行政院國家科學委員會專題研究計畫 期中進度報告
膽道閉鎖嬰兒及輪狀病毒感染誘發肝外膽管阻塞鼷鼠之基
因表現(1/3)
計畫類別： 個別型計畫 計畫編號： NSC93-2314-B-002-129- 執行期間： 93 年 08 月 01 日至 94 年 07 月 31 日 執行單位： 國立臺灣大學醫學院一般醫學科 計畫主持人： 許宏遠 計畫參與人員： 江政倫 報告類型： 精簡報告 處理方式： 本計畫可公開查詢 中 華 民 國 94 年 5 月 27 日行政院國家科學委員會補助專題研究計畫
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膽道閉鎖嬰兒及輪狀病毒感染誘發肝外膽管阻塞鼷鼠之基因表現
Gene expression profiles in infants with biliary atresia and in mice with rotavirus induced extrahepatic biliary obstruction
計畫類別：█ 個別型計畫 □ 整合型計畫 計畫編號：NSC93－2314－B－002－129－ 執行期間： 2004 年 08 月 01 日至 2005 年 07 月 31 日 計畫主持人：許宏遠 共同主持人： 計畫參與人員： 江政倫 成果報告類型(依經費核定清單規定繳交)：■精簡報告 □完整報告 本成果報告包括以下應繳交之附件： □赴國外出差或研習心得報告一份 □赴大陸地區出差或研習心得報告一份 □出席國際學術會議心得報告及發表之論文各一份 □國際合作研究計畫國外研究報告書一份 處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、列管計 畫及下列情形者外，得立即公開查詢 □涉及專利或其他智慧財產權，□一年□二年後可公開查詢
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膽道閉鎖嬰兒及輪狀病毒感染誘發肝外膽管阻塞鼷鼠之基因表現
Gene expression profiles in infants with biliary atresia and in mice with rotavirus induced extrahepatic biliary obstruction
Hong-Yuan Hsu, M.D. Department of Primary Care Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
Supported by grant NSC 93-2314-B-002-129 from National Science Council of Taiwan
中文摘要： 膽道閉鎖症（BA）是一種肝外膽管及肝內膽管呈漸進性、發炎性的損傷，最終 導致肝硬化及肝衰竭。這種動態的發炎壞死的膽管病變其致病機轉不明。利用基因 微陣列（DNA microarray）是解開膽管閉鎖症所牽涉複雜的基因調控網路一種有力 的方法。本研究以膽道閉鎖嬰兒在接受肝臟移植時作全肝切除所取下之肝組織作分 析並以活體捐肝手術時，來自捐贈者之正常肝組織作對照組，以了解膽道閉鎖嬰兒 其肝組織中生理相關之基因表現之輪廓。我們所用的基因微陣列法是由台大基因體 醫學中心所發展的人類寡核苷晶片（含 14208 基因產物）來作分析。作數據過濾及 群集分析後我們發現與正常肝組織比較，BA 嬰兒肝組織中主要有與免疫及發炎作 用相關之基因過度表現。其中轉譯產物之訊號強度在 2 倍以上的相關基因有 chemokine （c-c motif）ligand 2，osteopontin，cyclin B2，腫瘤壞死因子受器 superfamily and member 10a。其中 osteopontin 係一種細胞外基質蛋白，有類似細胞素之功能且 且為細胞性免疫之調節者。在 BA 嬰兒肝組織中也有一些基因其表現顯著低於正常 肝組織，包括與 hydrolase 活性、免疫反應或細胞素與其受體互動及 B 細胞調控之 免疫性有關之基因。其中 rynodine receptor 1 與 B 細胞生長及 B 細胞調控之免疫性 有關。這些初步結果顯示 osteopontin 之過度表現（或類似幫助者 T 細胞第一型細胞 素反應）可能在 BA 的致病機轉中扮演某種角色，我們仍繼續對 BA 早期之肝組織 及其他新生兒膽汁滯流症作分析，以了解 BA 之分子致病機轉。 關鍵語：基因表現，膽道閉鎖，基因微陣列，發炎作用。
SUMMARY
Biliary atresia (BA) is a progressive, destructive and inflammatory process of extra- and inrahepatic bile ducts resulting in liver cirrhosis or failure. The pathogenesis underlying this dynamic necro-inflammmatory destructive cholangiopathy remains unknown. DNA microarray is potentially able to delineate the complex gene regulation networks involved in the genesis of BA. To understand the physiologically relevant expression profiles of genes in livers of BA , two samples of liver taken by total hepatectomy from BA infants receiving liver transplantation and two nondiseased liver tissue were studied by using an oligonucleotide based human known gene chips containing 14208 gene products. By data filtering, we have identified those genes that were differentially expressed with a > 2 fold changes in BA infants compared with non-diseased control. Further cluster analysis showed a predominant activation of immunity/inflammation genes within the liver of infants with BA. Those with two fold increase or above in the signal intensity of transcripts including genes encoding chemokine (C-C motif) ligand 2, osteopontin, cyclin B2, tumor necrosis factor receptor superfamily and member 10a. Among them, osteopontin is an extracellular matrix protein with important cytokine-like functions (a regulator of cell-mediated [Th-1] immunity). Some genes with known function displaying lower signal intensity in BA than in healthy controls were found and relevant to hydrolase activity, immune response or cytokine-cytokine receptor interaction and B cell growth and B cell-mediated immunity( rynodine receptor 1). These preliminary results suggest that overexpression of osteopontin, namely, the Th-1-like cytokine response, is involved in disease pathogenesis of biliary atresia. Further studies including microarrays of cDNA from livers of infants with early stage BA and from diseased control with neonatal intrahepatic cholestasis are currently under investigation in our laboratory.
Key words: gene expression profile, microarray, biliary atresia
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INTRODUCTION
Extra-hepatic biliary atresia (BA) is the leading cause of liver diseases resulting in death in pediatric population.1 The incidence of BA is higher in Chinese than in Caucasians. BA is the end result of a progressive destructive, inflammatory of extrahepatic as well as intrahepatic bile ducts, resulting in fibrosis and obliteration of the biliary tracts, biliary cirrhosis and finally liver failure requiring liver transplantation.2 The pathogenesis underlying this dynamic necro-inflammatory destructive cholangitis remains unknown. Evidence to date supports several possible pathogenetic mechanisms for the development of BA.2 Several studies suggested that biliary epithelial injury of this disease possibly resulting from the interplay between genetic susceptibility and environmental exposure (viruses, toxic agents and metabolic insults) at perinatal period.1-3 Immunohistochemical studies demonstrated the infiltration of CD4+, CD8+ lymphocytes and natural killer (CD56+) cells predominated in the liver and extrahepatic bile ducts and an increase in CD68+ macrophage infiltration in portal tracts and biliary remnant tissue in BA patients with poor outcome after the portoenterostomy procedure.5-7 These findings suggested that inflammation involving several kinds of immune cells produce biliary epithelial injury such as pyknosis and necrosis of biliary epithelial cells and also promote hepatic fibrosis and cirrhosis in infants with BA. The mechanisms underlying recruitment of inflammatory cells, immunologic dysregulation, ductular apoptosis and progressive hepatic fibrosis in BA remains unclear. DNA microarrays is potentially able to delineate the complex gene regulation networks involved in the the genesis of BA.
METHODS
Liver tissue was also taken from two BA infants with advanced stages of chronic cholestasis at the time of total hepatectomy for liver transplantation. Liver samples from normal, age-matched infants were not obtained owing to ethical consideration. Liver tissue was also obtained from two healthy adults who were living related donor in liver transplantation and served as controls.
For DNA microarrays, total RNA was extracted using TriReagent (Molecular Research Center, Inc.) according to the manufacturer’s protocol. The structural integrity of the isolated RNA was confirmed by electrophoresis of the RNA through a 1.2% gel. Purity of RNA was ascertained by inclusion of samples with an absorbance ratio 260/280 nm >1.8.Each sample was fluorescence-labeled and hybridized to oligonucleotide based human known gene chips (developed in National Taiwan University Center for Genomic Medicine) containing 14208 gene products. (control genes: 768; known genes: 13400). The indirect-labeling method was performed according to J. Hasseman's standard operating procedure for aminoallyl labeling of RNAfor microarrays from The Institute For Genomic Research. The hybridization method was performed according to the protocol described by Ideker et al.8 The hybridized slides were then washed briefly at room temperature with 0.5x SSC and 0.01%SDS to remove the coverslip, followed by three successive roomtemperature washes with 0.5x SSC, 0.1x SSC, and 0.01x SSC for2 min for each wash. Slides were dried by centrifugation andimmediately scanned. Three independent time-course experiments were performed. The data obtained from experimental repeats were consistent. The slides were scanned with a GenePix 4000 scanner (Axon Instruments, Inc., Foster City, Calif.) as previously described.9 and analyzed using Genepix Pro 4.0 (Axon Instruments) and a customdesigned gene array list file generated by the Bioinformaticsand Molecular Analysis Section (BIMAS) at the Center for InformationTechnology (CIT), NIH. The localized raw expression ratios were calculated following background subtraction, and the expressionratios were normalized using the expression ratios obtainedfor the control cellular housekeeping genes to obtain a calibrated expression ratio (CalRatio).10,11 CalRatio data were subsequentlyuploaded into the Center for Cancer Research Microarray DataBase (mAdb) for spot filteringand for clustering analysis using M. Eisen's Cluster and Tree Viewsoftware. CalRatios were log2 transformed and clustered using Pearson's correlation coefficient with the
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RESULTS
Data filtering and cluster analysis identified those transcripts with significant differences in mean signal intensity between BA patients and healthy controls and generated a profile of genes overexpressed or underexpressed in livers of biliary atresia.. We identified those genes uniquely expressed in biliary atresia compared with healthy controls. The reproducibility of the signal intensity was confirmed for two genes by RT-PCR. If we consider the genes with increased signal intensity in BA, a major biological process can be identified in this disease, i.e. immunity and inflammation. The relevant genes included chemokine (C-C motif) ligand 2, and genes encoding osteopontin, cyclin B2, tumor necrosis factor receptor superfamily and member 10a. Among them, the 3.4 fold increase in the signal intensity for osteopontin was noted. This is an extracellular matrix cell adhesion protein which is also a novel substrate for MMP-3 and MMP-7. MMP-cleaved osteopontin has increased activities in cell migration, cell survival, and most notably, cytokine-like functions such as a central regulator for the T-helper 1 (Th-1) commitment of lymphocytes in vivo Thus, an activation of a cell-mediated immune response might occur in affected livers. On the other hand, several genes with known function displaying lower signal intensity in BA than in healthy controls are protein tyrosine phosphatase receptor type A (relevant to hydrolase activity and inflammatory response), complement component 9 (relevant to cytolysis and immune response), interleukin 2 receptor, gamma (relevant to cytokine-cytokine receptor interaction) and rynodine receptor 1 (relevant to B cell differentiation, proliferation and B cell-mediated immunity). The significance of the lower expression of several immunity/inflammation genes in BA such as a suppressed B lymphocyte functions within the liver in late stage of BA needs further studies.
DISCUSSION
The results revealed a transcriptional profile in the liver of infants with biliary atresia with coordinated activation or suppression of specific genes which differentiates this disease from normal healthy individuals. An increased expression of osteopontin at the time when biliary cirrhosis develops in infants with biliary atresia suggests that Th-1 like cytokines play a significant role in the pathogenesis of biliary atresia. Direct comparison in gene expression with age-matched healthy infants was not possible due to ethical reasons in obtaining liver biopsy samples from healthy, age-matched infants. It has been shown that T-helper phenotype in circulating mononuclear cells can be skewed toward a Th-2 phenotype at birth, but this functional commitment may depend directly on a timely exposure to antigens.12,13
Bezerra et al.14 analyzed a large-scale gene expression in the livers of infants with BA or neonatal intrahepatic cholestasis. They found a predominant and coordinated activation of immunity/inflammation genes within the livers of infants with BA. Most of the genes showed differential lymphocyte function, with activation of oeteopontin, a regulator of cell-mediated (T-helper 1) immunity in T-helper lymphocytes and suppression of immunoglobulin genes in early stages of disease.
Our preliminary results, therefore, add further support to the view that a dominant proinflammatory differentiation of lymphocytes in affected livers may have a key role in the pathogenesis of BA. Further studies including microarrays of cDNA from livers of infants with early stage BA and from diseased control with neonatal intrahepatic cholestasis are currently under investigation in our laboratory. In addition, the disease model of rotavirus-induced hepatobiliary injury and obstruction in young mice may be a promising experimental alternative in the future.15,16
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REFERENCES
1. Balistreri, WF, et al. Biliary atresia: current concepts and research directions. Summary of a symposium. Hepatology 1996;23:1682-1692
2. Sokol, RJ, Mack C, Narkewicz, MR, Karrer, FM. Pathogenesis and outcome of biliary atresia: current concepts. J Pediatr Gastroenterol Nutr 2003;37:4-21
3. Yoon PW, Bresee JS, Olney RS, James LM, Khoury MJ. Epidemiology of biliary atresia: a population-based study. Pediatrics 1997;99:376-82
4. Riepenhoff-Talty M, Gouvea V, Evans MJ, Svensson L, Hoffenberg E, Sokol RJ, Uhnoo I, et al. Detection of group C rotavirus in infants with extrahepatic biliary atresia. J Infect Dis 1996:174:8-15
5. Ohya T, Fujimoto T, Shimomura H, Miyano T. Degeneration of intrahepatic bile duct with lymphocyte infiltration into biliary epithelial cells in biliary atresia. J Peditr Surg 1995;30:515-518
6. Ahmed AF, Ohtani H, Nio M, Funaki N, Shimaoka S, Nagura H, Ohi R. CD8+ T cells infiltrating into bile ducts in biliary atresia do not appear function as cytotoxic T cells: a clinicopathological analysis. J Pathol 2001;193:383-389
7. Davenport M, Gonde C, Redkar R, Koukoulis G, Tredger M, Mieli-Vergani G, Portmann B, Howard ER. Immunohistochemistry of the liver and biliary tree in extrahepatic biliary atresia. J Pediatr Surg 2001;36:1017-1025
8. Ideker T, S Ybarra, S Grimmond. Hybridization and posthybridization washing, P.228-239. In D Bowtell and J Sambrook (ed.), DNA microarrays, 1st ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003
9. Paulose-Murphy M, Ha NK, Xiang C, et al. Transcription program of human herpesvirus type 8 (Kaposi’s sarcoma-associated herpesvirus). J Virol; 2001:75:4843-53
10. Bittner M, Meltzer P, Chen Y, et al. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 2000;406:536-40
11. Chen Y, Kamat V, Dougherty R, et al. Ratio statistics of gene expression levels and applications to microarray data analysis. Bioinformatics 2002;18:1207-1215
12. Prescott SL, Macaubes C, Yabuhara A, et al. Developing patterns of T cell memory to environmental allergens in the first two years of life. Int Arch Allergy Immunolol 1997;113:75-79
13. Marchant A, Aaby P, Newborns develop a Th1-type immune response to Mycobacterium bovis bacillus Calmette-Guerin vaccination. J Immunol 1999;163:2249-55
immunity in children with biliary atresia. Lancet 2002;360:1653-9
15. Riepenhoff-Talty M, Schaekel K, Clark HF, et al. Group A rotaviruses produce extrahepatic biliary obstruction in orally inoculated newborn mice. Peditr Res 1993;33:394-99
16. Petersen C, Biermanns D, Kuske M, et al. New aspects in a murine model for extrahepatic biliary atresia. J Peditr Surg 1997;32:1190-95
