肝衰竭患者血浆对永生化人肝细胞增殖和肝细胞功能蛋白表达的影响
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摘要
目的探讨肝衰竭患者血浆对永生化人肝细胞(immortalized human hepatocytes,IHH)增殖和肝细胞功能蛋白谷氨酰胺合成酶(glutamine synthetase,GS)、谷胱甘肽-S-转移酶(glutathione-S-transferase,GST)、尿苷二磷酸葡萄糖醛酸基转移酶(uridine diphosphate glucuronosyltransferase,UGT)和白蛋白(albumin,Alb)表达的影响。方法 IHH细胞分为观察组和对照组,对照组细胞采用含体积分数5%胎牛血清的培养基进行培养,观察组细胞采用含体积分数5%肝衰竭患者血浆的培养基进行培养,应用倒置显微镜观察2组细胞生长状态,采用CCK8法测定2组细胞第1、3、5、7、9天增殖情况,并绘制生长曲线;采用Western blot法检测2组细胞中肝细胞功能蛋白GS、GST、UGT、Alb及肝细胞生长因子(hepatocyte growth factor,HGF)蛋白表达水平;采用实时荧光定量PCR检测2组细胞中HGF mRNA表达水平。结果2组细胞形态正常,呈梭形贴壁生长,观察组细胞第5、7、9天时细胞增殖吸光度值(1.44±0.08、1.87±0.11、1.95±0.06)明显高于对照组(1.26±0.06、1.62±0.08、1.78±0.07)(P<0.05);观察组细胞Alb表达水平(1.32±0.07)高于对照组(0.94±0.05)(P<0.05),GS、GST、UGT表达水平与对照组比较差异无统计学意义(P>0.05);观察组细胞HGF mRNA和蛋白(7.940±0.203、1.17±0.05)表达水平均高于对照组(1.020±0.135、0.97±0.04)(P<0.05)。结论肝衰竭患者血浆可促进IHH细胞增殖,上调肝细胞功能蛋白Alb表达,同时对GS、GST、UGT蛋白表达无明显影响;其促增殖机制可能与HGF表达上调有关。
Objective To investigate the influence of the plasma from liver failure patients on the proliferation of immortalized human hepatocytes(IHH)and the expressions of hepatocyte function proteins as glutamine synthetase(GS),glutathione-S-transferase(GST),uridine diphosphate glucuronosyltransferase(UGT)and albumin(Alb)in patients with liver failure.Methods IHH cells were divided into observation group and control group.Control group was cultured with the medium containing 5% fetal bovine serum,and observation group was cultured with the medium containing 5% plasma from liver failure patients.The cell growth was observed by inverted microscope.The proliferation was determined by CCK8 assay on the 1 st,3 rd,5 th,7 th and 9 th days to draw growth curve.The expressions of GS,GST,UGT,Alb and hepatocyte growth factor(HGF)protein were detected by Western blot method.The expression of HGF mRNA was tested by real-time fluorescence quantitative PCR in both groups.Results The morphology was normal and in spindle-shaped adherent growth in two groups.The absorbance values of cell proliferation were significantly higher in observation group(1.44±0.08,1.87±0.11,1.95±0.06)than those in control group(1.26±0.06,1.62±0.08,1.78±0.07)on the 5 th,7 th and 9 th days(P<0.05).The Alb level was significantly higher in observation group(1.32±0.07)than that in control group(0.94±0.05)(P<0.05).There were no significant differences in the expressions of GS,GST and UGT proteins between two groups(P>0.05).The mRNA and protein of HGF were significantly higher in observation group(7.940±0.203,1.17±0.05)than those in control group(1.020±0.135,0.97±0.04)(P<0.05).Conclusion The plasma from liver failure patients can promote the proliferation of IHH and up-regulate the expression of Alb,while it shows no significant effect on the expressions of GS,GST and UGT proteins.The proliferation mechanism may be correlated with the up-regulation of HGF expression.
Objective To investigate the influence of the plasma from liver failure patients on the proliferation of immortalized human hepatocytes(IHH)and the expressions of hepatocyte function proteins as glutamine synthetase(GS),glutathione-S-transferase(GST),uridine diphosphate glucuronosyltransferase(UGT)and albumin(Alb)in patients with liver failure.Methods IHH cells were divided into observation group and control group.Control group was cultured with the medium containing 5% fetal bovine serum,and observation group was cultured with the medium containing 5% plasma from liver failure patients.The cell growth was observed by inverted microscope.The proliferation was determined by CCK8 assay on the 1 st,3 rd,5 th,7 th and 9 th days to draw growth curve.The expressions of GS,GST,UGT,Alb and hepatocyte growth factor(HGF)protein were detected by Western blot method.The expression of HGF mRNA was tested by real-time fluorescence quantitative PCR in both groups.Results The morphology was normal and in spindle-shaped adherent growth in two groups.The absorbance values of cell proliferation were significantly higher in observation group(1.44±0.08,1.87±0.11,1.95±0.06)than those in control group(1.26±0.06,1.62±0.08,1.78±0.07)on the 5 th,7 th and 9 th days(P<0.05).The Alb level was significantly higher in observation group(1.32±0.07)than that in control group(0.94±0.05)(P<0.05).There were no significant differences in the expressions of GS,GST and UGT proteins between two groups(P>0.05).The mRNA and protein of HGF were significantly higher in observation group(7.940±0.203,1.17±0.05)than those in control group(1.020±0.135,0.97±0.04)(P<0.05).Conclusion The plasma from liver failure patients can promote the proliferation of IHH and up-regulate the expression of Alb,while it shows no significant effect on the expressions of GS,GST and UGT proteins.The proliferation mechanism may be correlated with the up-regulation of HGF expression.
引文
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[13]Lee S,Han J,Choi D.Cell sources,liver support systems and liver tissue engineering:alternatives to liver transplantation[J].Int J Stem Cells,2015,8(1):36-47.
[14]Ramboer E,De C,De K,et al.Development and characterization of a new human hepatic cell line[J].EXCLI J,2015,14(1):875.
[15]Ramboer E,Vanhaecke 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.
[16]房青,高福云,徐梅,等.永生化人肝细胞治疗小鼠急性肝衰竭的实验研究[J].中国医药生物技术,2010,5(4):252-256.
[17]Filippi C,Keatch S,Rangar D,et al.Improvement of C3Acell metabolism for usage in bioartificial liver support systems[J].J Hepatol,2014,41(4):599-605.
[18]赵丽莉,唐淑珍,陈煜,等.慢性重型肝炎及肝衰竭患者血浆对C3A细胞活力和蛋白合成功能的影响[J].中国血液净化,2005,4(5):257-260.
[19]王玉萍,楚天骄,党秋红,等.乳腺癌中肝细胞生长因子及其受体C-Met和血管内皮生长因子表达的意义[J].实用诊断与治疗杂志,2006,20(5):336-338.
[20]Noji S,Tashiro K,Koyama E,et al.Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers,as revealed by in situ hybridization[J].Biochem Biophys Res Commun,1990,173(1):42-47.
[21]Cai H,Zhou Y,Jia W,et al.Effects of SNPs and alternative splicing within HGF gene on its expression patterns in Qinchuan cattle[J].J Anim Sci Biotechnol,2016,6(2):134-140.
[22]Ahn SY,Kim J,Kim MA,et al.Increased HGF expression induces resistance to c-MET tyrosine kinase inhibitors in gastric cancer[J].Anticancer Res,2017,37(3):1127-1138.
[23]Aasrum M,Brusevold IJ,Christoffersen T,et al.HGFinduced DNA synthesis in hepatocytes is suppressed by p38[J].Growth Factors,2016,34(5/6):217-223.
[2]Wenum M,Chamuleau R,Gulik T,et al.Bioartificial livers in vitro and in vivo:tailoring biocomponents to the expanding variety of applications[J].Expert Opin Biol Ther,2014,14(12):1745-1760.
[3]Cardoso FS,Marcelino P,Bagulho L,et al.Acute liver failure:an up-to-date approach[J].J Crit Care,2017,39:25-30.
[4]Davies NA,Ba1ares R.A new horizon for liver support in acute liver failure[J].J Hepatol,2015,63(2):388-398.
[5]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,2008,3(1):11-18.
[6]中华医学会感染病学分会肝衰竭与人工肝学组,中华医学会肝病学分会重型肝病与人工肝学组.肝衰竭诊治指南[J].实用肝脏病杂志,2013,16(3):210-216.
[7]Shalimar,Acharya SK.Management in acute liver failure[J].J Clin Exp Hepatol,2015,5(1):104-115.
[8]刘树人,罗显荣,张雁,等.肝功能衰竭患者医院感染的临床特点分析[J].中华实用诊断与治疗杂志,2011,25(11):1053-1055.
[9]Aron J,Agarwal B,Davenport A.Extracorporeal support for patients with acute and acute on chronic liver failure[J].Expert Rev Med Devices,2016,13(4):367-380.
[10]Nicolas C,Hickey R,Chen H,et al.Concise review:liver regenerative medicine:from hepatocyte transplantation to bioartificial livers and bioengineered grafts[J].Stem Cells,2017,35(1):42-50.
[11]Lee K,Stadlbauer V,Jalan R.Extracorporeal liver support devices for listed patients[J].Liver Transpl,2016,22(6):839-848.
[12]van Wenum M,Adam AA,Hakvoort TB,et al.Selecting cells for bioartificial liver devices and the importance of a 3Dculture environment:a functional comparison between the HepaRG and C3Acell lines[J].Int J Biol Sci,2016,12(8):964-978.
[13]Lee S,Han J,Choi D.Cell sources,liver support systems and liver tissue engineering:alternatives to liver transplantation[J].Int J Stem Cells,2015,8(1):36-47.
[14]Ramboer E,De C,De K,et al.Development and characterization of a new human hepatic cell line[J].EXCLI J,2015,14(1):875.
[15]Ramboer E,Vanhaecke 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.
[16]房青,高福云,徐梅,等.永生化人肝细胞治疗小鼠急性肝衰竭的实验研究[J].中国医药生物技术,2010,5(4):252-256.
[17]Filippi C,Keatch S,Rangar D,et al.Improvement of C3Acell metabolism for usage in bioartificial liver support systems[J].J Hepatol,2014,41(4):599-605.
[18]赵丽莉,唐淑珍,陈煜,等.慢性重型肝炎及肝衰竭患者血浆对C3A细胞活力和蛋白合成功能的影响[J].中国血液净化,2005,4(5):257-260.
[19]王玉萍,楚天骄,党秋红,等.乳腺癌中肝细胞生长因子及其受体C-Met和血管内皮生长因子表达的意义[J].实用诊断与治疗杂志,2006,20(5):336-338.
[20]Noji S,Tashiro K,Koyama E,et al.Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers,as revealed by in situ hybridization[J].Biochem Biophys Res Commun,1990,173(1):42-47.
[21]Cai H,Zhou Y,Jia W,et al.Effects of SNPs and alternative splicing within HGF gene on its expression patterns in Qinchuan cattle[J].J Anim Sci Biotechnol,2016,6(2):134-140.
[22]Ahn SY,Kim J,Kim MA,et al.Increased HGF expression induces resistance to c-MET tyrosine kinase inhibitors in gastric cancer[J].Anticancer Res,2017,37(3):1127-1138.
[23]Aasrum M,Brusevold IJ,Christoffersen T,et al.HGFinduced DNA synthesis in hepatocytes is suppressed by p38[J].Growth Factors,2016,34(5/6):217-223.