have shown that the expression of ERβprotein decreased greatly in mucous layer of sigmoid colon of patients with STC though immunohistochemistry method, which indicated ERβwas related to the pathogenesis of STC.Interstitial cells of Cajal (ICC) have been shown to be the pacemaker cells of the intestine and have been implied in the pathogenesis of a number of gastrointestinal motility dysfunction including idiopathic slow transit constipation. A major breakthrough in this field was the discovery that the tyrosine kinase receptor c-kit are critical in the normal development, maturation, and maintenance of phenotype of ICC, and ICC can be reliably identified by c-kit immunohistochemical technique. Blockade of c-kit with neutralized antibody could induce transdifferentiation of ICC to a smooth muscle phenotype, and intestinal slow waves disappear. A loss-of-function mutation of c-kit results in depletion of stem cells and ICC, while its gain-of-function mutation results in their oncogenesis. Several studies indicate that ICC decreased in the colon of patients with STC, and the expression of c-kit mRNA was also reduced. So, abnormal expression of c-kit gene may disturb the SCF/c-kit signal pathway, and contribute to the alteration of ICC in STC. However, the mechanism is unknown.Recent study suggested the blockade of ER signaling can lead to the decreased expression of c-kit mRNA, and ER may regulate c-kit signal pathway. So, we want to investigate further the expression of ERβin patients and rats with STC in transcription or translation phase. we study the changes of c-kit and ICC though blocking the ERβsignal pathway, and explore whether ERβhas any effects on ICC.Objective: The aims of this study were:(1) To investigate the sexual homones in patients and rats with STC, and to determine if sexual homones are abnormal.(1) To detect the expression of ERβprotein and its isoform mRNA within the colon of patients and rats with STC, and to determine if the expression of ERβare abnormal.(2) To explore the motility of rat colon though blocking the ERβsignal pathway, and further to investigate the expression of c-kit and ICC in the colon of experimental group, and to determine whether ERβhas any effects on ICC.Methods:Twenty patients with STC and Twenty age-matched controls were studied. The sexual homones were detected by Chemiluminescence. The distribution and expression of ERβwere observed with immunohistochemistry. Western blotting and Revers-transcriptional polymerase chain reaction (RT-PCR) technique were employed to determine the expression of ERβprotein and ERβisoform mRNA. An experimental rat model of STC was established with Rhubarb. Chemiluminescence was used to detect sexual homones. Frequency and amplitude of colonic slow waves were examined though electrophysiology. Western blotting and RT-PCR technique were employed to investigate the expression of ERβprotein and ERβisoform mRNA. Though blocking ERβpathway with tamoxifen (TAM), frequency and amplitude of colonic slow waves were examined though electrophysiology in both groups. Western blotting and RT-PCR technique were employed to determine the expression of c-kit protein and c-kit mRNA. The distribution and configuration of ICC were observed with immunohistochemistry. With an immunofluorescence staining, ICC were examined with fluorescence microscope and the area occupied by ICC were calculated with an image analysis system.Results:(1) Chemiluminescence displayed that FSH, LH, E2, PRL, P and Testo in patients with STC had no differences with that of control group (P>0.05).(2) Immunohistochemistry displayed that ERβwas detected in mucous layer, myenteric nerve plexus and submucous nerve plexus, but not detected in muscular layer in the sigmoid colon of patients with STC and the controls. ERβwas mainly expressed in cytoplasm. The expression of ERβin each of the three regions in STC group decreased significantly compared with the control group (P<0.05).(3) The RT-PCR results indicated that the ERβ1, ERβ2 and ERβ5 mRNA were detected in the sigmoid colon of patients with STC and the controls, but ERβ3 and ERβ4 mRNA were not detected. In comparison with the control group, the expression of ERβprotein and ERβ1, ERβ2 and ERβ5 mRNA of STC group significantly declined (P<0.01).(4) Western blotting technique showed that ERβprotein reduced markedly in the sigmoid colon of patients with STC compared with the controls (P<0.01).(5) An experimental rat model of STC was established with Rhubarb for 3 months. The frequency and amplitude of colonic slow waves were decreased in vitro (P<0.05).(6) Chemiluminescence displayed that FSH, E2, LH, P and Testo in rats with STC had no differences with that of control group (P>0.05), and PRL was too little to be detected.(7) The RT-PCR results indicated that the rERβ1 and rERβ2 mRNA were detected in the two groups,but rERβ1δ3, rERβ2δ3 and rERβ1δ4 mRNA were not detected. In comparison with the control group, the expression of rERβ1 and rERβ2 mRNA in the colon of rats with STC significantly declined (P<0.01).(8) Western blotting technique showed that ERβprotein in the colon of rats with STC reduced markedly compared with the controls (P<0.01).(9) Though blocking the ERβsignal pathway with TAM, the frequency and amplitude of slow waves of the experimental group were reduced significantly in vitro and in vivo compared with the control group(P<0.05). The RT-PCR and Western blot results indicated that the expression of c-kit mRNA and c-kit protein also decreased significantly (P<0.01). Immunohistochemistry displayed that ICC were located in the four regions (ICC-LM, ICC-MP, ICC-CM, ICC-SMB), and ICC in the experimental group appeared blunted and shorter compared with the controls. Immunofluorescence staining showed that the percentage of the area occupied by ICC in SMB, MP regions were significantly decreased while compared with the controls (p<0.01), and ICC in CM and LM regions almost can not be find.Conclusions:(1) Our studies revealed the sexual homones in patients and rats with STC had no differences with that in controls, which indicated that the serum sexual homones level had no direct relationship with STC.(2) The reduction of ERβin myenteric nerve plexus and submucous nerve plexus may relate to the pathogenesis of STC.(3) Abnormal expression of ERβgene in the colon of patients and rats with STC at transcription or translation phase may relate to the pathogenesis of STC.(4) Abnormal expression of ERβgene may disturb the c-kit signal pathway, and contribute to the alteration of ICC.更多还原