巨噬细胞游走抑制因子参与肝星状细胞活化的实验研究
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摘要
目的探讨大鼠肝星状细胞(HSC)活化过程中表达巨噬细胞游走抑制因子(MIF)的变化,以及MIF特异的反义寡核苷酸(MIF-ASONs)对活化的原代HSC表达MIF和α-平滑肌肌动蛋白(α-SMA)的影响。方法检测分离培养不同时间(1、3、5、7和10 d)的HSC,以及转染MIF-ASONs后的HSC中MIF和α-SMA的表达。基因表达用RT-PCR,蛋白表达用酶联免疫吸附(ELISA)、免疫荧光(IF)或免疫细胞化学(ICC)检测。结果随着培养时间延长,原代HSC形态逐渐改变,脂滴减少消失,自行激活;MIF基因和蛋白表达逐渐增加(P<0.05),且MIF与α-SMA mRNA表达正相关(r=0.944,P<0.01);活化的原代HSC转染MIF-ASONs后MIF与α-SMA基因表达和MIF蛋白表达均比对照组低(P<0.05)。结论 HSC活化过程中,MIF表达逐渐增加并与α-SMA正相关;MIF-ASONs可抑制HSC活化,提示MIF是HSC活化过程中一种重要的细胞因子,可能参与肝纤维化的发生。
Objecitve To investigate the role of macrophage migration inhibitory factor(MIF) in activation of primary hepatic stellate cells(HSC),and to explore the blocking effect of specific antisense oligonucleotides(ASONs) targeting MIF on the activation of primary HSCs.Methods Rat primary HSC were isolated from SD rats by in situ perfusion of collagenase and pronase and single-step Nycodenz density gradient centrifugation,and then cultured and activated.The expression of α-smooth muscle actin(α-SMA),the marker of HSC activation,was assessed by RT-PCR and immunocytochemistry(ICC).The expression of MIF was detected by RT-PCR,enzyme-linked immuno sorbent assay(ELISA),immunofluorescence(IF) or ICC.Completely activated primary HSC were transfected with MIF-specific antisense oligonucleotide(MIF-ASONs),mis-sense oligonucleotides of MIF,or blank liposome(negative-control).Results Quiescent HSC expressed very low level of MIF,while MIF gene and protein expression increased significantly during the process of HSC activation.Positive correlation was found between the expression of α-SMA and MIF(r=0.944,P<0.01).Compared with the control,activated primary HSC transfected with MIF-ASONs showed lower MIF and α-SMA expressions at gene and protein profiles(P<0.05).Conclusion The expression of MIF was up-regulated in the process of HSC activation.Activation in culture of primarily isolated rat HSC could be inhibited by MIF-ASONs,suggesting at least in part that,MIF might be a novel and important factor involved in HSC activation and hepatic fibrosis.
Objecitve To investigate the role of macrophage migration inhibitory factor(MIF) in activation of primary hepatic stellate cells(HSC),and to explore the blocking effect of specific antisense oligonucleotides(ASONs) targeting MIF on the activation of primary HSCs.Methods Rat primary HSC were isolated from SD rats by in situ perfusion of collagenase and pronase and single-step Nycodenz density gradient centrifugation,and then cultured and activated.The expression of α-smooth muscle actin(α-SMA),the marker of HSC activation,was assessed by RT-PCR and immunocytochemistry(ICC).The expression of MIF was detected by RT-PCR,enzyme-linked immuno sorbent assay(ELISA),immunofluorescence(IF) or ICC.Completely activated primary HSC were transfected with MIF-specific antisense oligonucleotide(MIF-ASONs),mis-sense oligonucleotides of MIF,or blank liposome(negative-control).Results Quiescent HSC expressed very low level of MIF,while MIF gene and protein expression increased significantly during the process of HSC activation.Positive correlation was found between the expression of α-SMA and MIF(r=0.944,P<0.01).Compared with the control,activated primary HSC transfected with MIF-ASONs showed lower MIF and α-SMA expressions at gene and protein profiles(P<0.05).Conclusion The expression of MIF was up-regulated in the process of HSC activation.Activation in culture of primarily isolated rat HSC could be inhibited by MIF-ASONs,suggesting at least in part that,MIF might be a novel and important factor involved in HSC activation and hepatic fibrosis.
引文
1 Safadi R,Friedman SL.Hepatic fibrosis--role of hepaticstellate cell activation.MedGenMed,2002;4(3):27.
2 Saile B,Matthes N,Knittel T,et al.Transforming growthfactorβand tumor necrosis factorαinhibit both apoptosis andproliferation of activated rat hepatic stellate cells.Hepatology,1999;30(1):196-202.
3 Foo NP,Lin SH,Lee YH,et al.α-Lipoic acid inhibits liverfibrosis through the attenuation of ROS-triggered signaling inhepatic stellate cells activated by PDGF and TGF-β.Toxicology,2011;282(1):39-46.
4 Bennett B,Bloom BR.Studies on the migration inhibitoryfactor associated with delayed-type hypersensitivity:cytodynamics and specificity.Transplantation,1967;5(4):996-1000.
5 Sivaram G,Tiwari SK,Bardia A,et al.Macrophage migrationinhibitory factor,Toll-like receptor 4,and CD14polymorphisms with altered expression levels in patients withulcerative colitis.Hum Immunol,2012;73(2):201-205.
6 Wong BLW,Zhu SL,Huang XR,et al.Essential role formacrophage migration inhibitory factor in gastritis induced byHelicobacter pylori.Am J Pathol,2009;174(4):1319-1328.
7 Liu XJ,Yang L,Luo F,et al.Association of differentiallyexpressed genes with activation of mouse hepatic stellate cellsby high-density cDNA mircoarray.World J Gastroenterol,2004;10(11):1600-1607.
8 Sato Y,Murase K,Kato J,et al.Resolution of liver cirrhosisusing vitamin A-coupled liposomes to deliver siRNA against acollagen-specific chaperone.Nat Biotechnol,2008;26(4):431-442.
9 Svegliati-Baroni G,Saccomanno S,Goor H,et al.Involvement of reactive oxygen species and nitric oxide radicalsin activation and proliferation of rat hepatic stellate cells.Liver,2008;21(1):1-12.
10 Reeves HL,Friedman SL.Activation of hepatic stellate cells-akey issue in liver fibrosis.Front Biosci,2002;7(4):808-826.
11 Zheng YX,Yang M,Rong TT,et al.CD74and macrophagemigration inhibitory factor as therapeutic targets in gastriccancer.World J Gastroenterol,2012;18(18):2253-2261.
12 Zhao YM,Wang L,Dai Z,et al.Validity of plasmamacrophage migration inhibitory factor for diagnosis andprognosis of hepatocellular carcinoma.Int J Cancer,2011;129(10):2463-2472.
2 Saile B,Matthes N,Knittel T,et al.Transforming growthfactorβand tumor necrosis factorαinhibit both apoptosis andproliferation of activated rat hepatic stellate cells.Hepatology,1999;30(1):196-202.
3 Foo NP,Lin SH,Lee YH,et al.α-Lipoic acid inhibits liverfibrosis through the attenuation of ROS-triggered signaling inhepatic stellate cells activated by PDGF and TGF-β.Toxicology,2011;282(1):39-46.
4 Bennett B,Bloom BR.Studies on the migration inhibitoryfactor associated with delayed-type hypersensitivity:cytodynamics and specificity.Transplantation,1967;5(4):996-1000.
5 Sivaram G,Tiwari SK,Bardia A,et al.Macrophage migrationinhibitory factor,Toll-like receptor 4,and CD14polymorphisms with altered expression levels in patients withulcerative colitis.Hum Immunol,2012;73(2):201-205.
6 Wong BLW,Zhu SL,Huang XR,et al.Essential role formacrophage migration inhibitory factor in gastritis induced byHelicobacter pylori.Am J Pathol,2009;174(4):1319-1328.
7 Liu XJ,Yang L,Luo F,et al.Association of differentiallyexpressed genes with activation of mouse hepatic stellate cellsby high-density cDNA mircoarray.World J Gastroenterol,2004;10(11):1600-1607.
8 Sato Y,Murase K,Kato J,et al.Resolution of liver cirrhosisusing vitamin A-coupled liposomes to deliver siRNA against acollagen-specific chaperone.Nat Biotechnol,2008;26(4):431-442.
9 Svegliati-Baroni G,Saccomanno S,Goor H,et al.Involvement of reactive oxygen species and nitric oxide radicalsin activation and proliferation of rat hepatic stellate cells.Liver,2008;21(1):1-12.
10 Reeves HL,Friedman SL.Activation of hepatic stellate cells-akey issue in liver fibrosis.Front Biosci,2002;7(4):808-826.
11 Zheng YX,Yang M,Rong TT,et al.CD74and macrophagemigration inhibitory factor as therapeutic targets in gastriccancer.World J Gastroenterol,2012;18(18):2253-2261.
12 Zhao YM,Wang L,Dai Z,et al.Validity of plasmamacrophage migration inhibitory factor for diagnosis andprognosis of hepatocellular carcinoma.Int J Cancer,2011;129(10):2463-2472.