肝窦内皮细胞对肝细胞生物学特性影响的体外研究
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摘要
目的:探讨永生化人肝窦内皮细胞(IHLESC)能否提高肝细胞的代谢、解毒及合成功能。方法:设立永生化人肝细胞(IHH)单独培养组和IHLESC与IHH共培养组,评价两种不同培养方式对肝细胞代谢、解毒和合成功能的影响;将两组细胞分别接种于微载体上,分别与肝衰竭患者血浆共孵育,比较两组IHH的解毒功能。结果:相比于单独培养组,共培养组IHH密切接触,聚集成团。共培养组IHH的谷氨酰胺合成酶(GS)、谷胱甘肽-S-转移酶(GST)、鸟苷二磷酸葡萄糖醛酸基转移酶1A1(UGT1A1)、白蛋白(ALB)表达量均显著高于单独培养组(P<0.01)。共培养组血浆总胆红素的下降及血浆尿素的上升均比单独培养组显著(P<0.01)。结论:用IHH与IHLSEC在微载体上共培养制备成新型生物人工肝的核心材料,可提高生物人工肝细胞的代谢、解毒及合成功能。
Objective: To investigate whether human liver sinusoidal endothelial cells can improve metabolism,detoxification and synthesis via co-culture of immortalized human hepatocytes( IHH) with immortalized human liver sinusoidal endothelial cells( IHLSEC). Methods: Two experimental groups were set up: IHH single culture,and IHLSEC and IHH co-culture groups. Metabolism,detoxification and synthesis in both cell groups were compared.Sequentially,the two groups cultured in micro-carriers were respectively incubated with plasma from patients with liver failure to compare detoxification function between the two groups. Results: Compared to the single culture group,IHH in the co-culture group had tight contact between cells,which were clumped together. The expression of GS,GST,UGT1 A1,and ALB in the co-culture group was significantly higher than the other( P < 0. 01). Coculture group decreased plasma urea and increased plasma total bilirubin more significantly than the single group( P <0. 01). Conclusion: The new bioartificial liver,constituted of IHLSEC and IHH attached to micro-carriers,may have better metabolism,detoxification,synthesis,and functionality.
Objective: To investigate whether human liver sinusoidal endothelial cells can improve metabolism,detoxification and synthesis via co-culture of immortalized human hepatocytes( IHH) with immortalized human liver sinusoidal endothelial cells( IHLSEC). Methods: Two experimental groups were set up: IHH single culture,and IHLSEC and IHH co-culture groups. Metabolism,detoxification and synthesis in both cell groups were compared.Sequentially,the two groups cultured in micro-carriers were respectively incubated with plasma from patients with liver failure to compare detoxification function between the two groups. Results: Compared to the single culture group,IHH in the co-culture group had tight contact between cells,which were clumped together. The expression of GS,GST,UGT1 A1,and ALB in the co-culture group was significantly higher than the other( P < 0. 01). Coculture group decreased plasma urea and increased plasma total bilirubin more significantly than the single group( P <0. 01). Conclusion: The new bioartificial liver,constituted of IHLSEC and IHH attached to micro-carriers,may have better metabolism,detoxification,synthesis,and functionality.
引文
[1]PODOLL A S,DEGOLOVINE A,FINKEL K W.Liver support systems-a review[J].ASAIO J,2012,58(5):443-449.
[2]O'GRADY J.Timing and benefit of liver transplantation in acute liver failure[J].ASAIO J,2014,60(3):663-670.
[3]BERNAL W,JALAN R,QUAGLIA A,et al.Acute-on-chronic liver failure[J].Lancet,2015,386(10003):1576-1587.
[4]LEE S Y,KIM H J,CHOI D.Cell sources,liver support systems and liver tissueengineering:alternatives to liver transplantation[J].Int J Stem Cells,2015,8(1):36-47.
[5]DING Y,SHI X.Bioartificial liver devices:perspectives on the state of the art[J].Front Med,2011,5(1):15-19.
[6]RAMBOER E,VANHAEKE T,ROGIERS V,et al.Immortalized human hepatic cell lines for in vitro testing and research purposes[J].Methods Mol Biol,2015,1250:53-76.
[7]PAN X P,WANG Y N,YU X P,et al.Efficient generation of functional hepatocyte-like cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells[J].Hepatobiliary Pancreatic Dis Int,2016,15(2):173-179.
[8]ZHOU M,ZHAO F,LI J,et al.Long-term maintenance of human fetal hepatocytes and prolonged susceptibility to HBV infection by co-culture with non-parenchymal cells[J].J Virol Methods,2014,195:185-193.
[9]BHATIA S N,BALIS U J,YARMUSH M L,et al.Effect of cell-cell interactions in preservation of cellular phenotype:cocultivation of hepatocytes and nonparenchymal cells[J].FASEB J,1999,13(14):1883-1900.
[10]LOU J N,MILI N,DECRIND C,et al.An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung[J].In Vitro Cell Dev Biol Anim,1998,34(7):529-536.
[11]张然星,李宓,张文健,等.永生化人肝细胞系的建立和生物学特性研究[J].中国医药生物技术,2008(1):11-18.
[12]SALMON P,OBERHOLZER J,OCCHIODORO T,et al.Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes[J].Mol Ther,2000,2(4):404-414.
[13]张本厚,张文健,娄晋宁,等.永生化人肝窦内皮细胞株的形态和功能特性研究[J].中华外科杂志,2010,48(1):48-52.
[14]FRACZEK J,BOLLEYN J,VANHAECKE T,et al.Primary hepatocyte cultures for pharmaco-toxicological studies:at the busy crossroad of various anti-dedifferentiation strategies[J].Arch Toxicol,2013,87(4):577-610.
[15]Mc CUSKEY R S.The hepatic microvascular system in health and its response to toxicants[J].Anat Rec(Hoboken),2008,291(6):661-671.
[16]BALE S S,GOLBERG I,JINDAL R,et al.Long-term coculture trategies for primary hepatocytes and liver sinusoidal endothelial cells[J].Tissue Eng,Part C:Methods,2015,21(4):413-422.
[17]PAN X,WANG Y,YU X,et al.Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes[J].Int J Med Sci,2015,12(3):248-255.
[18]NGUYEN T V,UKAIRO O,KHETANI S R,et al.Establishment of a hepatocyte-kupffer cell coculture model for assessment of proinflammatory cytokine effects on metabolizing enzymes and drug transporters[J].Drug Metab.Dispos.,2015,43(5):774-785.
[19]SHANG Y,TAMAI M,ISHII R,et al.Hybrid sponge comprised of galactosylated chitosan and hyaluronic acid mediates the co-culture of hepatocytes and endothelial cells[J].J Biosci Bioeng,2014,117(1):99-106.
[20]QIN H H,FILIPPI C,SUN S,et al.Hypoxic preconditioning potentiates the trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes[J].Stem Cell Res Ther,2015,6(1):1-12.
[2]O'GRADY J.Timing and benefit of liver transplantation in acute liver failure[J].ASAIO J,2014,60(3):663-670.
[3]BERNAL W,JALAN R,QUAGLIA A,et al.Acute-on-chronic liver failure[J].Lancet,2015,386(10003):1576-1587.
[4]LEE S Y,KIM H J,CHOI D.Cell sources,liver support systems and liver tissueengineering:alternatives to liver transplantation[J].Int J Stem Cells,2015,8(1):36-47.
[5]DING Y,SHI X.Bioartificial liver devices:perspectives on the state of the art[J].Front Med,2011,5(1):15-19.
[6]RAMBOER E,VANHAEKE T,ROGIERS V,et al.Immortalized human hepatic cell lines for in vitro testing and research purposes[J].Methods Mol Biol,2015,1250:53-76.
[7]PAN X P,WANG Y N,YU X P,et al.Efficient generation of functional hepatocyte-like cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells[J].Hepatobiliary Pancreatic Dis Int,2016,15(2):173-179.
[8]ZHOU M,ZHAO F,LI J,et al.Long-term maintenance of human fetal hepatocytes and prolonged susceptibility to HBV infection by co-culture with non-parenchymal cells[J].J Virol Methods,2014,195:185-193.
[9]BHATIA S N,BALIS U J,YARMUSH M L,et al.Effect of cell-cell interactions in preservation of cellular phenotype:cocultivation of hepatocytes and nonparenchymal cells[J].FASEB J,1999,13(14):1883-1900.
[10]LOU J N,MILI N,DECRIND C,et al.An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung[J].In Vitro Cell Dev Biol Anim,1998,34(7):529-536.
[11]张然星,李宓,张文健,等.永生化人肝细胞系的建立和生物学特性研究[J].中国医药生物技术,2008(1):11-18.
[12]SALMON P,OBERHOLZER J,OCCHIODORO T,et al.Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes[J].Mol Ther,2000,2(4):404-414.
[13]张本厚,张文健,娄晋宁,等.永生化人肝窦内皮细胞株的形态和功能特性研究[J].中华外科杂志,2010,48(1):48-52.
[14]FRACZEK J,BOLLEYN J,VANHAECKE T,et al.Primary hepatocyte cultures for pharmaco-toxicological studies:at the busy crossroad of various anti-dedifferentiation strategies[J].Arch Toxicol,2013,87(4):577-610.
[15]Mc CUSKEY R S.The hepatic microvascular system in health and its response to toxicants[J].Anat Rec(Hoboken),2008,291(6):661-671.
[16]BALE S S,GOLBERG I,JINDAL R,et al.Long-term coculture trategies for primary hepatocytes and liver sinusoidal endothelial cells[J].Tissue Eng,Part C:Methods,2015,21(4):413-422.
[17]PAN X,WANG Y,YU X,et al.Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes[J].Int J Med Sci,2015,12(3):248-255.
[18]NGUYEN T V,UKAIRO O,KHETANI S R,et al.Establishment of a hepatocyte-kupffer cell coculture model for assessment of proinflammatory cytokine effects on metabolizing enzymes and drug transporters[J].Drug Metab.Dispos.,2015,43(5):774-785.
[19]SHANG Y,TAMAI M,ISHII R,et al.Hybrid sponge comprised of galactosylated chitosan and hyaluronic acid mediates the co-culture of hepatocytes and endothelial cells[J].J Biosci Bioeng,2014,117(1):99-106.
[20]QIN H H,FILIPPI C,SUN S,et al.Hypoxic preconditioning potentiates the trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes[J].Stem Cell Res Ther,2015,6(1):1-12.