GPC1在胆道闭锁患儿肝组织中表达水平及临床意义的初步探索

目的初步探讨GPC1在胆道闭锁患儿发病过程中的临床意义。方法选取深圳市儿童医院2016年7月至2018年2月收治的胆道闭锁患儿35例作为观察组,同期选取本院收治的先天性胆总管扩张症患儿42例作为对照组。通过免疫组化结果了解GPC1在胆道闭锁肝脏中的分布情况;通过qPCR及Western Blot对胆道闭锁(BA)及先天性胆总管扩张症(CC)患儿肝脏中的CGPC1蛋白水平、GPC1 mRNA水平进行检测。结果本研究结果显示GPC1主要分布在胆道闭锁肝脏胆管细胞顶部,观察组和对照组患儿GPC1 mRNA表达水平(0.65±0.05 vs. 2.54±0. 12,P0.05)、蛋白表达量(1.31±0.14 vs. 2.83±0.25,P0.05)比较,差异有统计学意义。结论 GPC1 mRNA和蛋白产物的低表达可能与胆道闭锁的发病密切相关。     

Toll样受体在胆道闭锁肝脏中的表达

目的 检测胆道闭锁(biliary atresia,BA)患儿肝脏组织中Toll样受体(TLRs)的表达情况,探讨其与胆道闭锁发生的关系.方法 应用RT-PCR和Westen blot方法检测13例BA患儿、7例胆总管扩张(CC)患儿和8例正常对照儿童(CO)肝脏组织中TLRs mRNA和蛋白的表达.结果 BA患儿肝脏组织TLR7表达明显高于CC组患儿和CO组患儿(P＜0.05);而TLR3的表达在BA组患儿和CC组患儿之间差异无统计学意义,略高于CO组患儿(P＞0.05).TLR4的表达在BA组患儿和CC组患儿之间差异无统计学意义(P＞0.05),但高于CO组患儿(P＜0.05).结论 胆道闭锁患儿肝组织中TLR3、TLR7、TLR4表达异常,可能在胆道闭锁胆管损伤中发挥作用.     

PDCD5在胆道闭锁患儿胆管组织中的表达及意义

目的研究促凋亡基因PDCD5在胆道闭锁及胆总管囊肿患儿胆管组织中的表达规律,探讨促凋亡基因PDCD5在胆道闭锁发病机制中的作用。方法分别取18例胆道闭锁（Biliary Atresia,BA）和11例胆总管囊肿（Choledochal cyst,CC）患儿的胆管组织,应用蛋白质印迹（westong blot）与荧光实时定量（quantitative real—time PCR,qRT-PCR）的方法分别检测PDCD5在胆道闭锁与胆总管囊肿患儿胆管组织中的表达,并进行定位和定量分析与比较。结果PDCD5在CC组胆管组织中的CT值为（6．7292±1．169）,高于BA组的相应CT值（4．0125±1．0735）,P〈0．05。应用2-△△CT计算出CC与BA中PDCD5基因表达量的比值为1：6．57。PDCD5在BA组胆管组织中mRNA转录水平明显高于对照组;Western blot结果显示,PDCD5在BA组胆管组织中蛋白表达量明显高于CC组。结论PDCD5在胆道闭锁患儿胆管组织中的蛋白和基因表达水平均增强。PDCD5可能参与胆道闭锁胆管上皮细胞凋亡,从而参与胆道闭锁的炎性反应,在胆道闭锁的发病机制中发挥重要作用。     

胆道闭锁与婴儿肝炎综合征肝活检病理对比分析

目的对比分析胆道闭锁、婴儿肝炎综合征患儿肝组织活检病理表现,明确胆道闭锁与婴儿肝炎综合征的相关性及不同病理表现。方法收集2004年1月至2014年1月在本院因黄疸保守治疗效果不佳而疑为胆道畸形并行胆道探查、胆道造影患儿的肝活检标本32例,其中胆道闭锁25例,婴儿肝炎综合征7例,分别就两者肝活检HE染色切片肝细胞淤胆、变性,毛胆管淤胆,汇管区胆管增生,胆管内胆栓,汇管区炎性细胞浸润,肝脏纤维化程度进行比较。结果虽然婴儿肝炎综合征胆道造影存在胆道形态异常,但同年龄段胆道闭锁与婴儿肝炎综合征患儿肝细胞淤胆、变性,毛胆管淤胆,汇管区炎性细胞浸润等情况比较无明显差异;胆道闭锁患儿汇管区胆管增生,肝脏纤维化程度明显高于婴儿肝炎综合征组(P0.05)。结论婴儿肝炎综合征与胆道闭锁病理表现明显不同;汇管区胆管增生、肝纤维化程度是鉴别胆道闭锁与婴儿肝炎综合征的主要病理依据;婴儿肝炎综合征能否最终发展为胆道闭锁尚需进一步随访研究。     

热休克蛋白47的表达与胆道闭锁肝纤维化及预后的相关性研究

目的 研究热休克蛋白47的表达与胆道闭锁患儿肝脏纤维化程度及预后的相关性.方法 选择30例胆道闭锁、10例胆汁淤积症和10例胆总管囊肿患儿入组,分别取肝脏组织以及外周血,通过RT-PCR、Western-blot、ELISA以及病理切片来评估不同水平HSP47的表达情况及其与肝脏纤维化相关性.结果 三组HSP47在mRNA水平(0.915±0.730比0.066±0.037比0.314±0.150,P＜0.05)、蛋白水平(3.061 81±0.504 763比1.358 018±0.373 174比1.23649±0.350 173,P＜0.05)以及血清水平(63.38±17.11比57.87±14.83比45.78±11.23,P＜0.05)的表达胆道闭锁组均比胆汁淤积组以及胆总管囊肿组高,差异有统计学意义,且不同水平表达具有一致性(mRNA比蛋白水平:r=0.836,P＜0.05;蛋白水平比血清水平:r=0.989,P＜0.01;mRNA比血清水平:r=0.883,P＜0.05).胆道闭锁血清HSP47的表达与肝脏组织光镜下纤维化程度呈正相关关系(r=0.669,P＜0.01).结论 肝脏组织HSP47的表达与血清HSP47的表达水平具有一致性,能够评估肝脏纤维化程度;血清HSP47的高表达可能作为胆道闭锁预后不良的一个监测指标.     

Notch信号通路活化协同巨噬细胞对胆道闭锁肝纤维化的作用研究

目的探究Notch信号通路活化及相关炎性反应分子机制在胆道闭锁患者肝纤维化进展中的作用。方法选取2019年1月至2020年11月于天津市儿童医院确诊的20例胆道畸形患者(15例胆道闭锁,5例胆总管扩张)作为研究对象,收集临床资料及术中留取的肝活检组织样本,使用免疫组化方法检测患者肝组织内Notch信号通路的效应分子HES-1,M2型巨噬细胞标志物CD163及细胞因子IL-6、PDGF-AA的表达并进行半定量分析。结果与胆总管扩张组相比,HES-1(0.044±0.017 vs.0.143±0.027)、CD163(0.089±0.020 vs.0.259±0.111)、IL-6(0.070±0.007 vs.0.216±0.028)、PDGF-AA(0.155±0.019 vs.0.220±0.044)在胆道闭锁患者肝组织中的表达明显增加,且差异有统计学意义(P<0.05);HES-1的表达与CD163的表达呈正相关(r_s=0.687,P<0.01);HES-1、CD163、IL-6、PDGF-AA与肝纤维化程度成明显正相关(r_s=0.791,r_s=0.912,r_s=0.920,r_s=0.887,P<0.001)。结论Notch信号通路参与了胆道闭锁肝纤维化进程,并与M2型巨噬细胞有协同作用。     

胆道闭锁患者肝内胆管γδT细胞和调节性T细胞浸润及意义

目的研究胆道闭锁(Biliary atresia,BA)患者肝组织中γδT细胞和调节性T细胞(Foxp3+Treg)的比例变化。方法采用免疫组织化学方法和流式细胞术观察和检测胆道闭锁患儿组(BA组)23例和对照组(CG组)12例肝组织中γδT细胞分布情况以及γδT细胞和Foxp3~+Treg细胞比例关系。结果免疫组织化学染色显示BA组肝脏汇管区胆管周围有大量γδT细胞和一定程度的Foxp3~+Treg细胞浸润。流式细胞术显示胆道闭锁肝组织中γδT细胞与Foxp3~+Treg细胞比例明显高于对照组(P0.05),且γδTT细胞与Foxp3~+Treg细胞比例呈显著负相关(P0.05)。结论胆道闭锁患儿肝组织中γδT细胞增多,或抑制Foxp3~+Treg细胞增值,促进了胆管的进行性炎症损伤。     

胆道闭锁患儿肝组织细胞因子及相关基因表达的研究

目的 研究胆道闭锁(BA)患儿肝脏组织中上皮-间充质转化过程中特异性细胞因子的表达及胚胎期发育相关基因的表达激活.旨在探讨BA肝脏纤维化等系列改变的分子机制.方法 取经手术证实为BA患儿的8例肝活检组织(年龄2～3个月),以无消化道疾病尸检婴儿8例肝脏组织做正常对照(年龄0～2个月),RT-PCR半定量法检测胆管上皮细胞特异性因子CK19,纤维化早期指标Ⅰ型胶原蛋白COL1A1,发育相关基因Notch基因受体HES1,TGF-β及其阳性信号分子Smad3的表达水平变化.结果 BA患儿肝脏组织中CK-19,COL1A1、HES1、TGF-β及Smad3等基因表达水平均明显提高,实验组及对照组差异显著.结论 BA患儿肝脏组织胶原组织增生,同时有胆管上皮细胞增生,胚胎期肝脏组织发育相关基因Notch信号通路系统重新激活,TGF-β及其阳性信号分子Smad3在这一过程中发挥作用.     

新生儿肝外胆道囊性病变的诊断与治疗

目的了解新生儿肝外胆道囊性病变的不同类型及其临床特点,掌握其鉴别诊断的要点。方法分析9例新生儿肝外胆道囊性病变患儿的临床表现、肝功能、B超、CT、光镜、术中胆道造影的资料。结果本组胆道闭锁和新生儿胆总管囊肿女性多见,以梗阻性黄疸为主要表现,肝功能检查结果提示梗阻性黄疸。二者肝脏的组织学改变相似。但B超和CT检查发现,胆道闭锁组囊肿与胆囊大小均小于胆总管囊肿组。术中胆道造影显示,Ⅰ型胆道闭锁组肝内胆管变形、连续性中断;Ⅲ型胆道闭锁肝内胆管无法显示;胆总管囊肿组肝内胆管形态正常或略有被动扩张。结论多种胆道畸形都可以表现为新生儿肝外胆道囊肿,B超和术中胆道造影可以帮助鉴别这类疾病并且为治疗提供帮助。     

新生儿肝外胆道囊性病变的诊断与治疗

目的了解新生儿肝外胆道囊性病变的不同类型及其临床特点，掌握其鉴别诊断的要点。方法分析9例新生儿肝外胆道囊性病变患儿的临床表现、肝功能、B超、CT、光镜、术中胆道造影的资料。结果本组胆道闭锁和新生儿胆总管囊肿女性多见，以梗阻性黄疸为主要表现，肝功能检查结果提示梗阻性黄疸。二者肝脏的组织学改变相似。但B超和CT检查发现，胆道闭锁组囊肿与胆囊大小均小于胆总管囊肿组。术中胆道造影显示，Ⅰ型胆道闭锁组肝内胆管变形、连续性中断；Ⅲ型胆道闭锁肝内胆管无法显示；胆总管囊肿组肝内胆管形态正常或略有被动扩张。结论多种胆道畸形都可以表现为新生儿肝外胆道囊肿，B超和术中胆道造影可以帮助鉴别这类疾病并且为治疗提供帮助。     

Clinical and pathological characteristics of cystic lesions of extrahepatic bile duct in neonates

Aim: To investigate the differences in clinical and pathological manifestations between biliary atresia with extrahepatic biliary cyst and choledochal cyst in neonates. Methods: Symptoms and clinical signs in 5 neonates with biliary atresia with extrahepatic biliary cyst (4 of type I and 1 of type III) and 17 neonates with choledochal cyst were recorded. The levels of serum alkaline phosphatase, bilirubin, direct bilirubin, transaminase, gamma-glutamyl transpeptidase were analysed. Width and length of gallbladder and choledochal cyst were measured on B-mode ultrasound before surgery. Intrahepatic or extrahepatic biliary ducts were visualized with intraoperative cholangiography. The pathologic features in specimens of the liver were studied with light- and electron transmission microscopy. Results: All malformations occurred more commonly in girls, and obstructive jaundice was the main manifestation in both groups. Laboratory tests showed similar results for all patients in this study. With regard to pathological features, no significant difference was seen in either light microscopy or transmission electron microscopy, but it was shown with ultrasound that the length and width of the cysts and the gallbladder in neonates with biliary atresia were all shorter than the measurements in patients with choledochal cyst. The intrahepatic bile ducts could not be visualized on intraoperative cholangiography in type III biliary atresia. Deformation of the biliary ducts within the liver and stricture of the portal bile duct were the predominant features in type I biliary atresia, while the bile duct within the liver was normal or dilated in neonates with choledochal cyst.

Conclusions: Cystic lesions of the extrahepatic bile duct might be a common manifestation of biliary atresia and choledochal cyst. Intraoperative cholangiography is a precise and effective technique in the differential diagnosis of those lesions and helps decide on the most rational method of treatment.     

不同年龄胆总管囊肿的临床与肝纤维化特点探讨

目的 探讨不同年龄胆总管囊肿的临床与肝脏纤维化特点与相互关系。方法 分析12例婴儿及36例幼儿、儿童胆总管囊肿的临床资料;肝组织HE染色观察纤维化程度和炎性细胞浸润情况,免疫组化染色观察细胞角化蛋白(AE1/AE3)、人类组织相容性抗原DR(HLA—DR)表达,并与18例胆道闭锁作对照。结果 婴儿胆总管囊肿黄疸出现率12/12,显著高于幼儿儿童组8/36(P<0.01);肝纤维化程度、小叶周边AE1/AE3阳性细胞和HLA—DR在肝脏表达显著高于非婴儿组(P<0.01),低于胆道闭锁组(P<0.01)。肝纤维化程度与黄疸及肝小叶周边AE1/AE3表达分级呈正相关(P<0.05)。结论 婴儿胆总管囊肿以黄疸为主要临床表现,其肝纤维化重于幼儿及儿童组,胆道梗阻和胆小管增生可能是其发生肝纤维化的原因。     

Foregut atresias and bile duct anomalies: rare,infrequent or common?!

The association of foregut atresias and bile duct anomalies is reportedly rare. We encountered five referrals within 2 years where the secondary diagnosis was missed at operation. Four patients initially presented on antenatal scans as a foregut atresia whereas the fifth presented at nine years with abdominal pain due to a choledochal cyst. The biliary anomalies (cholecysto-hepatic duct, liver cyst and choledochal cysts) in the first four presented as postoperative jaundice during infancy whereas the fifth patient developed subacute intestinal obstruction due to congenital duodenal stenosis at fifteen years. In the patients with duodenal atresia neither did the preoperative X ray reveal any distal bowel gas nor did the subsequent intraoperative cholangiograms reveal bifid common bile duct or pancreato-biliary malunion. Atresias were corrected by primary repair (duodenoduodenostomy for congenital duodenal obstruction in four patients and disconnection/ligation of tracheo-oesophageal fistula with oesophageal anastomosis in one patient). The biliary anomalies were corrected by excision of the abnormal bile ducts (choledochal cyst/liver cyst/cholecystectomy) with Roux en Y hepaticojejunostomy. All patients are asymptomatic and liver function and biliary dilatation has normalised. The association of foregut atresias and bile duct anomalies is not as rare as previously reported. Antenatal ultrasound suggesting either a foregut or a biliary anomaly should alert one to the association. Full radiological and/or imaging investigation may be indicated prior to corrective surgery of the primary anomaly.     

Expression of osteopontin correlates with portal biliary proliferation and fibrosis in biliary atresia

The acquired or perinatal form of biliary atresia is a Th1 fibro-inflammatory disease affecting both the extrahepatic and intrahepatic bile ducts. Osteopontin (OPN) is a Th1 cytokine implicated in several fibro-inflammatory and autoimmune diseases. We examined the expression of OPN in acquired biliary atresia in comparison to normal liver and several pediatric cholestatic liver diseases. We also assessed OPN expression by cultured human bile duct epithelial cells. We found that liver OPN mRNA and protein expression were significantly increased in biliary atresia versus normal and other cholestatic diseases. OPN expression in biliary atresia was localized to epithelium of proliferating biliary structures (ductules and/or ducts) and bile plugs contained therein. No portal biliary OPN expression could be demonstrated in normal liver, syndromic biliary atresia, biliary obstruction not due to biliary atresia, and idiopathic neonatal hepatitis. OPN expression by human bile duct epithelial cells in culture was responsive to IL-2 and TNF-alpha. Our results demonstrate an up-regulation of OPN expression by interlobular biliary epithelium in biliary atresia, which correlates with biliary proliferation and portal fibrosis. These findings suggest a role for OPN in the pathogenesis of biliary atresia.     

Non-correctable biliary atresia with large extrahepatic cyst: a report of two cases.

Biliary atresia constitutes a serious problem in early infancy, due to the concomitant cholestasis. The conventional classification falls into 3 main types: type I, atresia of the common bile duct; type II, atresia of the hepatic duct; and type III, atresia of the porta hepatis. We report 2 unusual cases of biliary atresia type III with a cystic structure that could be mistaken for correctable biliary atresia or choledochal cyst.     

Hyaluronic acid: Additional biochemical marker in the diagnosis of biliary atresia

BACKGROUND: The purpose of the present paper was to evaluate the value of biochemical markers, including conventional liver function tests, gamma-glutamyl transferase (GGT), and hyaluronic acid (HA), in the diagnosis of neonatal cholestasis. METHODS: Infants with neonatal jaundice were consecutively enrolled during 1 year period. The patients were diagnosed as having biliary atresia (BA) if there was either bile ductular proliferation in the portal tracts, atretic common bile duct/gallbladder, or evidence of bile duct obstruction demonstrated by liver pathology or intraoperative cholangiography, respectively. Serum HA was measured using an enzyme-linked immunosorbent assay-based test. RESULTS: A total of 25 patients diagnosed as having BA (n = 10), neonatal hepatitis (NH; n = 9), choledochal cyst (n = 3) and parenteral nutrition-induced cholestasis (n = 3), were studied. The age at diagnosis was not significantly different between groups. Only GGT and HA were significantly elevated in the patients with BA when compared to NH (P = 0.02, P = 0.03, respectively). In BA, the median value of serum HA was 514 ng/mL (range 19-4476 ng/mL), compared to 50 ng/mL (range 19-315 ng/mL) in NH. Additionally, the serum HA level was much higher in children with choledochal cyst. CONCLUSION: HA could be considered as a complementary biochemical marker for evaluating infants with prolonged jaundice.     

Hepatic fibrosis with choledochal cyst in infants and children - an immunohistochemical assessment.

METHODS: The study population was divided into 4 groups: 12 infants with choledochal cyst, aged 4 m to 12 m, were classified as the infant choledochal cyst (ICC) group; 36 children, aged 1 y to 14 y, were classified as the children with choledochal cyst (CCC) group; while 18 patients, aged 2 m to 5 m, with biliary atresia (BA) were included as positive controls; and 14 infants, aged 1 d to 3 y, who died from non-liver diseases served as negative controls (CON). Liver specimens were examined using H&E sections to score fibrosis by means of Ohkuma's classification, and immunohistochemical sections were evaluated by counting the cells positive for cytokeratin (CK) and human leukocyte antigen-DR (HLA-DR) to discover the pathogenic factors of fibrosis. RESULTS: Most ICC patients had clinical biliary obstruction. The liver fibrosis score was highest in the BA group (2.9 +/- 0.7). The fibrosis score in the ICC group was higher (2.5 +/- 0.9) than that of the CCC (1.5 +/- 1.2; p < 0.05) and of the CON (0.1 +/- 0.4; p < 0.01) groups. The densities of CK-positive cells were 164 +/- 80/HP, 253 +/- 165/HP, 70 +/- 57/HP and 23 +/- 12/HP in the BA, ICC, CCC and CON groups, respectively, and differed significantly (p < 0.01) with the exception of the ICC vs. the BA group. The densities of HLA-DR positive cells were 130 +/- 72/HP, 98 +/- 54/HP, 96 +/- 50/HP and 36 +/- 13/HP in the portal area in the BA, ICC, CCC and CON groups, respectively. The density was lowest in the CON group (p < 0.01). CONCLUSION: In patients with choledochal cyst, liver fibrosis is more common and severe in infants than in children. Obstruction of the bile duct and proliferation of bile duct cells were the main pathogenic factors for fibrosis, while HLA-DR mediating immuno-injury may play a limited role.