肌成纤维细胞在小鼠肝内胆管发育过程中的作用
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摘要
背景:研究表明肌成纤维细胞具有促进肝祖细胞分化为胆管细胞作用,但对于肌成纤维细胞在体内胆管发育中作用仍不清楚。目的:观察小鼠肝内胆管发育的特点,以及肌成纤维细胞在胆管发育过程中的作用。方法:SPF级C57小鼠购自重庆医科大学实验动物中心,实验方案经重庆医科大学动物实验伦理委员会批准,批准号为2017-182。采用14.5 d、16.5 d孕鼠及刚出生(0 d)和出生后10 d的小鼠,麻醉后取其胎鼠肝脏(E14.5、E16.5)和出生后小鼠肝脏(P0、P10),采用免疫组织化学技术观察α平滑肌肌动蛋白、Sox9及细胞角蛋白19在E14.5、E16.5小鼠胎肝和P0、P10小鼠肝脏中的表达。结果与结论:①E14.5小鼠胎肝中,α平滑肌肌动蛋白阳性肌成纤维细胞环绕门静脉周围,Sox9阳性胆管细胞紧靠α平滑肌肌动蛋白阳性肌成纤维细胞,并形成单层细胞排列的胆管板;②E16.5小鼠胎肝中,紧靠α平滑肌肌动蛋白阳性肌成纤维细胞的细胞角蛋白19阳性胆管细胞形成双层胆管细胞结构;③P0小鼠肝脏中,细胞角蛋白19阳性胆管细胞发育形成具有管腔的胆管结构,并且α平滑肌肌动蛋白阳性肌成纤维细胞开始包绕胆管;④P10小鼠肝脏中,胆管进一步发育扩张,门静脉周围未形成胆管的胆管细胞开始消失。并且α平滑肌肌动蛋白阳性肌成纤维细胞环绕胆管,胆管旁发育形成肝动脉;⑤结果表明了小鼠肝内胆管发育的特点,发现α平滑肌肌动蛋白阳性肌成纤维细胞与肝内胆管发育的关系密切,且在肝内胆管发育中发挥了重要作用。
BACKGROUND: Existing evidence has shown that myofibroblasts can promote hepatic progenitor cells to differentiate into bile duct cells. But,the role of myofibroblasts in the development of bile duct in vivo remains unclear.OBJECTIVE: To investigate the developmental features of intrahepatic bile duct in mice and role of myofibroblasts in the development of bile duct.METHODS: C57 mice, SPF grade were proved by the Laboratory Animal Center of Chongqing Medical University, and the study was approved by the Ethics Committee of Experimental Animal Ethics of Chongqing Medical University, approval number: 2017-182. The mice in gestational day 14.5 and 16.5, postnatal 0 and 10 days(P0, P10) mice were selected. After anesthesia, the liver was removed from fetal mice of embryonic day 14.5 and 16.5(E14.5, E16.5) and P0 and P10 mice. The expression levels of α smooth muscle actin, Sox9 and cytokeratin19 in liver were examined by immunocytochemical method.RESULTS AND CONCLUSION:(1) In the fetal liver of E14.5 mice, myofibroblasts positive for α smooth muscle actin were main located around the portal vein, which was adjacent to the bile duct cells positive for Sox9, and single layered ring called ductal plate formed.(2) In the fetal liver of E16.5 mice, the ductal plate positive for cytokeratin 19, which neared the myofibroblasts positive for α smooth muscle actin,became focally bilayered.(3) In the liver of P0 mice, the cholangiocytes positive for cytokeratin 19 formed bile ducts, which were surrounded by myofibroblasts positive for α smooth muscle actin.(4) In the liver of P10 mice, the bile duct expanded further, and cholangiocytes that could not form bile duct surrounding the portal vein disappeared. The bile duct was encircled by myofibroblasts positive for α smooth muscle actin,and a well-formed hepatic artery was found near the bile duct.(5) The developmental characteristics of intrahepatic bile ducts are systematic displayed. Myofibroblasts positive for α smooth muscle actin are closely related to intrahepatic bile duct development, and play critical role.
BACKGROUND: Existing evidence has shown that myofibroblasts can promote hepatic progenitor cells to differentiate into bile duct cells. But,the role of myofibroblasts in the development of bile duct in vivo remains unclear.OBJECTIVE: To investigate the developmental features of intrahepatic bile duct in mice and role of myofibroblasts in the development of bile duct.METHODS: C57 mice, SPF grade were proved by the Laboratory Animal Center of Chongqing Medical University, and the study was approved by the Ethics Committee of Experimental Animal Ethics of Chongqing Medical University, approval number: 2017-182. The mice in gestational day 14.5 and 16.5, postnatal 0 and 10 days(P0, P10) mice were selected. After anesthesia, the liver was removed from fetal mice of embryonic day 14.5 and 16.5(E14.5, E16.5) and P0 and P10 mice. The expression levels of α smooth muscle actin, Sox9 and cytokeratin19 in liver were examined by immunocytochemical method.RESULTS AND CONCLUSION:(1) In the fetal liver of E14.5 mice, myofibroblasts positive for α smooth muscle actin were main located around the portal vein, which was adjacent to the bile duct cells positive for Sox9, and single layered ring called ductal plate formed.(2) In the fetal liver of E16.5 mice, the ductal plate positive for cytokeratin 19, which neared the myofibroblasts positive for α smooth muscle actin,became focally bilayered.(3) In the liver of P0 mice, the cholangiocytes positive for cytokeratin 19 formed bile ducts, which were surrounded by myofibroblasts positive for α smooth muscle actin.(4) In the liver of P10 mice, the bile duct expanded further, and cholangiocytes that could not form bile duct surrounding the portal vein disappeared. The bile duct was encircled by myofibroblasts positive for α smooth muscle actin,and a well-formed hepatic artery was found near the bile duct.(5) The developmental characteristics of intrahepatic bile ducts are systematic displayed. Myofibroblasts positive for α smooth muscle actin are closely related to intrahepatic bile duct development, and play critical role.
引文
[1]Strazzabosco M, Fabris L. Development of the bile ducts:essentials for the clinical hepatologist. J Hepatol. 2012;56(5):1159-1170.
[2]De Assuncao TM,Jalan-Sakrikar N,Huebert RC.Regenerative medicine and the biliary tree. Semin Liver Dis. 2017;37(1):17-27.
[3]Boulter L,Lu WY,Forbes SJ.Differentiation of progenitors in the liver:a matter of local choice. J Clin Invest.2013;123:1867-1873.
[4]Si-Tayeb K,Lemaigre FP,Duncan SA.Organogenesis and development of the liver.Dev Cell. 2010;18:175-189.
[5]Clotman F,Jacquemin P,Plumb-Rudewiez N.Control of liver cell fate decision by a gradient of TGFβsignaling modulated by Onecut transcription factors. Genes Dev. 2005;19(16):1849-1854.
[6]Antoniou A,Raynaud P,Cordi S,et al.Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9. Gastroenterology. 2009;136(7):2325-2333.
[7]Hofmann JJ,Zovein AC,Koh H,et al.Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development:Insights into Alagille syndrome. Development. 2010;137(23):4061-4072.
[8]Wang W,Feng Y,Aimaiti Y.TGFβsignaling controls intrahepatic bile duct development may through regulating the Jagged1-NotchSox9 signaling axis.J Cell Physiol. 2018;233:5780-5791.
[9]Lemaigre FP.Development of the biliary tract. Mech Dev. 2003;120(1):81-87.
[10]Lemaigre FP. Notch signaling in bile duct development:new insights raise new questions. Hepatology 2008; 48(2):358-360
[11]Raynaud P,Carpentier R,Antoniou A,et al.Biliary differentiation and bile duct morphogenesis in development and disease. Int J Biochem Cell Biol. 2011;43(2):245-256.
[12]Wang Y,Yao H,Cui C.Paracrine Signals from Mesenchymal Cell Populations govern Expansion and Differentiation of Human Hepatic Stem cells to Adult Liver Fates.Hepatology.2010 Oct,52(4):1443-1454.
[13]Quondamatteo F,Knittel T,Mehde M,et al.Matrix metalloproteinases in early human liver development.Histochem Cell Biol.1999; 112(4):277-282.
[14]Pan XR,Jing YY,Liu WT,et al.Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts via activation of the Hedgehog signaling pathway.Cell Cycle.2017; 16(14):1357-1365.
[15]Chen J,Li X,Hu Y,et al.Gypenosides Ameliorate Carbon Tetrachloride-Induced Liver Fibrosis by Inhibiting the Differentiation of Hepatic Progenitor Cells into Myofibroblasts.Am J Chin Med. 2017;45(5):1061-1074.
[16]Carpentier R, Suner RE, van Hul N,et al.Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes,and adult liver progenitor cells. Gastroenterology. 2011;141(4):1432-1438,1438.e1-4.
[17]Ober EA,Lemaigre FP.Development of the liver:Insights into organ and tissue morphogenesis.J Hepatol. 2018;68(5):1049-1062.
[18]Fabris L,Cadamuro M, Libbrecht L,et al. Angiogenic growth factors secreted by liver epithelial cells modulate arterial vasculogenesis during human liver development. Hepatology.2008; 47(2):719-728.
[19]Lemaigre FP.Mechanisms of liver development:concepts for understanding liver disorders and design of novel therapies.Gastroenterology 2009(1);137:62-79.
[20]Gerard C,Tys J,Lemaigre FP.Gene regulatory networks in differentiation and direct reprogramming of hepatic cells.Semin Cell Dev Biol. 2017;66:43-50.
[21]Ogawa M,Ogawa S,Bear CE,et al.Directed differentiation of cholangiocytes from human pluripotent stem cell. Nat Biotechnol.2015;33(8):853-861.
[22]Kamiya A,Ito K, Yanagida A,et al.MEK-ERK Actibity Regulates the proliferative Activity of Fetal Hepatoblasts Through Accumalation of P16/19(cdkn2a). Stem Cells Dev.2015;24(21):2525-2535.
[23]Kaylan KB,Ermilova V,Yada RC.Combinatorial microenvironmental regulation of liver progenitor differentiation by Notch ligands,TGFbeta,and extracellular matrix. Sci Rep. 2016;6:23490.
[24]Lua I, Li Y, Zagory JA, et al.Characterization of hepatic stellate cells,portal fibroblasts,and mesothelial cells in normal and fibrotic livers.J Hepatol.2016;64(5):1137-1146.
[2]De Assuncao TM,Jalan-Sakrikar N,Huebert RC.Regenerative medicine and the biliary tree. Semin Liver Dis. 2017;37(1):17-27.
[3]Boulter L,Lu WY,Forbes SJ.Differentiation of progenitors in the liver:a matter of local choice. J Clin Invest.2013;123:1867-1873.
[4]Si-Tayeb K,Lemaigre FP,Duncan SA.Organogenesis and development of the liver.Dev Cell. 2010;18:175-189.
[5]Clotman F,Jacquemin P,Plumb-Rudewiez N.Control of liver cell fate decision by a gradient of TGFβsignaling modulated by Onecut transcription factors. Genes Dev. 2005;19(16):1849-1854.
[6]Antoniou A,Raynaud P,Cordi S,et al.Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9. Gastroenterology. 2009;136(7):2325-2333.
[7]Hofmann JJ,Zovein AC,Koh H,et al.Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development:Insights into Alagille syndrome. Development. 2010;137(23):4061-4072.
[8]Wang W,Feng Y,Aimaiti Y.TGFβsignaling controls intrahepatic bile duct development may through regulating the Jagged1-NotchSox9 signaling axis.J Cell Physiol. 2018;233:5780-5791.
[9]Lemaigre FP.Development of the biliary tract. Mech Dev. 2003;120(1):81-87.
[10]Lemaigre FP. Notch signaling in bile duct development:new insights raise new questions. Hepatology 2008; 48(2):358-360
[11]Raynaud P,Carpentier R,Antoniou A,et al.Biliary differentiation and bile duct morphogenesis in development and disease. Int J Biochem Cell Biol. 2011;43(2):245-256.
[12]Wang Y,Yao H,Cui C.Paracrine Signals from Mesenchymal Cell Populations govern Expansion and Differentiation of Human Hepatic Stem cells to Adult Liver Fates.Hepatology.2010 Oct,52(4):1443-1454.
[13]Quondamatteo F,Knittel T,Mehde M,et al.Matrix metalloproteinases in early human liver development.Histochem Cell Biol.1999; 112(4):277-282.
[14]Pan XR,Jing YY,Liu WT,et al.Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts via activation of the Hedgehog signaling pathway.Cell Cycle.2017; 16(14):1357-1365.
[15]Chen J,Li X,Hu Y,et al.Gypenosides Ameliorate Carbon Tetrachloride-Induced Liver Fibrosis by Inhibiting the Differentiation of Hepatic Progenitor Cells into Myofibroblasts.Am J Chin Med. 2017;45(5):1061-1074.
[16]Carpentier R, Suner RE, van Hul N,et al.Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes,and adult liver progenitor cells. Gastroenterology. 2011;141(4):1432-1438,1438.e1-4.
[17]Ober EA,Lemaigre FP.Development of the liver:Insights into organ and tissue morphogenesis.J Hepatol. 2018;68(5):1049-1062.
[18]Fabris L,Cadamuro M, Libbrecht L,et al. Angiogenic growth factors secreted by liver epithelial cells modulate arterial vasculogenesis during human liver development. Hepatology.2008; 47(2):719-728.
[19]Lemaigre FP.Mechanisms of liver development:concepts for understanding liver disorders and design of novel therapies.Gastroenterology 2009(1);137:62-79.
[20]Gerard C,Tys J,Lemaigre FP.Gene regulatory networks in differentiation and direct reprogramming of hepatic cells.Semin Cell Dev Biol. 2017;66:43-50.
[21]Ogawa M,Ogawa S,Bear CE,et al.Directed differentiation of cholangiocytes from human pluripotent stem cell. Nat Biotechnol.2015;33(8):853-861.
[22]Kamiya A,Ito K, Yanagida A,et al.MEK-ERK Actibity Regulates the proliferative Activity of Fetal Hepatoblasts Through Accumalation of P16/19(cdkn2a). Stem Cells Dev.2015;24(21):2525-2535.
[23]Kaylan KB,Ermilova V,Yada RC.Combinatorial microenvironmental regulation of liver progenitor differentiation by Notch ligands,TGFbeta,and extracellular matrix. Sci Rep. 2016;6:23490.
[24]Lua I, Li Y, Zagory JA, et al.Characterization of hepatic stellate cells,portal fibroblasts,and mesothelial cells in normal and fibrotic livers.J Hepatol.2016;64(5):1137-1146.