胆道闭锁中LDOC1基因调控NF-κB介导炎症及凋亡反应的研究
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摘要
胆道闭锁(Biliary Atresia, BA)是引起3个月以内小儿阻塞性黄疸的主要原因,以肝内和肝外胆管进行性炎症、各级胆管梗阻和破坏为特征,从而导致胆汁淤积以及进行性肝纤维化和肝硬化。许多患儿往往在胆道闭锁确诊时已存在严重的肝纤维化,并且很快发展成为肝硬化,最终导致肝功能衰竭。即使成功进行了Kasia手术并重建胆汁流出道,仍然有68%～80%胆道闭锁患儿在手术后因为各级胆管进行性梗阻及炎症损伤,导致胆汁淤积和肝纤维化加重,进展为肝功能衰竭,最终需要接受肝脏移植。肝内胆道上皮细胞炎症损伤、凋亡和肝纤维化、肝硬化是胆道闭锁的主要病理改变,其发生和发展机制尚不明确。近年来,国内外研究发现NF-κ B介导的炎症和凋亡反应是重要的致病因素。基于基因芯片筛查结果,我们课题组前期发现,癌症亮氨酸拉链下调因子1(LDOC1)在胆道闭锁肝脏组织中高表达,生物信息学研究提示该基因对胆道闭锁的发病起核心调控作用。但其内在机制仍不十分清楚。因此本研究拟以LDOC1为切入点,深入探讨LDOC1与NF-κB介导的炎症及凋亡反应之间的内在关系及其在胆道闭锁发病中的作用,旨在阐明LDOC1在胆道闭锁发病和发展过程中的可能机制。
第一部分
猕猴轮状病毒(RRV)致胆道闭锁(BA) Balb/c小鼠模型的建立及LDOC1基因在模型中的表达
目的：应用RRV感染Balb/c新生小鼠建立胆道闭锁动物模型,并在模型中检测LDOC1表达及NF-κB介导的炎症和凋亡反应。
方法：感染组(n=31)新生小鼠出生后24小时内腹腔注射RRV (20μl106PFU);对照组(n=30)新生小鼠出相同时间腹腔注射20μl细胞培养液(EME)。生后5日内死亡或拒乳小鼠被排除出研究(RRV感染组中5只小鼠因拒乳或死亡被排除出研究,1只于第10日死亡,仅纳入生长发育研究,未纳入成模率研究。对照组中4只小鼠因拒乳或5日内死亡被排除出研究)。对比观察2组小鼠体重增长变化、无毛区黄染情况至第15天取材；取材后检测2组小鼠总胆红素(TBI)、直接胆红素(DBI)、谷丙转氨酶(AST)和谷草转氨酶(ALT)数值差异；HE染色分析小鼠肝内外病理变化；免疫组化(IHC)检测2组小鼠肝脏内LDOC1和NF-κB的表达差异；酶联免疫吸附试验(ELISA)检测小鼠血清NF-κB下游炎症因子(IL-2、TNF-α)蛋白含量变化；利用末端脱氧核苷酸原位标记法(TUNEL法)检测肝脏组织中细胞凋亡率改变。对RRV感染小鼠组内出现完全肝外闭锁小鼠和未出现完全肝外闭锁小鼠上述指标同样进行对比。
结果：
(1)对照组RRV感染组小鼠第5天平均体重较对照组降低(RRV感染组小鼠平均体重为3.3±0.7g,对照组平均为5.0±0.6g),但两者无统计学差异(p=0.075>0.05)。从第7天至15天取材时,两组小鼠体重出现显著性差异,其中第儿天差异最大(RRV感染组平均体重5.11±0.8,对照组平均为13.46±1.3g,p=0.017<0.05)。
(2)RRV感染组小鼠从第5天开始出现无毛区皮肤黄染,且TBI、DBI和AST、ALT水平与对照组相比显著升高(50.2±15.4umol/L vs14.5±3.8umol, p=0.002<0.01；45.9±15.6umol/L vs12.5umol/L,p=0.005<0.01；102±13U/L vs41±8U/L,p=0.005<0.01;99±16U/L vs38±8U/L, p=0.002<0.01);HE检查发现RRV感染组中68.8%小鼠出现完全性的肝外胆道闭锁,所有小鼠肝脏内均出现不同程度的炎症反应和肝内小胆管增生。
(3)免疫组化分析发现,RRV感染组小鼠肝脏内LDOC1和NF-κB抗体阳性表达率(64.0%、76.0%),与对照组(26.9%、34.6%)相比显著升高(p=0.008<0.01；p=0.003<0.01)。ELISA法检测RRV感染组小鼠血清IL-2和TNF-α蛋白光密度均值较对照组明显升高(1.52±0.21vs1.39±0.54,p=0.041<0.05；1.63±0.41vs0.79±0.09,p=0.002<0.01)；RRV感染组小鼠肝脏组织凋亡细胞比率与对照组相比显著降低(29.9±4.6%vs39.9±5.8%,p=0.02<0.05)；相关研究分析发现感染组内LDOC1和NF-κB抗体阳性表达存在相关性(p=0.004<0.01)
(4)RRV感染组小鼠中,出现肝外胆道闭锁的小鼠平均体重增长较未闭锁小鼠缓慢,第13天和15天平均体重存在显著性差异(5.6±0.3vs7.7±0.4,p=0.044<0.05；7.3±0.9vs8.6±1.2,p=0.048<0.05)；闭锁小鼠TBI、DBI较未闭锁小鼠显著升高(54.2±13.3umol/L vs39.7±4.4umol, p=0.022<0.01；49.9±15.6umol/L vs30.5±6.7umol/L, p=0.005(0.01):但AST、ALT无明显统计学差异(103±13U/L vs97±14U/L,p=0.059>0.05；106±16U/L vs98±11U/L,p=0.89>0.05)。
(5)肝外胆道闭锁小鼠肝组织LDOC1和NF-κ B抗体阳性表达率与未闭锁小鼠相比显著升高(75.0%vs33.3%,p=0.001<0.01；81.2%vs33.3%,p=0.001<0.01)。血清ELISA检测结果发现肝外胆道闭锁小鼠IL-2和TNF-α蛋白光密度均值高于未闭锁小鼠(1.66±0.21vs1.41±0.33,p=0.047<0.05；1.71±0.41vs1.39±0.28,p=0.038<0.05)；两者细胞凋亡率无统计性差异(40.3±5.9%vs37.8±4.8%,p=0.068>0.05)。
结论：
(1) RRV感染新生小鼠可成功制备出与人类胆道闭锁临床和病理相似的动物模型。
(2) NF-κB介导的炎症、凋亡反应在RRV感染致胆道闭锁动物模型中起重要作用。
(3) LDOC1在该动物模型中的高表达,并与NF-κB表达的相关性提示LDOC1可能通过调控NF-κB介导的炎症和凋亡反应对胆道闭锁的发生产生影响,提示其参与胆道闭锁形成的可能性。
第二部分LDOC1基因沉默在RRV致胆道闭锁小鼠模型中的作用研究
目的：腺病毒介导的RNA干扰(RNAi)方法沉默RRV感染致胆道闭锁小鼠肝脏组织内LDOC1基因的表达,研究LDOC1对NF-κB介导的炎症及凋亡反应是否具有调节作用,探索该基因沉默对模型小鼠的潜在治疗作用。
方法：构建LDOC1基因沉默的重组复制缺陷型腺病毒,对胆道闭锁动物模型进行干预。免疫荧光及Western-blot法明确肝脏组织中LDOC1基因的沉默情况。动物分3组：正常对照组(对照组n=26)；RRV+RNAi无效干扰组(MOCK组,n=28);LDOCl+RNAi组(LDOCl干扰组,n=29)。生后5日内死亡或拒乳小鼠被排除出研究。基因沉默后,观察各组小鼠生长发育和肝功能变化；免疫组化、RT-PCR及Western-blot检测NF-κB和TGF-β表达改变；ELISA检测血清炎症因子IL-2和TNF-α含量改变。并检测LDOC1基因沉默后肝组织内凋亡变化。
结果：
(1)免疫荧光显示LDOC1腺病毒干扰载体广泛分布于肝实质中,Western-blot检测发现,腺病毒载体转染后肝脏在内LDOC1蛋白表达显著下降(对照组,MOCK组、LDOC1干扰组中LDOC1蛋白相对表达量均值分别为0.83±0.02、0.74±0.03,0.23±0.02;LDOC1干扰组vs MOCK组,p=0.005<0.01；LDOC1干扰组vs对照组,p=0.003<0.01)。
(2) LDOC1干扰组小鼠各时间点平均体重均低于对照组；从第7天开始至第15天取材,2组小鼠平均体出现显著性差异(第7天：6.12±0.29g vs8.21±1.01,p=0.038<0.05；第15天：11.30±1.20vs15.90±1.23,p=0.012<0.05)；LDOC1干扰组从第9天开始平均体重(7.12±0.59)显著高于MOCK组平均体重(4.59±1.02,p=0.029<0.05),趋势持续至取材。
(3) LDOC1干扰组小鼠血清总胆红素(39.9±9.9umol/L)和直接胆红素(35.7±7.8umol/L)均显著高于对照组(13.1±2.1umol/L,p=0.004；7.2±2.1umol/L,p=0.003)；但低于MOCK组(88.1±11.2umol/L,P=0.001,76.0±12.3umol/L,p=0.001)；AST和ALT的检测结果显示出相同趋势。
(4)免疫组化检测发现LDOC1干扰组小鼠肝组织NF-κB抗体的阳性表达率明显低于MOCK组(54.1%vs76.0%,p=0.023<0.05),而与对照组相比无明显差异(54.1%vs34.6%,p=0.721>0.05)。RT-PCR检测发现,NF-κB mRNA在3组动物肝组中的相对表达量均值依次为：0.22±0.02、1.12±0.03、0.32±0.03,LDOC1干扰组显著低于MOCK组(p=0.002<0.05),但与对照组无统计学差异(p=0.61>0.05)。Western-blot检测发现,3组动物蛋白相对表达量也显示出相同的趋势：对照组,MOCK组、LDOC1干扰组中LDOC1蛋白相对表达量均值分别是0.68±0.02、1.23±0.03、0.71±0.02；LDOC1干扰组显著低于MOCK组(p=0.021<0.01),但与对照组无统计学差异(p=0.763>0.05)。
(5) LDOC1基因沉默后免疫组化检测发现,TGF-β抗体在对照组、MOCK组和LDOC1干扰组小鼠肝脏组织中的阳性表达率依次为：42.3%,88.0%和45.8%;LDOC1干扰组TGF-β阳性表达率低于MOCK组(p=0.031<0.05),与对照组无明显差异(p=0.643>0.05)。LDOC1干扰组TGF-β mRNA的相对表达量均值(0.49±0.03)低于MOCK组(1.07±0.03,p=0.005<0.01),LDOC1干扰组与对照组结果无统计学差异(0.52±0.03,p=0.751>0.05)。对照组,MOCK组、LDOC1干扰组中TGF-β蛋白相对表达量的均值分别是0.29±0.01、0.88±0.03、0.21±0.02;LDOC1干扰组显著低于MOCK(p=0.001<0.01),与对照组无统计学差异(p=0.549>0.05)。
(6)凋亡检测发现,LDOC1干扰组小鼠肝脏内细胞凋亡率均值为48.1±4.9%,显著高于MOCK组(37.7±5.1%,p=0.042<0.05),也高于对照组(25.2±3.6%,p=0.008<0.01)。
(7) ELISA法检测LDOC1干扰组小鼠血清IL-2、TNF-α蛋白光密度均值为分别为1.08±0.04和0.87±0.41,与MOCK组相比显著下降(1.58±0.14,p=O。036<0.05；1.49±0.11,p=0.015<0.05)。与对照组相比无显著性差异(1.12±0.02,p=0.628>0.05；0.79±0.01,p=0.71>0.05)。
结论：
(1)腺病毒介导的LDOC1-RNAi载体可有效抑制RRV致胆道闭锁动物模型肝脏组织内LDOC1基因表达。
(2) LDOC1基因沉默可减轻胆道闭锁模型小鼠的胆汁淤积程度和肝脏损害。
(3) LDOC1基因沉默显著抑制胆道闭锁模型小鼠NF-κB表达,下游炎症因子IL-2和TNF-a含量也显著减少,细胞凋亡率升高。
(4) LDOC1基因沉默下调了RRV感染致胆道闭锁动物模型肝脏组织中TGF-β的表达,对小鼠纤维化形成具有一定调节作用。
第三部分LDOC1基因转染人胆道上皮细胞(HIBEC)的研究
目的：通过构建LDOC1基因慢病毒表达载体,将LDOC1转染入人胆道上皮细胞系(HIBEC),观察转染后胆道上皮细胞的NF-κB及其下游炎症因子的表达改变、胆道上皮细胞凋亡率和TGF-β的表达改变。在体外细胞水平探讨LDOC1在胆道闭锁发病过程中的可能机制。
方法：细胞分为3组：正常对照组(对照组),空载体转染组(MOCK组)和LDOC1基因转染组(LDOC1转染组)。使用免疫荧光、Western-blot及RT-PCR等方法检测LDOC1基因转染效率及转染后各组细胞NF-κB、TGF-β的表达差异,Hochest/PI法检测各组细胞间凋亡率差异。ELISA法检测组织液上清IL-2、TNF-α含量变化。
结果：
(1)HIBEC在光镜下呈梭形或偏平梭形。转染后免疫荧光检测可见大部分胆道上皮细胞胞浆内均出现GFP绿色荧光蛋白表达；流式细胞检测结果显示转染效率为99.8%。
(2)慢病毒载体转染后,免疫荧光显示LDOC1转染组LDOC1抗体荧光表达率高达90%以上；对照组、MOCK组、LDOC1转染组细胞中LDOC1mRNA的相对表达量依次为：0.29,0.27,1.98；3组细胞LDOC1蛋白相对表达量分别为：0.31、0.27、0.99。表达水平高低趋势与mRNA水平一致,LDOC1转染组表达最高。
(3)转染后LDOC1转染组NF-kBp65阳性表达率超过80%,高于MOCK组和对照组(40%和30%)；对照组、MOCK组、LDOC1转染组细胞中核p65蛋白相对表达量依次为：0.52、0.49、1.02。
(4)免疫荧光检测显示,3组细胞TGF-β抗体荧光阳性表达率均为50%左右。且RT-PCR和Western-blot检测结果显示出相同的趋势(TGF-β mRNA的相对表达量依次为：0.98,0.99,0.89；蛋白相对表达量分别为：0.58、0.61、0.71)。
(5)对照组、MOCK组和LDOCl转染组细胞凋亡率分别为21.9±2.8%、22.3±3.1%、2.8±0.7%,LDOCl转染组凋亡率显著低于MOCK组(p=0.035,<0.05)以及对照组(p=0.034,<0.05)。
(6) LDOC1转染组培养液上清IL-2、TNF-α蛋白光密度均值分别为1.01±0.07和0.71±0.09,与MOCK组(0.56±0.02,p=0.002<0.01；0.41±0.02,p=0.001<0.01)和对照组(0.41±0.03,p=0.006<0.01,0.39±0.01,p=0.001<0.01)相比显著升高。
结论：
(1) LDOC1基因慢病毒表达载体,可成功地将LDOC1转染入人胆道上皮细胞：LDOC1转染组LDOC1抗体荧光表达率、mRNA表达量和蛋白表达量明显升高。
(2) LDOC1基因表达上调可以促进胆道上皮细胞NF-κB p65入核,激活下游炎症因子IL-2和TNF-α表达,同时可抑制细胞凋亡,但在体外细胞系中LDOC1基因表达上调对TGF-β影响不大。
(3)体外细胞系研究结果进一步提示LDOC1可能通过调节NF-κB相关的炎症和反应凋亡而参与胆道闭锁的形成。
Biliary atresia (BA) is characterized by a progressive sclerosis of the extrahepatic biliary tree and occurs only within the first3months of life. It is the most common cause of obstructive jaundice in infants and accounts for over half of children who undergo liver transplantation. The disease occurs more often in girls than in boys with an incidence of1in10,000to15,000neonates. The underlying pathogenetic factors of this disease have so far not been fully clarified. A variety of prenatal or perinatal insults to the biliary tree may culminate in complete obliteration of the lumen of the extrahepatic biliary tract and also lead to progressive inflammatory injury, apoptosis and sclerosis of intrahepatic bile ducts, even after successful Kasai procedure.
For many years, there is substantial evidence that rotavirus play a key role in the pathogenesis of BA. NF-κ B protein are sequence-specific transcription faction that control a variety of important biologic decisions including the activation of inflammatory and innate immune response to the virus infection. Many researchers have indicated that after infection of RRV, abnormal expression of nuclear factor-K B(NF-κ B) play a very important role in the progress of duct damage in BA. Microarray screen in our previous research has found leucine zipper down-regulated in cancer1(LDOC1) express the most significant difference between BA liver tissue and control liver tissue. We also found the high expression of LDOC1in liver tissue of BA. Some researchers have shown that LDOC1is a novel regulator of NF-κ B and can affect the inflammation and apoptosis mediated by NF-κB. The aim of current paper is to study the relationship between LDOC1and inflammation/apoptosis mediated by NF-κB and educate the role of LDOC1in the pathogenesis.
Part One Expression of LDOC1in Rotavirus-induced Murine Biliary Atrsia
Aim:Intraperitoneal inoculation of rhesus rotavirus(RRV) was show to cause jaundice and atretic-appearing segments of extraphatic ducts in a murine model. The expression of LDOC1and inflammatory and apoptosis mediated by NF-κ B were investigated.
Methods:Within the first24hours of life,31the newborn mice were infected through intraperitoneal route with a volume of20μl containing106PFU RRV. Pup's body weight were recorded on day5,7,9,11,13and15after inoculation. The pups were sacrificed on day15, vaule of total bilirubin(TBI), direct bilirubin(DBI), AST(aspartate transaminase) and ALT(alaine transaminase) were tested and consecutive sections of liver specimens were stained with H&E for histopahologic analysis. The methods of immnohistochemical(IHC) staining was used to detect the expression of LDOC1and NF-κ B. The content of IL-2and TNF-α in serum and the apoptosis were detected by ELISA and TUNEL respectively.
Results:
(1) Five days after inoculation, average body weight in the pups with RRV infection is lower than those without RRV infection. However, there exist no significant difference between them(3.46±1.3g VS5.11±0.8, p=0.017<0.05). Significant diffidence were found from days7to days15(p<0.05).
(2) The pups with RRV infection had a higher level of TBI, DBI, AST and ALT comparing with those without RRV infection.(50.2±15.4umol/L VS14.5±3.8, p=0.002<0.01;45.9±15.6umol/L VS12.5umol/L, p=0.005<0.01;102±13U/L VS41±8U/L, p=0.005<0.01;99±16U/L VS38±8U/L, p=0.002<0.01;). In H&E examine,16(68.8%) pups appear total atretic of extrahepatic duct and all pups show inflammation and hyperplastic of intrahepatic duct.
(3) Under IHC staining, the liver tissue of RRV infected pups show higher expression of LDOC1and NF-κ B comparing with those in anther group. It also happened between pups with or without atretic-appearing segments.
Conclusion:
(1) Intraperitoneal inoculation with RRV can produce biliary atresia in new born balb/c mice.
(2) Inflammatory and apoptosis mediated by NF-κB play a very important role in the RRV-induced murine atresia biliary.
(3) The high expression of LDOC1indicated that it may be involved in pathogenesis of biliary atresia.
(4) The correlation between LDOC1and NF-κ B suggest that LDOC1may control the expression the pathogenesis of BA.
Part Two
The Effects of Gene Silence of LDOC1on Biological Characteristics of RRV-induced Murine Biliary Atresia
Aim:In order to investigate the relationship between LDOC1and NF-κB, adenovirus-delivered shRNA was used to silencing the expression of LDOC1in RRV-induced murine biliray atresia. The expression of LDOC1and inflammation and apoptosis mediated by NF-κ B were studied.
Methods:After constructing adenovirus-delivered shRNA and intervene the expression of LDOC1in RRV-induced murine biliary atresia. Three groups of pups were involved in this part:control group(n=26), MOCK group(n=28) and LDOC1siRNA group(n=29). Body weight and liver function were investigated. Methods of IHC, RT-PCR and western-blot were used to study the expression of NF-κ B and TGF-β. Inflammatory factors and apoptosis were also investigated in this section.
Results:
(1) Immunofluorescence examine shows that GTP existed uniformly in the liver. Western-blot test indicated that LDOC1protein expression were greatly inhibited(the relative protein content of control group, MOCK group and LDC1siRNA group is0.83±0.02.0.74±0.03.0.23±0.02respectively;LDOC1siRNA VS MOCK group, p=0.005<0.01; LDOC1siRNA group VS control, p=0.003<0.01).
(2) On days7, the average body weight (6.12±0.29g) in LDOC1siRNA group is lower than those in control group(8.21±1.01, p=0.038<0.05) and showed the same tendency. However, from days9, the body weight in this group is much higher than those in MOCK group.
(3) After the silence of LDOC1, comparing with MOCK group, expression of NF-κ B tested by IHC decreased greatly in LODC1siRNA group.(54.1%VS76.0%, p=0.023<0.05). There showed no difference between LODC1siRNA group and control group.(54.1%VS34.6%, p=0.721>0.05) The mRNA level of NF-κB in the three groups were0.22±0.02.1.12±0.03.0.32±0.03. the mRNA level in LDOC1group is lower than those in MOCK group.(p=0.002<0.05) and no apparent difference with control group(p=0.61>0.05). Same results were found in Western-blot test, the relative protein content is much lower than those in MOCK group(0.71±0.02VS1.23±0.03, p=0.02<0.01) and very close to control group.(0.71±0.02VS0.68±0.02, p=0.763>0.05).
(4) Positive express of TGF-β tested by IHC in three group were42.3%,88.0%and45.8%respectively. Positive rate in LDOC1siRNA group was lower than those in MOCK group(p=0.0.031<0.05)and showed no difference with control group.(p=0.643>0.05). The mRNA level of TGF-β in the three groups were0.52±0.03,1.07±0.03and0.49±0.03. The mRNA level in LDOC1siRNA group is lower than those in MOCK group.(p=0.005<0.01) and no apparent difference with control group(p=0.751>0.05). Same results were found in Western-blot test, the relative protein content is much lower than those in MOCK group(0.21±0.02VS0.88±0.03, p=0.001<0.01) and very close to control group.(0.21±0.02VS0.29±0.01, p=0.001<0.01).
(5) The apoptosis rate in LDOC1siRNA group is higher than those in MOCK group (48.1±4.9%VS37.7±5.1%, p=0.042<0.05) and control group(48.1±4.9%VS25.2±3.6%, p=0.008<0.01).
(6) The relative optical density of IL-2and TNF-α were1.08±0.04and0.87±0.41and lower than those in MOCK group (1.58±0.14, p=0.036<0.05;1.49±0.11, p=0.015<0.05). However, they were very similar with those in control group.(1.12±0.02, p=0.628>0.05;0.79±0.01, p=0.71>0.05)
Conclusion:
(1) Adenovirus-delivered shRNA can effectively inhibited the expression of LDOC1in liver tissue.
(2) Silence of LDOC1can abate the cholestasis and damage in murine biliray atresia.
(3) Silence of LDOC1can greatly lower the expression of NF-κ B and inflammatory factors mediated by NF-κ B. It also promotes the level of apoptosis in liver tissue.
(4) Silence of LDOC1can potentially inhibits the formation of liver fibrosis by down-regulating the expression of TGF-β.
Part Three
Effect of gene transfection of LDOC1on Human Intrahepatic Biliary-epithelial cells(HIBEC)
Aim:In order to further investigate the relation between LDOC1and NF-κ B, a lentivirus vector was used to upregulate the expression of LDOC1in HIBEC. The expression of LDOC1and inflammatory and apoptosis mediated by NF-κ B were studied.
Methods:HIBEC were divided into three groups:control group, MOCK group and LDOC1tansfection group. Methods of Immunofluorescence, RT-PCR,Western-blot and flow cytometry were used to investigate the effectiveness of transfenction and different expression of NF-κ B and TGF-β in three groups. Apoptosis was detected by Hochest/PI staining. IL-2and TNF-α were studied by ELISA.
Results
(1) Green fluorescence of GFP was detected in majority of Cytoplasm and transfection efficiency was99.8%by examine of flow cytometry.
(2) After transfection of lentivirus, more than90%cell in LDOC1transfection group show red fluorescence of LDOC1antibody;LDOC1transfection group had the higher expression of LDOC1comparing with the other two groups. LDOC1mRNA expression in three group were0.29,0.27and1.98respectively. There existed same tendency in the test of Western-blot.(0.3、0.27、0.99).
(3) More than80%HIBEC in LDOC1transfection group had the expression of NF-kB p65, higher than other two groups.(40%,30%) Relative protein content in three groups were0.52、0.49、1.02respectively.
(4) In three groups, expression of TGF-β was very close (50%) and results of RT-PCR and Western-blot showed the same tendency.(mRNA:0.98,0.99,0.89; protein:0.58、0.61、0.71)
(5) Apoptosis rates in three groups were21.9±2.8%、22.3±3.1%、2.8±0.7%respectively. Apoptosis rate in LDOC1transfection group was much lower than those in other two groups.(p=0.035<0.05, p=0.034<0.05)
(6) In ELISA test, the relative optical density of IL-2and TNF-α in LDOC1transfection group were1.01±0.0and0.71±0.09. Relative optical density value in LDOC1group were much higher than other two groups (IL-2:0.56±0.02, p=0.002<0.01;0.41±0.02, p=0.001<0.01; TNF-α:0.41±0.03, p=0.006<0.01,0.39±0.01, p=0.001<0.01)
Conclusion:
(1) Up-regulation of LDOC1in HIBEC can increase the expression of NF-κB p65, promote the activation of IL-2and TNF-α, and inhibit cell apoptosis.
(2) According to the results in all three parts, LDOC1may play a key role in the pathogenesis of BA.
第一部分
猕猴轮状病毒(RRV)致胆道闭锁(BA) Balb/c小鼠模型的建立及LDOC1基因在模型中的表达
目的：应用RRV感染Balb/c新生小鼠建立胆道闭锁动物模型,并在模型中检测LDOC1表达及NF-κB介导的炎症和凋亡反应。
方法：感染组(n=31)新生小鼠出生后24小时内腹腔注射RRV (20μl106PFU);对照组(n=30)新生小鼠出相同时间腹腔注射20μl细胞培养液(EME)。生后5日内死亡或拒乳小鼠被排除出研究(RRV感染组中5只小鼠因拒乳或死亡被排除出研究,1只于第10日死亡,仅纳入生长发育研究,未纳入成模率研究。对照组中4只小鼠因拒乳或5日内死亡被排除出研究)。对比观察2组小鼠体重增长变化、无毛区黄染情况至第15天取材；取材后检测2组小鼠总胆红素(TBI)、直接胆红素(DBI)、谷丙转氨酶(AST)和谷草转氨酶(ALT)数值差异；HE染色分析小鼠肝内外病理变化；免疫组化(IHC)检测2组小鼠肝脏内LDOC1和NF-κB的表达差异；酶联免疫吸附试验(ELISA)检测小鼠血清NF-κB下游炎症因子(IL-2、TNF-α)蛋白含量变化；利用末端脱氧核苷酸原位标记法(TUNEL法)检测肝脏组织中细胞凋亡率改变。对RRV感染小鼠组内出现完全肝外闭锁小鼠和未出现完全肝外闭锁小鼠上述指标同样进行对比。
结果：
(1)对照组RRV感染组小鼠第5天平均体重较对照组降低(RRV感染组小鼠平均体重为3.3±0.7g,对照组平均为5.0±0.6g),但两者无统计学差异(p=0.075>0.05)。从第7天至15天取材时,两组小鼠体重出现显著性差异,其中第儿天差异最大(RRV感染组平均体重5.11±0.8,对照组平均为13.46±1.3g,p=0.017<0.05)。
(2)RRV感染组小鼠从第5天开始出现无毛区皮肤黄染,且TBI、DBI和AST、ALT水平与对照组相比显著升高(50.2±15.4umol/L vs14.5±3.8umol, p=0.002<0.01；45.9±15.6umol/L vs12.5umol/L,p=0.005<0.01；102±13U/L vs41±8U/L,p=0.005<0.01;99±16U/L vs38±8U/L, p=0.002<0.01);HE检查发现RRV感染组中68.8%小鼠出现完全性的肝外胆道闭锁,所有小鼠肝脏内均出现不同程度的炎症反应和肝内小胆管增生。
(3)免疫组化分析发现,RRV感染组小鼠肝脏内LDOC1和NF-κB抗体阳性表达率(64.0%、76.0%),与对照组(26.9%、34.6%)相比显著升高(p=0.008<0.01；p=0.003<0.01)。ELISA法检测RRV感染组小鼠血清IL-2和TNF-α蛋白光密度均值较对照组明显升高(1.52±0.21vs1.39±0.54,p=0.041<0.05；1.63±0.41vs0.79±0.09,p=0.002<0.01)；RRV感染组小鼠肝脏组织凋亡细胞比率与对照组相比显著降低(29.9±4.6%vs39.9±5.8%,p=0.02<0.05)；相关研究分析发现感染组内LDOC1和NF-κB抗体阳性表达存在相关性(p=0.004<0.01)
(4)RRV感染组小鼠中,出现肝外胆道闭锁的小鼠平均体重增长较未闭锁小鼠缓慢,第13天和15天平均体重存在显著性差异(5.6±0.3vs7.7±0.4,p=0.044<0.05；7.3±0.9vs8.6±1.2,p=0.048<0.05)；闭锁小鼠TBI、DBI较未闭锁小鼠显著升高(54.2±13.3umol/L vs39.7±4.4umol, p=0.022<0.01；49.9±15.6umol/L vs30.5±6.7umol/L, p=0.005(0.01):但AST、ALT无明显统计学差异(103±13U/L vs97±14U/L,p=0.059>0.05；106±16U/L vs98±11U/L,p=0.89>0.05)。
(5)肝外胆道闭锁小鼠肝组织LDOC1和NF-κ B抗体阳性表达率与未闭锁小鼠相比显著升高(75.0%vs33.3%,p=0.001<0.01；81.2%vs33.3%,p=0.001<0.01)。血清ELISA检测结果发现肝外胆道闭锁小鼠IL-2和TNF-α蛋白光密度均值高于未闭锁小鼠(1.66±0.21vs1.41±0.33,p=0.047<0.05；1.71±0.41vs1.39±0.28,p=0.038<0.05)；两者细胞凋亡率无统计性差异(40.3±5.9%vs37.8±4.8%,p=0.068>0.05)。
结论：
(1) RRV感染新生小鼠可成功制备出与人类胆道闭锁临床和病理相似的动物模型。
(2) NF-κB介导的炎症、凋亡反应在RRV感染致胆道闭锁动物模型中起重要作用。
(3) LDOC1在该动物模型中的高表达,并与NF-κB表达的相关性提示LDOC1可能通过调控NF-κB介导的炎症和凋亡反应对胆道闭锁的发生产生影响,提示其参与胆道闭锁形成的可能性。
第二部分LDOC1基因沉默在RRV致胆道闭锁小鼠模型中的作用研究
目的：腺病毒介导的RNA干扰(RNAi)方法沉默RRV感染致胆道闭锁小鼠肝脏组织内LDOC1基因的表达,研究LDOC1对NF-κB介导的炎症及凋亡反应是否具有调节作用,探索该基因沉默对模型小鼠的潜在治疗作用。
方法：构建LDOC1基因沉默的重组复制缺陷型腺病毒,对胆道闭锁动物模型进行干预。免疫荧光及Western-blot法明确肝脏组织中LDOC1基因的沉默情况。动物分3组：正常对照组(对照组n=26)；RRV+RNAi无效干扰组(MOCK组,n=28);LDOCl+RNAi组(LDOCl干扰组,n=29)。生后5日内死亡或拒乳小鼠被排除出研究。基因沉默后,观察各组小鼠生长发育和肝功能变化；免疫组化、RT-PCR及Western-blot检测NF-κB和TGF-β表达改变；ELISA检测血清炎症因子IL-2和TNF-α含量改变。并检测LDOC1基因沉默后肝组织内凋亡变化。
结果：
(1)免疫荧光显示LDOC1腺病毒干扰载体广泛分布于肝实质中,Western-blot检测发现,腺病毒载体转染后肝脏在内LDOC1蛋白表达显著下降(对照组,MOCK组、LDOC1干扰组中LDOC1蛋白相对表达量均值分别为0.83±0.02、0.74±0.03,0.23±0.02;LDOC1干扰组vs MOCK组,p=0.005<0.01；LDOC1干扰组vs对照组,p=0.003<0.01)。
(2) LDOC1干扰组小鼠各时间点平均体重均低于对照组；从第7天开始至第15天取材,2组小鼠平均体出现显著性差异(第7天：6.12±0.29g vs8.21±1.01,p=0.038<0.05；第15天：11.30±1.20vs15.90±1.23,p=0.012<0.05)；LDOC1干扰组从第9天开始平均体重(7.12±0.59)显著高于MOCK组平均体重(4.59±1.02,p=0.029<0.05),趋势持续至取材。
(3) LDOC1干扰组小鼠血清总胆红素(39.9±9.9umol/L)和直接胆红素(35.7±7.8umol/L)均显著高于对照组(13.1±2.1umol/L,p=0.004；7.2±2.1umol/L,p=0.003)；但低于MOCK组(88.1±11.2umol/L,P=0.001,76.0±12.3umol/L,p=0.001)；AST和ALT的检测结果显示出相同趋势。
(4)免疫组化检测发现LDOC1干扰组小鼠肝组织NF-κB抗体的阳性表达率明显低于MOCK组(54.1%vs76.0%,p=0.023<0.05),而与对照组相比无明显差异(54.1%vs34.6%,p=0.721>0.05)。RT-PCR检测发现,NF-κB mRNA在3组动物肝组中的相对表达量均值依次为：0.22±0.02、1.12±0.03、0.32±0.03,LDOC1干扰组显著低于MOCK组(p=0.002<0.05),但与对照组无统计学差异(p=0.61>0.05)。Western-blot检测发现,3组动物蛋白相对表达量也显示出相同的趋势：对照组,MOCK组、LDOC1干扰组中LDOC1蛋白相对表达量均值分别是0.68±0.02、1.23±0.03、0.71±0.02；LDOC1干扰组显著低于MOCK组(p=0.021<0.01),但与对照组无统计学差异(p=0.763>0.05)。
(5) LDOC1基因沉默后免疫组化检测发现,TGF-β抗体在对照组、MOCK组和LDOC1干扰组小鼠肝脏组织中的阳性表达率依次为：42.3%,88.0%和45.8%;LDOC1干扰组TGF-β阳性表达率低于MOCK组(p=0.031<0.05),与对照组无明显差异(p=0.643>0.05)。LDOC1干扰组TGF-β mRNA的相对表达量均值(0.49±0.03)低于MOCK组(1.07±0.03,p=0.005<0.01),LDOC1干扰组与对照组结果无统计学差异(0.52±0.03,p=0.751>0.05)。对照组,MOCK组、LDOC1干扰组中TGF-β蛋白相对表达量的均值分别是0.29±0.01、0.88±0.03、0.21±0.02;LDOC1干扰组显著低于MOCK(p=0.001<0.01),与对照组无统计学差异(p=0.549>0.05)。
(6)凋亡检测发现,LDOC1干扰组小鼠肝脏内细胞凋亡率均值为48.1±4.9%,显著高于MOCK组(37.7±5.1%,p=0.042<0.05),也高于对照组(25.2±3.6%,p=0.008<0.01)。
(7) ELISA法检测LDOC1干扰组小鼠血清IL-2、TNF-α蛋白光密度均值为分别为1.08±0.04和0.87±0.41,与MOCK组相比显著下降(1.58±0.14,p=O。036<0.05；1.49±0.11,p=0.015<0.05)。与对照组相比无显著性差异(1.12±0.02,p=0.628>0.05；0.79±0.01,p=0.71>0.05)。
结论：
(1)腺病毒介导的LDOC1-RNAi载体可有效抑制RRV致胆道闭锁动物模型肝脏组织内LDOC1基因表达。
(2) LDOC1基因沉默可减轻胆道闭锁模型小鼠的胆汁淤积程度和肝脏损害。
(3) LDOC1基因沉默显著抑制胆道闭锁模型小鼠NF-κB表达,下游炎症因子IL-2和TNF-a含量也显著减少,细胞凋亡率升高。
(4) LDOC1基因沉默下调了RRV感染致胆道闭锁动物模型肝脏组织中TGF-β的表达,对小鼠纤维化形成具有一定调节作用。
第三部分LDOC1基因转染人胆道上皮细胞(HIBEC)的研究
目的：通过构建LDOC1基因慢病毒表达载体,将LDOC1转染入人胆道上皮细胞系(HIBEC),观察转染后胆道上皮细胞的NF-κB及其下游炎症因子的表达改变、胆道上皮细胞凋亡率和TGF-β的表达改变。在体外细胞水平探讨LDOC1在胆道闭锁发病过程中的可能机制。
方法：细胞分为3组：正常对照组(对照组),空载体转染组(MOCK组)和LDOC1基因转染组(LDOC1转染组)。使用免疫荧光、Western-blot及RT-PCR等方法检测LDOC1基因转染效率及转染后各组细胞NF-κB、TGF-β的表达差异,Hochest/PI法检测各组细胞间凋亡率差异。ELISA法检测组织液上清IL-2、TNF-α含量变化。
结果：
(1)HIBEC在光镜下呈梭形或偏平梭形。转染后免疫荧光检测可见大部分胆道上皮细胞胞浆内均出现GFP绿色荧光蛋白表达；流式细胞检测结果显示转染效率为99.8%。
(2)慢病毒载体转染后,免疫荧光显示LDOC1转染组LDOC1抗体荧光表达率高达90%以上；对照组、MOCK组、LDOC1转染组细胞中LDOC1mRNA的相对表达量依次为：0.29,0.27,1.98；3组细胞LDOC1蛋白相对表达量分别为：0.31、0.27、0.99。表达水平高低趋势与mRNA水平一致,LDOC1转染组表达最高。
(3)转染后LDOC1转染组NF-kBp65阳性表达率超过80%,高于MOCK组和对照组(40%和30%)；对照组、MOCK组、LDOC1转染组细胞中核p65蛋白相对表达量依次为：0.52、0.49、1.02。
(4)免疫荧光检测显示,3组细胞TGF-β抗体荧光阳性表达率均为50%左右。且RT-PCR和Western-blot检测结果显示出相同的趋势(TGF-β mRNA的相对表达量依次为：0.98,0.99,0.89；蛋白相对表达量分别为：0.58、0.61、0.71)。
(5)对照组、MOCK组和LDOCl转染组细胞凋亡率分别为21.9±2.8%、22.3±3.1%、2.8±0.7%,LDOCl转染组凋亡率显著低于MOCK组(p=0.035,<0.05)以及对照组(p=0.034,<0.05)。
(6) LDOC1转染组培养液上清IL-2、TNF-α蛋白光密度均值分别为1.01±0.07和0.71±0.09,与MOCK组(0.56±0.02,p=0.002<0.01；0.41±0.02,p=0.001<0.01)和对照组(0.41±0.03,p=0.006<0.01,0.39±0.01,p=0.001<0.01)相比显著升高。
结论：
(1) LDOC1基因慢病毒表达载体,可成功地将LDOC1转染入人胆道上皮细胞：LDOC1转染组LDOC1抗体荧光表达率、mRNA表达量和蛋白表达量明显升高。
(2) LDOC1基因表达上调可以促进胆道上皮细胞NF-κB p65入核,激活下游炎症因子IL-2和TNF-α表达,同时可抑制细胞凋亡,但在体外细胞系中LDOC1基因表达上调对TGF-β影响不大。
(3)体外细胞系研究结果进一步提示LDOC1可能通过调节NF-κB相关的炎症和反应凋亡而参与胆道闭锁的形成。
Biliary atresia (BA) is characterized by a progressive sclerosis of the extrahepatic biliary tree and occurs only within the first3months of life. It is the most common cause of obstructive jaundice in infants and accounts for over half of children who undergo liver transplantation. The disease occurs more often in girls than in boys with an incidence of1in10,000to15,000neonates. The underlying pathogenetic factors of this disease have so far not been fully clarified. A variety of prenatal or perinatal insults to the biliary tree may culminate in complete obliteration of the lumen of the extrahepatic biliary tract and also lead to progressive inflammatory injury, apoptosis and sclerosis of intrahepatic bile ducts, even after successful Kasai procedure.
For many years, there is substantial evidence that rotavirus play a key role in the pathogenesis of BA. NF-κ B protein are sequence-specific transcription faction that control a variety of important biologic decisions including the activation of inflammatory and innate immune response to the virus infection. Many researchers have indicated that after infection of RRV, abnormal expression of nuclear factor-K B(NF-κ B) play a very important role in the progress of duct damage in BA. Microarray screen in our previous research has found leucine zipper down-regulated in cancer1(LDOC1) express the most significant difference between BA liver tissue and control liver tissue. We also found the high expression of LDOC1in liver tissue of BA. Some researchers have shown that LDOC1is a novel regulator of NF-κ B and can affect the inflammation and apoptosis mediated by NF-κB. The aim of current paper is to study the relationship between LDOC1and inflammation/apoptosis mediated by NF-κB and educate the role of LDOC1in the pathogenesis.
Part One Expression of LDOC1in Rotavirus-induced Murine Biliary Atrsia
Aim:Intraperitoneal inoculation of rhesus rotavirus(RRV) was show to cause jaundice and atretic-appearing segments of extraphatic ducts in a murine model. The expression of LDOC1and inflammatory and apoptosis mediated by NF-κ B were investigated.
Methods:Within the first24hours of life,31the newborn mice were infected through intraperitoneal route with a volume of20μl containing106PFU RRV. Pup's body weight were recorded on day5,7,9,11,13and15after inoculation. The pups were sacrificed on day15, vaule of total bilirubin(TBI), direct bilirubin(DBI), AST(aspartate transaminase) and ALT(alaine transaminase) were tested and consecutive sections of liver specimens were stained with H&E for histopahologic analysis. The methods of immnohistochemical(IHC) staining was used to detect the expression of LDOC1and NF-κ B. The content of IL-2and TNF-α in serum and the apoptosis were detected by ELISA and TUNEL respectively.
Results:
(1) Five days after inoculation, average body weight in the pups with RRV infection is lower than those without RRV infection. However, there exist no significant difference between them(3.46±1.3g VS5.11±0.8, p=0.017<0.05). Significant diffidence were found from days7to days15(p<0.05).
(2) The pups with RRV infection had a higher level of TBI, DBI, AST and ALT comparing with those without RRV infection.(50.2±15.4umol/L VS14.5±3.8, p=0.002<0.01;45.9±15.6umol/L VS12.5umol/L, p=0.005<0.01;102±13U/L VS41±8U/L, p=0.005<0.01;99±16U/L VS38±8U/L, p=0.002<0.01;). In H&E examine,16(68.8%) pups appear total atretic of extrahepatic duct and all pups show inflammation and hyperplastic of intrahepatic duct.
(3) Under IHC staining, the liver tissue of RRV infected pups show higher expression of LDOC1and NF-κ B comparing with those in anther group. It also happened between pups with or without atretic-appearing segments.
Conclusion:
(1) Intraperitoneal inoculation with RRV can produce biliary atresia in new born balb/c mice.
(2) Inflammatory and apoptosis mediated by NF-κB play a very important role in the RRV-induced murine atresia biliary.
(3) The high expression of LDOC1indicated that it may be involved in pathogenesis of biliary atresia.
(4) The correlation between LDOC1and NF-κ B suggest that LDOC1may control the expression the pathogenesis of BA.
Part Two
The Effects of Gene Silence of LDOC1on Biological Characteristics of RRV-induced Murine Biliary Atresia
Aim:In order to investigate the relationship between LDOC1and NF-κB, adenovirus-delivered shRNA was used to silencing the expression of LDOC1in RRV-induced murine biliray atresia. The expression of LDOC1and inflammation and apoptosis mediated by NF-κ B were studied.
Methods:After constructing adenovirus-delivered shRNA and intervene the expression of LDOC1in RRV-induced murine biliary atresia. Three groups of pups were involved in this part:control group(n=26), MOCK group(n=28) and LDOC1siRNA group(n=29). Body weight and liver function were investigated. Methods of IHC, RT-PCR and western-blot were used to study the expression of NF-κ B and TGF-β. Inflammatory factors and apoptosis were also investigated in this section.
Results:
(1) Immunofluorescence examine shows that GTP existed uniformly in the liver. Western-blot test indicated that LDOC1protein expression were greatly inhibited(the relative protein content of control group, MOCK group and LDC1siRNA group is0.83±0.02.0.74±0.03.0.23±0.02respectively;LDOC1siRNA VS MOCK group, p=0.005<0.01; LDOC1siRNA group VS control, p=0.003<0.01).
(2) On days7, the average body weight (6.12±0.29g) in LDOC1siRNA group is lower than those in control group(8.21±1.01, p=0.038<0.05) and showed the same tendency. However, from days9, the body weight in this group is much higher than those in MOCK group.
(3) After the silence of LDOC1, comparing with MOCK group, expression of NF-κ B tested by IHC decreased greatly in LODC1siRNA group.(54.1%VS76.0%, p=0.023<0.05). There showed no difference between LODC1siRNA group and control group.(54.1%VS34.6%, p=0.721>0.05) The mRNA level of NF-κB in the three groups were0.22±0.02.1.12±0.03.0.32±0.03. the mRNA level in LDOC1group is lower than those in MOCK group.(p=0.002<0.05) and no apparent difference with control group(p=0.61>0.05). Same results were found in Western-blot test, the relative protein content is much lower than those in MOCK group(0.71±0.02VS1.23±0.03, p=0.02<0.01) and very close to control group.(0.71±0.02VS0.68±0.02, p=0.763>0.05).
(4) Positive express of TGF-β tested by IHC in three group were42.3%,88.0%and45.8%respectively. Positive rate in LDOC1siRNA group was lower than those in MOCK group(p=0.0.031<0.05)and showed no difference with control group.(p=0.643>0.05). The mRNA level of TGF-β in the three groups were0.52±0.03,1.07±0.03and0.49±0.03. The mRNA level in LDOC1siRNA group is lower than those in MOCK group.(p=0.005<0.01) and no apparent difference with control group(p=0.751>0.05). Same results were found in Western-blot test, the relative protein content is much lower than those in MOCK group(0.21±0.02VS0.88±0.03, p=0.001<0.01) and very close to control group.(0.21±0.02VS0.29±0.01, p=0.001<0.01).
(5) The apoptosis rate in LDOC1siRNA group is higher than those in MOCK group (48.1±4.9%VS37.7±5.1%, p=0.042<0.05) and control group(48.1±4.9%VS25.2±3.6%, p=0.008<0.01).
(6) The relative optical density of IL-2and TNF-α were1.08±0.04and0.87±0.41and lower than those in MOCK group (1.58±0.14, p=0.036<0.05;1.49±0.11, p=0.015<0.05). However, they were very similar with those in control group.(1.12±0.02, p=0.628>0.05;0.79±0.01, p=0.71>0.05)
Conclusion:
(1) Adenovirus-delivered shRNA can effectively inhibited the expression of LDOC1in liver tissue.
(2) Silence of LDOC1can abate the cholestasis and damage in murine biliray atresia.
(3) Silence of LDOC1can greatly lower the expression of NF-κ B and inflammatory factors mediated by NF-κ B. It also promotes the level of apoptosis in liver tissue.
(4) Silence of LDOC1can potentially inhibits the formation of liver fibrosis by down-regulating the expression of TGF-β.
Part Three
Effect of gene transfection of LDOC1on Human Intrahepatic Biliary-epithelial cells(HIBEC)
Aim:In order to further investigate the relation between LDOC1and NF-κ B, a lentivirus vector was used to upregulate the expression of LDOC1in HIBEC. The expression of LDOC1and inflammatory and apoptosis mediated by NF-κ B were studied.
Methods:HIBEC were divided into three groups:control group, MOCK group and LDOC1tansfection group. Methods of Immunofluorescence, RT-PCR,Western-blot and flow cytometry were used to investigate the effectiveness of transfenction and different expression of NF-κ B and TGF-β in three groups. Apoptosis was detected by Hochest/PI staining. IL-2and TNF-α were studied by ELISA.
Results
(1) Green fluorescence of GFP was detected in majority of Cytoplasm and transfection efficiency was99.8%by examine of flow cytometry.
(2) After transfection of lentivirus, more than90%cell in LDOC1transfection group show red fluorescence of LDOC1antibody;LDOC1transfection group had the higher expression of LDOC1comparing with the other two groups. LDOC1mRNA expression in three group were0.29,0.27and1.98respectively. There existed same tendency in the test of Western-blot.(0.3、0.27、0.99).
(3) More than80%HIBEC in LDOC1transfection group had the expression of NF-kB p65, higher than other two groups.(40%,30%) Relative protein content in three groups were0.52、0.49、1.02respectively.
(4) In three groups, expression of TGF-β was very close (50%) and results of RT-PCR and Western-blot showed the same tendency.(mRNA:0.98,0.99,0.89; protein:0.58、0.61、0.71)
(5) Apoptosis rates in three groups were21.9±2.8%、22.3±3.1%、2.8±0.7%respectively. Apoptosis rate in LDOC1transfection group was much lower than those in other two groups.(p=0.035<0.05, p=0.034<0.05)
(6) In ELISA test, the relative optical density of IL-2and TNF-α in LDOC1transfection group were1.01±0.0and0.71±0.09. Relative optical density value in LDOC1group were much higher than other two groups (IL-2:0.56±0.02, p=0.002<0.01;0.41±0.02, p=0.001<0.01; TNF-α:0.41±0.03, p=0.006<0.01,0.39±0.01, p=0.001<0.01)
Conclusion:
(1) Up-regulation of LDOC1in HIBEC can increase the expression of NF-κB p65, promote the activation of IL-2and TNF-α, and inhibit cell apoptosis.
(2) According to the results in all three parts, LDOC1may play a key role in the pathogenesis of BA.
引文
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12. Mack CL, Tucker RM, Lu BR, Sokol RJ, Fontenot AP, Ueno Y, et al. Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia. Hepatology.2006,44:1231-1239.
13. Harada K, Sato Y, Itatsu K, Isse K, Ikeda H, Yasoshima M, et al. Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia. Hepatology 2007,46(5):1146-1154.
14. Harada K, Ohba K, Ozak i S, Isse K, Hirayama T, Wada A, et al. Peptide antibiotic human beta-defensin-1 and-2 contribute to antimicrobial, defense of the intrahepatic biliary tree. Hepatology 2004,40 (2):925-932.
15. Minnick KE, Kreisberg R, Dillon PW. Soluble ICAM-1 (sICAM-1) in biliary atresia and its relationship to disease activity. J Surg Res.1998,76(2):53-56.
16. Leifeld L, Ramakers J, Schneiders AM, et al. Intrahepatic MxAexpression is correlated with interferon-alpha expression in chronic and fulminant hepatitis. J Pathol.2001,194:478-483.
17. Sokol RJ, Mack C. Etiopathogenesis of biliary atresia. Semin LiverDis. 2001,21(2):517-524.
18. Domiati-Saad R, Dawson DB, Margraf LR, et al. Cytomegalovirus and human herpesvirus 6, but not human papillomavirus, are present in neonatal giant cell hepatitis and extrahepatic biliary atresia. Pediatr Dev Pathol.2000,3(1):367-373.
19. Fischler B, Ehrnst A, Forsgren M, et al. The viral association of neonatal cholestasis in Sweden:a possible link between cytomegalovirus infection and extrahepatic biliary atresia. J Pediatr Gastroenterol Nutr.1998,27(2):57-64.
20. Drut R, Drut RM, Gomez MA, et al. Presence of human papillomavirus in extrahepatic biliary atresia. J Pediatr Gastroenterol Nutr. 1998,27 (3):530-535.
21. Riepenhoff-Talty M, Gouvea V, Evans MJ, et al. Detection of group C rotavirus in infants with extrahepatic biliary atresia.J Infect Dis. 1996,174(2):8-15
22. Tyler KL, Sokol RJ, Oberhaus SM, et al. Detection of reovirus RNA in hepatobiliary tissues from patients with extrahepatic biliary atresia and choledochal cysts. Hepatology.1998,27(2):1475-1482.
23. A-Kader HH, Nowicki MJ, Kuramoto KI, et al. Evaluation of the role of hepatitis C virus in biliary atresia. Pediatr Infect Dis J. 1994,13(1):657-659.
24. Brown WR, Sokol RJ, Levin MJ, et al. Lack of correlation between infection with reovirus 3 and extrahepatic biliary atresia or neonatal hepatitis. J Pediatr.1988,113(2):670-676.
25. Bobo L, Ojeh C, Chiu D, et al. Lack of evidence for rotavirus by polymerase chain reaction/enzyme immunoassay of hepatobiliary samples from children with biliary atresia. Pediatr Res.1997,41(1): 229-234.
26. Steele MI, Marshall CM, Lloyd RE, et al. Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease. Hepatology.1995,21(5):697-702.
27. Petersen C, Biermanns D, Kuske M, et al. New aspects in a murine model for extrahepatic biliary atresia. J Pediatr Surg. 1997,32 (6):1190-1195.
28. Kenichi Harada, Yasunori Sato, Kumiko Isse et al. Induction of innate immuneresponse and absence of subsequent tolerance to dsRNA in biliary epithelial cells relate to the pathogenesis of biliary atresia. Liver International 2008 7(15):614-621.
29. Magd A. Kotb. Review of historical cohort:ursodeoxycholic acid in extrahepatic biliary atresia Journal of Pediatric Surgery 2008,43 (4), 1321-1327.
30. Yoon JH,Gwak GY, Kim W, et al. Bile acid-mediated induction of cyclooxygenase-2 and Mcl-1 in hepatic stellate cells Biochemical and Biophysical Research Communications 342(4):1108-13, 2006 Apr 21 342(4):1108-1113,2006 Apr 21
31. Haude CG, Christelle G, Chantal C, et al. Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices. Hepatology,2001,45(3):112-118.
32. Petersen C, Grasshoff S, Luciano L. Diverse morphology of biliary atresia in an animal model. J Hepatol.1998,28(4):603-607.
33. Bezerra JA. The next challenge in pediatric cholestasis:deciphering the pathogenesis of biliary atresia. J Pediatr Gastroenterol Nutr.2006,43(suppl 1):S23-S29.
34. Feng J, Li M, Cai T, Tang H, Gu W. Rotavirus-induced murine biliary atresia is mediated by nuclear factor-kappaB. J Pediatr Surg 2005,40:630-6.
35. Petersen C, Bruns E, Kuske M, et al. Treatment of extrahepatic biliary atresia with interferon-alpha in a murine infectious model. Pediatr Res.1997,42(2):623-628.
36.王昕荣,陈素华,乔福元等.子宫巨细胞病毒感染与妊娠结局的相关性研究.中华医学杂志2004,84(17)：1445-1446.
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2. Joanne B, Craig Z, Marquelle K, et al. Biliary atresia-a fifteen-year review of clinical and pathologic factors associated with Liver Transplantation. J Pediatric Surg.2004,39(6):800-803.
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1. Gossey S, Otte JB, De Meyer R, et al. A histological study of extrahepaticbiliary atresia. Acta Paediatr Belg.2004,30(3):85-90.
2. Landing BH. Considerations of the pathogenesis of neonatal hepatitis, biliary atresia and choledochal cyst-the concept of infantile obstructive cholangiopathy. Prog Pediatr Surg.2001,6(2):113-139.
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4. Bezerra JA, Tiao G, Ryckman FC, et al. Genetic induction of proinflammatory immunity in children with biliary atresia. Lancet. 2002,360(3):1653-1659.
5. Davenport M, Gonde C, Redkar R, et al. Immunohistochemistry of the liver and biliary tree in extrahepatic biliary atresia. J Pediatr Surg. 2001,36(6):1017-1025.
6. Mack CL, Tucker RM, Sokol RJ, et al. Biliary atresia is associated with CD4+Thl cell-mediated portal tract inflammation. Pediatr Res. 2004,56 (4):79-87.
7. Kobayashi H, Puri P,O'Briain DS, et al. Hepatic overexpression of MHC class II antigens and macrophage-associated antigens (CD68) in patients with biliary atresia of poor prognosis. J Pediatr Surg. 2006,32 (4):590-593.
8. Shinkai M, Shinkai T, Puri P, et al. Increased CXCR3 expression associated with CD3-positive lymphocytes in the liver and biliary remnant in biliary atresia. J Pediatr Surg.2006,41(2):950-954.
9. Mack CL, Falta MT, Sullivan AK, et al. Oligoclonal expansions of CD4+ and CD8+T-cells in the target organ of patients with biliary atresia. Gastroenterology.2007,133(1):278-287.
10. Muraji T, Hosaka N, Irie N, et al. Maternal Microchimerism in Underlying Pathogenesis of Biliary Atresia:Quantification and Phenotypes of Maternal Cells in the Liver Pediatrics 2008,121(2):517-521.
11. Al-Masri AN, Flemming P, Rodeck B, et al. Expression of the interferon-induced Mx proteins in biliary atresia. J Pediatr Surg.2006,41 (2):1139-1143.
12. Mack CL, Tucker RM, Lu BR, Sokol RJ, Fontenot AP, Ueno Y, et al. Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia. Hepatology.2006,44:1231-1239.
13. Harada K, Sato Y, Itatsu K, Isse K, Ikeda H, Yasoshima M, et al. Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia. Hepatology 2007,46(5):1146-1154.
14. Harada K, Ohba K, Ozak i S, Isse K, Hirayama T, Wada A, et al. Peptide antibiotic human beta-defensin-1 and-2 contribute to antimicrobial, defense of the intrahepatic biliary tree. Hepatology 2004,40 (2):925-932.
15. Minnick KE, Kreisberg R, Dillon PW. Soluble ICAM-1 (sICAM-1) in biliary atresia and its relationship to disease activity. J Surg Res.1998,76(2):53-56.
16. Leifeld L, Ramakers J, Schneiders AM, et al. Intrahepatic MxAexpression is correlated with interferon-alpha expression in chronic and fulminant hepatitis. J Pathol.2001,194:478-483.
17. Sokol RJ, Mack C. Etiopathogenesis of biliary atresia. Semin LiverDis. 2001,21(2):517-524.
18. Domiati-Saad R, Dawson DB, Margraf LR, et al. Cytomegalovirus and human herpesvirus 6, but not human papillomavirus, are present in neonatal giant cell hepatitis and extrahepatic biliary atresia. Pediatr Dev Pathol.2000,3(1):367-373.
19. Fischler B, Ehrnst A, Forsgren M, et al. The viral association of neonatal cholestasis in Sweden:a possible link between cytomegalovirus infection and extrahepatic biliary atresia. J Pediatr Gastroenterol Nutr.1998,27(2):57-64.
20. Drut R, Drut RM, Gomez MA, et al. Presence of human papillomavirus in extrahepatic biliary atresia. J Pediatr Gastroenterol Nutr. 1998,27 (3):530-535.
21. Riepenhoff-Talty M, Gouvea V, Evans MJ, et al. Detection of group C rotavirus in infants with extrahepatic biliary atresia.J Infect Dis. 1996,174(2):8-15
22. Tyler KL, Sokol RJ, Oberhaus SM, et al. Detection of reovirus RNA in hepatobiliary tissues from patients with extrahepatic biliary atresia and choledochal cysts. Hepatology.1998,27(2):1475-1482.
23. A-Kader HH, Nowicki MJ, Kuramoto KI, et al. Evaluation of the role of hepatitis C virus in biliary atresia. Pediatr Infect Dis J. 1994,13(1):657-659.
24. Brown WR, Sokol RJ, Levin MJ, et al. Lack of correlation between infection with reovirus 3 and extrahepatic biliary atresia or neonatal hepatitis. J Pediatr.1988,113(2):670-676.
25. Bobo L, Ojeh C, Chiu D, et al. Lack of evidence for rotavirus by polymerase chain reaction/enzyme immunoassay of hepatobiliary samples from children with biliary atresia. Pediatr Res.1997,41(1): 229-234.
26. Steele MI, Marshall CM, Lloyd RE, et al. Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease. Hepatology.1995,21(5):697-702.
27. Petersen C, Biermanns D, Kuske M, et al. New aspects in a murine model for extrahepatic biliary atresia. J Pediatr Surg. 1997,32 (6):1190-1195.
28. Kenichi Harada, Yasunori Sato, Kumiko Isse et al. Induction of innate immuneresponse and absence of subsequent tolerance to dsRNA in biliary epithelial cells relate to the pathogenesis of biliary atresia. Liver International 2008 7(15):614-621.
29. Magd A. Kotb. Review of historical cohort:ursodeoxycholic acid in extrahepatic biliary atresia Journal of Pediatric Surgery 2008,43 (4), 1321-1327.
30. Yoon JH,Gwak GY, Kim W, et al. Bile acid-mediated induction of cyclooxygenase-2 and Mcl-1 in hepatic stellate cells Biochemical and Biophysical Research Communications 342(4):1108-13, 2006 Apr 21 342(4):1108-1113,2006 Apr 21
31. Haude CG, Christelle G, Chantal C, et al. Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices. Hepatology,2001,45(3):112-118.
32. Petersen C, Grasshoff S, Luciano L. Diverse morphology of biliary atresia in an animal model. J Hepatol.1998,28(4):603-607.
33. Bezerra JA. The next challenge in pediatric cholestasis:deciphering the pathogenesis of biliary atresia. J Pediatr Gastroenterol Nutr.2006,43(suppl 1):S23-S29.
34. Feng J, Li M, Cai T, Tang H, Gu W. Rotavirus-induced murine biliary atresia is mediated by nuclear factor-kappaB. J Pediatr Surg 2005,40:630-6.
35. Petersen C, Bruns E, Kuske M, et al. Treatment of extrahepatic biliary atresia with interferon-alpha in a murine infectious model. Pediatr Res.1997,42(2):623-628.
36.王昕荣,陈素华,乔福元等.子宫巨细胞病毒感染与妊娠结局的相关性研究.中华医学杂志2004,84(17)：1445-1446.
37. Kohsaka T, Yuan ZR, Guo SX, et al. The significance of humanjagged 1 mutations detected in severe cases of extrahepatic biliary atresia. Hepatology.2002,36(6):904-912.
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