SOX10在先天性巨结肠相关性小肠结肠炎发病中的作用

目的 探讨SOX10、潘氏细胞发育及分泌防御素-5与先天性巨结肠相关性小肠结肠炎的关系.方法 收集50例先天性巨结肠病变肠管,根据术前是否发生小肠结肠炎分为HAFC组(n=14)和HD组(n=36),并以20例正常结肠标本作对照组.采用免疫组织化学方法观察结肠中防御素-5蛋白质表达、潘氏细胞发育情况以及SOX10蛋白质表达情况.采用实时荧光定量PCR技术检测防御素-5 mRNA及Sox 10 mRNA表达情况.结果 防御素-5在正常肠管中不表达,HAEC组和HD组在肠腺隐窝基底处呈不同程度阳性表达.但前者阳性区域平均光密度值明显增高(0.33±0.039比0.10±0.031,P＜0.05),HAEC组防御素-5mRNA亦呈显著增高趋势(2.72±0.80比0.78±0.21,P＜0.05).对结肠组织同层切片进行潘氏细胞特异性产物溶菌酶免疫组化染色发现,对照组肠管中除1例存在弱阳性外其他均无阳性表达.HD组和HAEC组结肠中同样在隐窝基底处存在溶菌酶阳性细胞,可鉴别为化生的潘氏细胞,但HAEC组在发生率(78.6%)和细胞个数(2.97±0.80)明显高于HD组(27.8%,0.43±0.85)(P＜0.05).SOX10免疫产物主要在结肠神经节细胞膜及胞浆中表达,对照组、HAEC组、HD组阳性区域的平均光密度值递减(0.75±0.041,0.61±0.048,0.35±0.025),差异具有统计学意义(P＜0.05),同时RT-PCR检测显示Sox10 mRNA在各组中的表达与蛋白质水平呈平行结果.结论 SOX10可能通过影响潘氏细胞发育及分泌防御素-5在先天性巨结肠相关性小肠结肠炎的发生发展中起一定作用.

Abstract：

Objective To study the expression of SOX10 and Human α-defensins-5(HD-5 )in Hirschsprung's disease associated enterocolitis(HAEC) and expore the possible relationship between SOX10 and HAEC.Methods Fifty pathological colons of Hirschsprung's disease (HD) were divided into HAEC group (n = 14) and HD group(n = 36) according to the presence of preoperative enterocolitis,Twenty normal colons as control group.The protein and mRNA expression of HD-5 and SOX 10 were measured by immunohistochemical staining and real-time quantitative PCR Results Normal colons did not express HD-5 but positive expression of HD-5 was detected at the base of the crypts of Lieberkuhn in HAEC and HD groups in different degree.The mean optical density of HD-5 immunohistochemical staining (0.33 ± 0.039 vs 0.10 ± 0.031 )and HD-5 mRNA expression (2.72 ± 0.80 vs 0.78 ± 0.21 ) in HAEC group was apparently higher than those in HD group(P＜0.05).The expression of lysozyme,a specific product by Paneth Cell,on sequential sections was negative in control group except one sample.In HAEC group and HD group,positive expression of lysozyme can be seen in the crypts of Lieberkuhn,where the cells can be identified as Metaplastic Paneth cell,but the incidence (78.6% vs 27.8%) and the number of cells 2.97 ± 0.80 vs 0.43 ± 0.85) in HAEC group were obviously higher than those in HD group(2.97 ± 0.80 vs 0.43 ± 0.85) (P＜0.05).SOX10 was mainly located in the plasmalemma and cytoplasm of ganglion cell and its mean optical density in control group (0.75 ±0.041 ),HAEC group (0.61 ± 0.048)and HD group(0.35 ± 0.025) were decrement,the difference between three groups were statistically significant (P＜0.05).Meanwhile,Sox10mRNA detected by real-time quantitative PCR indicated the parallel result.Conclusions SOX10 may be an important factor in the pathogenesis and development of HAEC.     

胎羊单侧输尿管不完全性梗阻后肾脏足细胞表型转化及PAX2、VEGF的表达

Objective To investigate the phenotypic change of the podocytes and paired box gene 2 (PAX2) and vascular endothelial growth factor (VEGF) expressions in fetal lambs' kidneys with unilateral partial ureteral obstruction. Methods Sixteen fetal lambs were randomly assigned to the obstruction and control group. The obstruction group included 12 lambs, whose superior segment ureters were tied with splited silastic tube to induce unilateral partial ureteral obstruction between 75and 85 gestational days. The control group had 4 fetal lambs subjected to sham operation. After birth,the kidneys of the lambs were harvested to study the phenotypic change of the podocytes as well as expressions of PAX2 and VEGF in the kidneys. Results Nine out of the 12 lambs of the obstruction group were delivered alive. All 4 sham operated lambs were delivered alive. Renal cortex cysts of varying sizes, interstitial fibrosis, a decrease in glomeruli number, and severe podocyte foot process fusion (4. 20± 1.08% vs 86. 79 ± 1.66%) were found in the obstructed kidneys compared with the control kidneys. The PAX2 expression in obstructed kidneys was significantly higher than that of the control kidneys (1.43 ± 0. 09 vs 2. 44 ± 0. 09, P＜0. 05). The VEGF expression was significantly decreased compared with the control kidneys (0. 80 ± 0. 15 vs 0. 33 ± 0. 14, P＜0. 05). Conclusions The changes of the phenotype and PAX2 and VEGF expressions may play roles in the pathogenesis of obstructive nephropathy.     

Wnt2、Wnt5b和Wnt9a在先天性巨结肠症和肛门直肠畸形中的表达及意义

目的 研究Wnt2、Wnt5b和Wnt9a在先天性巨结肠症(Hirschsprung disease,HD)和肛门直肠畸形中(anorectal malformations,ARMs)的表达情况,以期探讨其在HD和ARMs发生发展中的作用.方法 利用荧光实时定量PCR(quantitative real-time PCR,qRT-PCR)和蛋白质印迹(western blot)技术检测HD狭窄肠段和正常肠段肠管组织及ARMs直肠末端(高位、中位和低位组)和死于非胃肠道疾病(正常对照组)患儿各30例中Wnt2、Wnt5b和Wnt9a表达,对其表达进行定位和定量,并比较分析.结果 Wnt2、Wnt5b和Wnt9a在HD狭窄段肠管中mRNA转录水平的表达分别为(10.19±0.27、10.38±0.19和11.43±0.51),均低于正常段肠管的相应表达水平(18.06±1.07、17.98±1.12、19.99±0.76)(P＜0.05);Wnt2、Wnt5b和Wnt9a在ARMs高位组中mRNA转录水平的表达分别为(14.27±0.31、13.69±0.45、13.07±0.67),均低于正常对照组(24.98±2.03、26.43±1.77、28.11±1.44)(P＜0.05),中位组和低位组之间mRNA转录水平比较无统计学意义.western b1ot结果显示,Wnt2、Wnt5b和Wnt9a在HD狭窄段肠管中蛋白表达水平分别为0.19±0.09、0.26±0.07、0.23±0.06,明显低于正常段肠管0.43±0.13、0.57±0.08、0.62±0.06,P＜0.05;Wnt2,Wnt5b和Wnt9a在ARMs高位组中蛋白表达水平分别为0.15±0.11、0.17±0.04、0.13±0.05明显低于正常对照组0.51±0.03、0.63±0.05、0.59±0.05,P＜0.05,而中位组和低位组之间蛋白表达水平无明显差异且与正常对照组比较无统计学意义(P＞0.05).结论 Wnt2、Wnt5b和Wnt9a在HD狭窄段和ARMs的高位组表达水平减低,可能在先天性消化道畸形的肠道发育中具有重要作用.

Abstract：

Objective To investigate the expressions of Wnt2,WntSb and Wnt9a in tissue samples from the patients with Hirschsprung disease (HD) and anorectal malformations (ARMs).Methods Ninety individuals were recruited in this study,including 30 with HD,30 with ARMs,and 30 controls died of non-gastrointestinal diseases.According to the position of malformations,the 30 ARMs patients were subgrouped into high,intermediate and low ARMs groups.The terminal rectum from the ARMs group and the stenotic and normal appearance intestines from HD patients were collected during operation.The expression levels of Wnt2,Wnt5b and Wnt9a were studied using quantitative Real-time PCR (qRT-PCR) and western blotting.Results In the HD patients,the mRNA of Wnt2,Wnt5b and Wnt9a in stenotic intestine were significantly lower than those in the normal intestine ( the relative quantification of mRNA 10.19 ± 0.27,10.38 ± 0.19,11.43 ± 0.51 vs 18.06 ± 1.07,17.98 ± 1.12,19.99 ± 0.76,P＜0.05).In the ARMs patients,the mRNA of Wnt2,Wnt5b and Wnt9a in high ARMs patients were significantly lower than those in control patients (the relative quantification of mRNA 14.27 ± 0.31,13.69 ± 0.45,13.07 ± 0.67 vs 24.98 ± 2.03,26.43 ± 1.77,28.11 ± 1.44,P＜0.05).No significant difference of the mRNA expression was found between the low ARMs patients and intermediate ARMs patients (P＞0.05).The protein expressions of Wnt2,Wnt5b and Wnt9a in these patients changed in the same manners with what were found in the mRNA studies,in which Wnt2,Wnt5b and Wnt9a were decreased in the stenotic intestine of HD patients and high ARMs patients.Conclusions The expression of Wnt2,Wnt5b and Wnt9a changes in the tissue samples from the patients with HD and ARMs,which may play a role in the pathogenesis of congenital malformation of the alimentary tract.     

先天性巨结肠同源病:临床诊断与手术指征的确立

Objective To determine the diagnostic criteria and surgical indications of allied diseases of Hirschsprung's disease (HAD) in children. Methods During the period from Jun. 2007 to Dec. 2009,65 consecutive patients were performed "radical surgery for megacolone" by this surgery team. Based on the clinical symptoms,anorectal manometry, AChE staining of rectal mucosa and barium enema,the preoperative suspected diagnosis of Hirschsprung's disease (HD) or HAD was made.Surgical indications were determined once the suspected HD diagnose was made. On the patients withsuspected HAD, strictly conservative treatments should be carried out for at least 3 months. If conservative treatments were ineffective or the symptoms were recurrent, surgical indication of HAD patients could be determined. Results The preoperative diagnosis was 38 patients with HD and 27 with HAD.However, the final pathologic diagnosis was 36 with HD and 29 with HAD. The misjudged preoperative diagnosis was made in 10. 5% (4/38) HD patients and 7. 4% (2/27) HAD patients. The HAD patients included 22 (75. 9%) hypoganglionosis (HG) cases and 7 (21.1 %) intestinal neuronal dysplasia (IND) cases. Among the 27 HAD patients, 27 (93. 1%) had anorectal inhibitory reflex, and varied and delayed reflection waveforms. AChE staining was positive in 12 (41.1%) patients. The results of anorectal manometry and AChE staining of the HAD patients were significantly different from those of the HD patients (P＜0. 01 ). Barium enema of the HAD patients appeared severe expanded rectum which occupied more than 90% space of the pelvic cavity. Redundant sigmoid colon was also found in more than 90% patients. All HAD patients had 24-hour barium retention. Subtotal colectomies were performed on 9 (31. 0%) HAD patients,and 8 (22. 2%) HD patients. Conclusions Anorectal manometry, AChE staining and barium enema are still important in determining the diagnosis and surgical indications for HAD. The efficacy of 3 months or longer conservative treatment is crucial to determine the surgical indications for HAD.     

小儿急性肠梗阻肠黏膜免疫屏障损伤及细菌移位的研究

目的 探讨小儿急性肠梗阻细菌移位及机制.方法 收集山西省儿童医院普外科2009年9月至2010年8月急性肠梗阻患儿共43例,依治疗方法分三组:肠切除肠吻合组(吻合组)22例,未切除肠管组14例,保守治疗组7例.另选择17例正常肠管及健康儿童10例分别作正常肠管、血清对照组.应用聚合酶链反应(PCR)定性检测细菌共有的16SrRNA和大肠杆菌特异性半乳糖苷酶基因BG;肠系膜淋巴结细菌培养;免疫组织化学方法检测肠黏膜固有层T细胞亚群、浆细胞计数;酶联免疫吸附试验(ELISA)定量检测血清TGF-β1浓度.结果 吻合组16SrRNA、BG阳性率分别为86.4%、72.7%,淋巴结培养阳性率77.3%;未切除肠管组16SrRNA、BG阳性率分别为64.3%、42.8%,淋巴结培养阳性率57.1%;保守治疗组16SrRNA、BG阳性率分别为57.1%、28.5%,正常肠管血清对照组16SrRNA、BG均未检出;吻合组肠黏膜固有层T细胞亚群、浆细胞数量均低于正常对照组;吻合组、未切除肠管组术前血清TGF-β1浓度均高于保守治疗组与对照组,术后7 d各组血清TGF-β1浓度与对照组比较无差异.结论 急性肠梗阻患儿肠黏膜固有层T细胞亚群、浆细胞均降低,引起肠黏膜免疫屏障破坏,同时TGF-β1水平升高,参与机体免疫抑制,免疫功能紊乱,加重黏膜免疫屏障破坏,增加机体易感性,促进了细菌移位.

Abstract：

Objective To investigate the mechanism of intestinal mucosal barrier damage and bacterial translocation in children with acute intestinal obstruction. Methods From September 2009 to August 2010, 43 patients with acute intestinal obstruction were treated at this center and recruited in this study. According to the treatment they underwent, they were divided into 3 groups: Group1 included 22 patients who were performed bowel resection and anastomosis. Group 2 had 14 patients who underwent surgeries but without bowel resection. Group 3 had 7 patients who were cured without surgery. The other 17 healthy bowels and 10 healthy children's serum were selected as normal controls.Polymerase chain reaction (PCR) was performed to examine β-lactosidase gene (BG) of E. coli and 16SrRNA gene. Bacteria in mesenteric lymph nodes were cultured. Immunohistochemical staining was used to indentify the T cell subsets and plasma cells in the lamina propria of mucosa. TGF-β1 in serum was quantified using enzyme-linked immunoabsorbent assay (ELISA). Results In group 1, 16SrRNA and BG were positive in 86. 4% and 72. 7% of the patients, respectively. Positive culture rate of E coli in mesenteric lymph nodes was 77. 3%. In group 2, 16SrRNA and BG positive rates were 64. 3% and 42. 8%, respectively. Positive culture rate of E coli in mesenteric lymph nodes was 57.1 %. In group 3,16SrRNA and BG positive rates were 57. 1 % and 28. 5%, respectively. 16SrRNA and BG were not detectable in normal control patients' serum and bowel. In the removed bowel, T cell subsets and plasma cells' population were significantly decreased compared with the control bowel. The serum TGF-β1 level of the patients underwent surgery was significantly higher than that of the healthy controls or the patients without surgery. But the difference was not significant between these 2 groups since 7 days after surgery. Conclusions In the children with acute intestinal obstruction, the reduction of T cell subsets and plasma cells in the lamina propria, increasing TGF-β1 in the serum are observed.These changes may be related to the damaged intestinal barrier and bacterial translocation.     

幼年大鼠溺水应激后胰岛素信号传导变化及外源胰岛素治疗反应

Objective To explore the alteration of insulin signal transduction in drowning-induced stress hyperglycemia and to investigate therapeutic effect of insulin on it.Methods Stress hyperglycemia model was induced by drowning.Thirty-two infant rats were randomized into control,drowning,air resuscitation and air-insulin resuscitation groups.Insulin was injected abdominally into the air-insulin resuscitation rats.Fasting serum glucose and insulin were determined routinely,and protein expressions and serine phosphorylation levels of insulin receptor substrate 1(IRS-1) in muscle tissue were detected by Western blot and immunoprecipitation.The expressions of glucose transporter 4(GLUT4) in the intracellular and plasma membranes of muscle tissue were detected by Western blot.Results In the drowning group,insulin resistance index(HOMA-IR) increased significantly as compared with that of the control group,but the level of IRS-1 had no significantly change as compared with the other three groups.As compared with that of the drowning group(0.71±0.12),IRS-1 serine phosphorylation levels of the two resuscitation groups decreased significantly(0.56±0.13 and 0.46±0.08 respectively).The intracellular GLUT4 expression in the two resuscitation groups(1.82±0.11 and 1.96±0.28 respectively)increased significantly in contrast to that of the drowning group(1.45±0.15),and the air-insulin resuscitation groups showed an especially high increase of intracellular GLUT4 expression.Conclusion During the drowning-induced stress hyperglycemia,the alteration of serine phosphorylation and GLUT4 distribution is one of the important mechanism of insulin resistance.Insulin may decrease the blood glucose through decreasing serine phosphorylation levels of IRS-1 and increasing the intracellular GLUT4 expressions.     

幼年大鼠溺水应激后胰岛素信号传导变化及外源胰岛素治疗反应

Objective To explore the alteration of insulin signal transduction in drowning-induced stress hyperglycemia and to investigate therapeutic effect of insulin on it.Methods Stress hyperglycemia model was induced by drowning.Thirty-two infant rats were randomized into control,drowning,air resuscitation and air-insulin resuscitation groups.Insulin was injected abdominally into the air-insulin resuscitation rats.Fasting serum glucose and insulin were determined routinely,and protein expressions and serine phosphorylation levels of insulin receptor substrate 1(IRS-1) in muscle tissue were detected by Western blot and immunoprecipitation.The expressions of glucose transporter 4(GLUT4) in the intracellular and plasma membranes of muscle tissue were detected by Western blot.Results In the drowning group,insulin resistance index(HOMA-IR) increased significantly as compared with that of the control group,but the level of IRS-1 had no significantly change as compared with the other three groups.As compared with that of the drowning group(0.71±0.12),IRS-1 serine phosphorylation levels of the two resuscitation groups decreased significantly(0.56±0.13 and 0.46±0.08 respectively).The intracellular GLUT4 expression in the two resuscitation groups(1.82±0.11 and 1.96±0.28 respectively)increased significantly in contrast to that of the drowning group(1.45±0.15),and the air-insulin resuscitation groups showed an especially high increase of intracellular GLUT4 expression.Conclusion During the drowning-induced stress hyperglycemia,the alteration of serine phosphorylation and GLUT4 distribution is one of the important mechanism of insulin resistance.Insulin may decrease the blood glucose through decreasing serine phosphorylation levels of IRS-1 and increasing the intracellular GLUT4 expressions.     

胰岛素对溺水后幼年大鼠应激性高血糖的疗效观察

Objective To explore the alteration of insulin signal transduction in drowning-induced stress hyperglycemia and to investigate therapeutic effect of insulin on it.Methods Stress hyperglycemia model was induced by drowning.Thirty-two infant rats were randomized into control,drowning,air resuscitation and air-insulin resuscitation groups.Insulin was injected abdominally into the air-insulin resuscitation rats.Fasting serum glucose and insulin were determined routinely,and protein expressions and serine phosphorylation levels of insulin receptor substrate 1(IRS-1) in muscle tissue were detected by Western blot and immunoprecipitation.The expressions of glucose transporter 4(GLUT4) in the intracellular and plasma membranes of muscle tissue were detected by Western blot.Results In the drowning group,insulin resistance index(HOMA-IR) increased significantly as compared with that of the control group,but the level of IRS-1 had no significantly change as compared with the other three groups.As compared with that of the drowning group(0.71±0.12),IRS-1 serine phosphorylation levels of the two resuscitation groups decreased significantly(0.56±0.13 and 0.46±0.08 respectively).The intracellular GLUT4 expression in the two resuscitation groups(1.82±0.11 and 1.96±0.28 respectively)increased significantly in contrast to that of the drowning group(1.45±0.15),and the air-insulin resuscitation groups showed an especially high increase of intracellular GLUT4 expression.Conclusion During the drowning-induced stress hyperglycemia,the alteration of serine phosphorylation and GLUT4 distribution is one of the important mechanism of insulin resistance.Insulin may decrease the blood glucose through decreasing serine phosphorylation levels of IRS-1 and increasing the intracellular GLUT4 expressions.     

改良Soave结合结肠切端缩口缝合术治疗先天性巨结肠疗效评价

目的 评价改良Soave结合结肠切端缩口缝合术治疗先天性巨结肠的术后疗效.方法 回顾性分析2005年7月至2009年8月在我院行改良Soave结合缩口缝合术的32例巨结肠患儿术后恢复情况,设为观察组,选取32例行单纯改良Soave根治术的患儿为对照组.观察术中扩张段肠管断端病理切片、术后复查肛诊、肛门镜、钡灌肠及直肠肛管测压并对二组患儿排便功能进行评分.结果 观察组中26例扩张段断端神经节细胞数量正常,无明显变性,6例神经节细胞数量减少,存在变性,对照组中前者28例,后者4例.肛诊及肛门镜示术后半年以上缩口缝合结肠黏膜皱褶基本消失.钡灌肠示结肠均无明显扩张,结肠形态恢复良好.观察组和对照组术后1年排便功能评分及直肠肛管测压中的肛管高压区长度、肛管静息压、直肠感觉阈值,分别为6.6±1.3、(2.0±0.7)cm、(53.1±3.9)mmHg、30～55 ml;6.8±1.2、(1.9±0.6)cm、(52.6±4.7)mmHg、35～60 ml,均无统计学差异.术后并发症观察组有吻合口狭窄1例,二组轻度污粪各1例.结论 改良Soave结合结肠切端缩口缝合巨结肠根治术后结肠及排便功能恢复满意,与单纯改良Soave术式疗效无差异.扩张但功能良好的近端结肠切端管径大于肛门口径时,可行结肠切端缩口缝合术,该术式可成为改良Soave手术较好的辅助手段.

Abstract：

Objective To evaluate the efficacy of modified Soave procedure combined with colon plication for the treatment of Hirschsprung's disease (HD) in children.Methods Between July 2005 and August 2009,32 HD patients of the plication group underwent modified Soave procedure combined with colon plication at this center.Another 32 patients who underwent modified Soave procedure were enrolled as the controls.The pathological examinations,follow-up,barium enema,anorectal manometry,and anorectal function were retrospectively reviewed and analyzed to evaluate the surgical efficacy.Results Of the 32 patients of the plication group,26 had normal ganglion cells at both ends of the dilated intestine,and decreased ganglia number and ganglia degeneration were found on the other 6 patients.However,among the 32 control patients,decreased ganglia number and ganglia degeneration was only found on 4 patients.Rectoscopy suggested that mucosa rugae caused by colon plication could eventually disappear half a year after the surgery.On patients' barium enema radiography,no dilated intestines were observed.No difference of the defecation function scores between the plication group and control group at 1 year after operation was found (defecation function score,6.6 ± 1.3 vs 6.8 ±1.2).Moreover,there was no difference of high pressure zone length (2.0 ± 0.7 cm vs 1.9 ± 0.6 cm),anal canal resting pressure (53.1 ± 3.9 mmHg vs 52 ± 4.7 mmHg) and sensation threshold (30～55 ml vs 35～60 ml) between the 2 groups 1 year after operation.Postoperative complications included anastomotic stenosis was noted on 1 patients of the placation group,1 of the plication group and 1 control patient had mild soiling.Conclusions Modified Soave procedure combined with colon plication improves the defecation function of the HD patients.     

鼻塞式呼吸机间歇指令通气联合肺表面活性物质治疗早产儿肺透明膜病的临床研究

Objective To evaluate the effect of nasal intermittent mandatory ventilation (NIMV)combined with pulmonary surfactant for the treatment of hyaline membrane disease in premature children and to compare the clinical efficacy with conventional mechanical ventilation and continuous positive airway pressure (CPAP). Methods Seventy-four babies with severe respiratory syndrome were given curosurf[100 mg/(kg·dose)],25 of them were given to NIMV, another 25 were given conventional intermittent mandatory ventilation (IMV), the last 24 patients were given conventional CPAP. Blood gas analysis parameters and incidences of respiratory complications including respiratory tract infection and chronic lung disease,frequent apnea and carbon dioxide retention, were compared among the three groups. Results After treatment of 1 h, symptoms and signs of the patients markedly improved. All the three groups showed an increase in arterial oxygen partial pressure, but arterial pressure of carbon dioxide and oxygen index decreased significantly by the time of 6,12,24 h after treatment and there were not significantly difference among the three groups. The rates of respiratory infection and chronic lung disease in NIMV group were less than that of conventional IMV group[(8% vs 36% ) ,(20% vs 72% )],the rates of frequent apnea and carbon dioxide retention in NIMV group were less than that of CPAP group[(8% vs 36% ), (20% vs 72% )]. Conclusion NIMV combined with pulmonary surfactant is a potentially efficient therapy for hyaline membrane disease in premature infants. NIMV treatment of hyaline membrane disease in premature infants can reduce or avoid ventilation-associated pneumonia,chronic lung disease and other complications,but also efficiently reduce the incidence of frequent apnea and carbon dioxide retention.     

Neurexin、Neuroligin基因在人类及鼠类动物肠神经系统的表达及意义

目的 探讨Neurexin和Neuroligin基因在人类及鼠类动物肠神经系统(ENS)是否有表达,以及对ENS突触结构与功能的作用,探查其在先天性巨结肠各段的表达特征及意义.方法 首先制备小鼠、大鼠、豚鼠、正常人体肠管肌间神经丛铺片,通过双重免疫荧光细胞染色,证明Ne-urexin、Neuroligin基因是否在肠神经系统表达,再采用免疫组织化学技术对20例先天性巨结肠狭窄段,移行段和扩张段肌间神经丛铺片标本染色,判断Neurexin、Neuroligin基因的表达情况.结果①Neurexin、Neuroligin蛋白在小鼠、大鼠、豚鼠、人体的肠肌间神经丛与神经细胞标记Hu在同一细胞表达;而Neurexin蛋白与突触前标记物synaptophysin,Neuroligin蛋白与突触后标记物PSD95也在同一神经组织出现;②Neurexin、Neuroligin蛋白在先天性巨结肠扩张段表达最多,移行段明显减少(P<0.05),病变段无表达;同一组织Neurexin和Neuroligin的表达无明显差异(P>0.05).结论 Neurexin、Neuroligin基因在鼠齿类动物及人体肠神经系统都有表达,不仅在胞体,而且在轴突、树突都有表达,Neurexin在突触前表达,Neuroligin在突触后表达.它们对肠神经系统突触的结构功能起重要作用,与一些胃、肠道动力性疾病有密切关系,是导致先天性巨结肠蠕动功能丧失的重要原因.     

SOX10mRNA在先天性巨结肠肠壁中的表达

目的研究性别决定区Y基因相关高可变区基因10（SRY related high mobilitvgroup—BoX gene10，SOX10）在先天性巨结肠（Hirschsprung’s disease，HD）肠壁中的表达情况，以进一步了解HD在分子基础上的发病机制。方法分别取12例先天性巨结肠病例的手术标本狭窄段、移行段及扩张段，随机取12例非巨结肠手术病例（如结肠造瘘或关瘘手术）作为对照组，提取平滑肌组织总RNA，应用逆转录多聚酶链反应（RT-PCR）扩增目的基因和看家基因片段，观察病变段和正常段SOX10mRNA的表达，并与看家基因（B—actin）在病变段和正常段的表达比较，进行统计学分析。结果SOX10mRNA在HD患儿痉挛段呈低表达，在扩张段及正常对照组呈高表达。痉挛段SOX10mRNA的表达量与移行段、扩张段及正常对照组比较，差异均有统计学意义；而移行段、扩张段及对照组比较，差异无统计学意义。结论HD患儿结肠SOX10mRNA的异常分布显示SOXIO基因是出生后肠神经系统维持正常功能所必需的，SOX10mRNA表达减少可引起肠管痉挛，肠腔狭窄，造成肠功能障碍。     

先天性巨结肠中HA117及SHH信号通路的表达及意义

目的 研究HA117及SHH信号通路(SHH、Ptch1、Gli1)在先天性巨结肠(Hirschsprung's disease,HD)中的表达情况,以探讨其与HD发生发展中的关系.方法 收集重庆医科大学附属儿童医院2014年至2015年经手术确诊为HD患儿的巨结肠组织标本30例,肠段分吻合段(正常对照组)、扩张段和痉挛段(实验组),运用实时荧光定量PCR (quantitative real-time PCR,qRT-PCR)、蛋白质印迹(Western blot)和免疫组织化学方法检测HA117、SHH信号途径在各段中的表达情况,对其进行定量和定性并比较.结果 qRT-PCR示痉挛段HA117mRNA表达水平为1.52±0.17,明显高于吻合段的0.28±0.04,差异具有统计学意义(P＜0.05).痉挛段SHH、Ptch1、Gli1mRNA表达水平分别为0.53±0.11、0.48±0.11、0.62±0.13,明显低于吻合段(1.23±0.26、1.20±0.22、1.71±0.46),差异具有统计学意义(P＜0.05);Western-Blot示SHH、Ptch1、Gli1蛋白相对表达水平与mRNA表达水平一致,差异均具有统计学意义(P＜0.05);免疫组织化学(DAB染色)结果提示:痉挛段SHH、Ptch1、Gli1染色明显弱于吻合段.结论 HA117及SHH信号通路可能与肠神经系统(ENS)发育有着密切的联系,这可能是HD病理发生的重要作用机制之一.     

先天性巨结肠Cajal间质细胞的研究

目的　本研究通过观察Cajal间质细胞 (interstitialcellofCajal,ICC)在先天性巨结肠患者狭窄段、移行段、扩张段中的分布情况 ,探讨ICC在先天性巨结肠发病中的作用。方法　收集我院1999～ 2 0 0 2年 2 6例先天性巨结肠患儿标本。短段型 2 4例 ,长段型 2例。于手术中分别选取扩张段 ,移行段及狭窄段肠壁的全层组织。采用SP法 (过氧化物酶标记的链霉卵白素法 )免疫组织化学技术 ,对 2 6例先天性巨结肠的狭窄段、移行段及扩张段标本分别进行c kit免疫组织化学反应 ,观察Ca jal间质细胞分布情况。 结果　发现ICC的密度从扩张段→移行段→狭窄段是逐渐减低的。ICC与肌间神经丛关系密切 ,在扩张段ICC分布在神经丛的周边部和内部 ,且数量相对较多 ,在狭窄段ICC偶见于神经丛的周边部 ,在神经丛内部未见该细胞。光学显微镜下比较同一例患者扩张段和狭窄段神经丛中Cajal间质细胞的数目不同 (t=2 3.0 4 ,P <0 .0 5 ) ,有统计学显著性差异。结论　ICC的分布异常与先天性巨结肠的发生有密切关系。我们推测胚胎基质的某种缺陷不仅损害了神经嵴细胞的移行 ,也影响ICC的分化和成熟。我们可以推论 ,与HD肠壁神经节缺失一样 ,ICC分布异常导致HD病变肠管慢波节律和兴奋传导异常 ,从而引起或加重HD的发病。     

5-羟色胺4受体在先天性巨结肠和同源病中的表达

目的探讨5-羟色胺4(5-HT4)受体在先天性巨结肠(HD)和先天性巨结肠同源病(HAD)中的表达,并研究能否将此检测应用于HD和HAD的术前鉴别诊断。方法共收集HD标本25例(HD组)、HAD标本20例(HAD组)和正常结肠标本15例(正常对照组)。应用免疫组织化学SP法染色,分析5-HT4受体表达的部位及肠管不同部位5-HT4受体表达水平强度的差异,采用t检验比较其表达是否存在差异性。结果 HD组5-HT4受体阳性指数在远端黏膜层较近端表达下降,肌间神经丛无表达;HAD组远端5-HT4受体阳性指数在黏膜层和肌间神经丛中均显著低于近端。与正常对照组比较,HD组和HAD组5-HT4受体阳性指数在黏膜及肌间神经丛表达均下降。结论 5-HT4受体在HD及HAD中的表达水平可作为术前鉴别诊断的参考指标之一,也为临床治疗提供新的参考指标。     

PERIPHERIN: A Novel Marker for the Immunohistochemical Study of Malformations of the Enteric Nervous System

Peripherin is a 57-kD type III intermediate filament that is a specific marker for peripheral neurons, including enteric ganglion cells (GCs). Hence antibodies to peripherin may be used to demonstrate abnormalities of the enteric nervous system (ENS). Serial longitudinal histologic sections of formalinfixed paraffin-embedded colons from 15 patients were immunostained for peripherin, neuron-specific enolase (NSE), neurofilaments, S-100, and synaptophysin. Ten patients had variable degrees of colonic aganglionosis (Hirschsprung's disease), three were premature infants, and two were controls. Peripherin labeling yielded the highest number of recognizable GCs. Overall, 56%, 78%, and 80% of the peripherin-positive GCs in the myenteric plexus and 55%, 75%, and 73% of the peripherinpositive GCs in the submucosal plexus were identified by staining for neurofilaments, NSE, and S-100, respectively. Intramucosal GCs were detected in 4 of 10 cases of Hirschsprung's disease (HD), none of which had been evident by routine histology. The other neuronal markers were less specific for intramucosal GCs than peripherin, because they also labeled enterochromaffin cells. Peripherin immunohistochemistry also allowed exact quantification of GC density expressed as GCs/mm colon, which is important for the diagnosis of HD-related disorders. In three cases of HD the GC density of the transition zone was markedly elevated compared with more proximal ganglionic bowel segments, consistent with neuronal intestinal dysplasia type B, and two cases of HD showed low GC density within the transition zone. Hence peripherin immunolabeling may prove to be a valuable aid in the diagnosis and classification of congenital malformations of the ENS.     

p14ARF/p16β与Oct-4、Nestin、GFAP在不同组织结构类型的神经母细胞瘤中的表达及意义

目的 探讨p14ARF＜/p16β与Oct-4、Nestin、GFAP在不同组织结构类型的神经母细胞瘤(组织结构良好型,FH型;组织结构不良型,UFH型)中的表达及其意义.方法 收集近6年来本院保存的儿童神经母细胞瘤石蜡标本33例,男17例,女16例;年龄范围1个月～15岁,平均年龄33.1个月;随访时间21～87个月,平均随访时间45.5个月,其中UFH神经母细胞瘤10例,平均肿瘤分期3期;FH神经母细胞瘤23例,平均肿瘤分期2.57期.利用组织芯片的高通量、低误差、经济省时等优点,将二组标本制作成组织芯片,再行免疫组织化学SP法检测p14ARF/p16β与Oct-4、Nestin、GFAP在神经母细胞瘤中的表达.结果 相对于FH神经母细胞瘤,UFH神经母细胞瘤中的p14ARF/p16β表达明显下降,差异有统计学意义(P<0.05);而Nestin和GFAP在二组肿瘤中表达均较低,Oct-4二组表达类似.二组之间差异无统计学意义(P0.05).UFH组失随访1例.平均随访时间34.5个月,随访时存活率66.6%;FH组失随访2例,平均随访时间49.5个月,随访时存活率80.9%,二组之间差异有统计学意义(P<0.05).结论 p14ARF/p16β基因的失活在肿瘤的发生中起着重要作用,UFH神经母细胞瘤中p14ARF/p16β的低表达表明了肿瘤分化较差,肿瘤分期较高;而Nestin、Oct-4和GFAP表达类似.表明了不同组织结构类型的神经母细胞瘤(FH型和UFH型)均具有一些最原始的神经干细胞的生物学特征,具有进一步分化的潜力.并使得神经母细胞瘤进展迅速,呈现恶性的组织表型.FH型预后相对较好,可能与患儿不同的年龄、肿瘤分期、是否转移和复发均有关.     

Altered differentiation of enteric neural crest-derived cells from endothelin receptor-B null mouse model of Hirschsprung’s disease

Purpose

Hirschsprung’s disease (HD) is caused by a failure of enteric neural crest-derived cells (ENCC) to colonize the bowel, resulting in an absence of the enteric nervous system (ENS). Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize ENCC with the green fluorescent protein, Venus. The aim of this study was to isolate Sox10-Venus⁺ cells, which are differentiated neurons and glial cells in the ENS, and analyze these cells using Sox10-Venus mice gut.

Methods

The mid-and hindgut of Sox10-Venus⁺/Ednrb ^+/+ and Sox10-Venus⁺/Ednrb ^?/? at E13.5 and E15.5 were dissected and cells were dissociated. Sox10-Venus⁺ cells were then isolated. Expression of PGP9.5 and GFAP were evaluated neurospheres using laser scanning microscopy.

Results

7 days after incubation, Sox10-Venus⁺ cells colonized the neurosphere. There were no significant differences in PGP9.5 expressions on E13.5 and E15.5. GFAP was significantly increased in HD compared to controls on E15.5 (P < 0.05).

Conclusions

Our results suggest increased glial differentiation causes an imbalance in ENCC lineages, leading to a disruption of normal ENS development in this HD model. Isolation of ENCC provides an opportunity to investigate the ENS with purity and might be a useful tool for modeling cell therapy approaches to HD.