【作者】 童卫东；

【导师】 刘宝华；

【作者基本信息】 第三军医大学 ， 外科学， 2004， 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,保守治疗对STC效果有限,严重者最终只有手术切除全(次全)结肠。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极其特殊的间质细胞,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,属受体酪氨酸激酶家族第三亚类,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。用中和抗体阻断c-kit与SCF结合可导致ICC数目减少,肠道慢波消失。c-kit基因失功能性突变(loss of function mutation)也可导致ICC缺失。国外有少量初步的研究发现STC患者结肠ICC明显减少,但ICC减少的原因和机制并不清楚,国内尚未见到类似的报道。那么,STC患者结肠ICC的形态和分布究竟发生了怎样的变化?这种异常改变是否与c-kit、SCF基因异常表达或者失功能性突变有关?迄今国内外未见报道。 目的: 1.研究STC患者乙状结肠ICC的分布、形态和数量变化,探讨STC结肠ICC形态和数量异常变化与细胞变性、凋亡、坏死是否有关。从转录和翻译水平探讨c-kit、SCF基因表达在STC结肠ICC异常变化中的作用。 2.初步检测STC患者c-kit基因第9～21外显子及部分相邻的内含子基因序列变异(突变)情况,检测SCF因第54号氨基酸多态位点在STC患者中的变化,为今后大规模的分子流行病学调查奠定基础。 方法: 收集我院1995年以来手术治疗的STC患者17例(基因测序部分包括了近期的6例,为23例),在常规光镜检查的基础上,采用免疫组织化学方法观察乙状结肠ICC的分布和形态变化;进一步采用免疫荧光染色结合激光共聚焦显微镜技术分析ICC的数量更多还原

【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. 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 and its ligand-stem cell factor (SCF) 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 from abroad indicate that ICC were decreased in the colon in patients with STC. However, the mechanism is unknown. Are ICC in the colon of patients with STC do decreased or with abnormalities in morphology? Whether those changes related to the abnormal expression or loss-of-function mutation of c-kit gene and SCF gene remain a matter for further investigation.Objective: The aims of this study were:(1) To determine the normal distribution of ICC within the human colon and to determine if ICC are abnormal within the colon in STC. To explore the correlation between abnormalities of ICC and degeneration, apoptosis, and necrosis. To investigate role of c-kit gene and SCF gene expression in the abnormalities of ICC in STC patients.(2) To screen a panel of 23 patients with STC for c-kit gene from exon 9 to exon 21,including some intron and the exon-intron boundaries, and to examine the variation of a single nucleotide polymorphisms (SNPs) at the codon 54 of SCF gene.Methods:Seventeen patients with STC (six latterly operated patients were enrolled in for DNA sequencing, so patients in the third part of this study was twenty-three.) and Seventeen age-matched controls were studied. Based on the routine microscope examination, the distribution and configuration of ICC were observed with immunohistochemistry. With an indirect immunofluorescence staining, ICC were examined with a laser scanning confocal microscope and the area occupied by ICC were calculated with an image analysis system. Apoptosis was detected with TUNEL. And the ultrastructure of ICC were observed by an electron microscope. Revers-transcriptional polymerase chain reaction (RT-PCR) and Western blotting technique was employed to determine the mRNA and protein expression of c-kit and SCF. Genomic DNA was isolated from frozen or paraffin embedded specimens. The exon of c-kit from 9 to 21 and the exon 8 of SCF were amplified. PCR products were purified and sequenced directly in both directions.Results:(1) H-E staining displayed that most patients have a chronic inflammation within the mucosal layer. However, no obvious abnormal presentation was determined within muscle layers, connective stroma and myenteric plexuses.(2) Immunohistochemistry displayed that ICC were located in the external muscle layers including myenteric plexus (MP) and submucosal border (SMB). Two types of kit-positive ICC were observed: bipolar cells and multipolar cells. Tissues from STC patients showed considerable decrease of ICC in each of the four regions (ICC-LM, ICC-MP, ICC-CM, ICC-SMB). The remaining ICC in the STC patients appeared blunted and shorter when compared with processes from ICC visualized in the controls.(3) Immunofluorescence staining combined with laser scanning confocal microscope showed that the percentage of the area occupied by ICC averaged 0.97%, 2.03%, 4.41%, and 1.59% in the SMB, CM, MP, and LM regions respectively, which were significantly decreased while compared with the controls (p<0.05).(4) Increased apoptosis were revealed within the mucosal and submucosal layer, while no differences were found within the external muscles layers, including myenteric plexusregions (p=0.122).(5) Electron microscope showed that ICC were relatively sparsity. The Space between ICC and smooth muscle enlarged. NO obvious apoptosis body was found.(6) The RT-PCR results indicated that the expression of c-kit mRNA significantly declined in the STC group (p=0.025), while the expression of SCF mRNA was found no difference compared with the controls (p=0.\22).(7) Both the expression of c-kit protein and SCF protein reduced markedly when compared with the controls, which clue to a relation with abnormal ICC in STC (p=0.020, ^=0.031).(8) Following base changes were detected in c-kit gene of STC patients within the region from exon 9 to exon 21: 75515T>C, 75794T>A, 81240OA, 81517OT, 85240A>G, 86548T>A. Among these variations, the base substitution at 75794T>A and the heterozygous mutation at 86548T>A are expected to affect exon-intron splicing, which are worthy of further exploration.(9) NO changes were detected at the codon 54 within exon 8 of SCF gene in all the subjects.Conclusions:(1) Our studies revealed decreased area of c-kit positice ICC in the STC colon, which may play an important role in the pathogenesis of STC.(2) Decreased or abnormal ICC found within the STC colon may not related to cell degeneration, apoptosis, or necrosis.(3) Abnormal expression of c-kit gene and SCF gene at transcription or translation phase may disturb the SCF/c-kit signal pathway, and contribute to the alteration of ICC in STC.(4) Following base changes were detected in c-kit gene of STC patients within the region from exon 9 to exon 21: 75515T>C, 75794T>A, 81240OA, 81517OT, 85240A>Q 86548T>A. Among them, the base substitution at 75794T>A and the heterozygous mutation at 86548T>A are expected to affect exon-intron splicing, which are worthy of further exploration. NO changes were detected at the codon 54 within exon 8 of SCF gene in all the subjects.更多还原