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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 effects of plasma in patients with liver failure on the proliferation of immortalized human hepatocytes (IHH) and the activities of hepatic cellular proteins glutamine synthetase (GS), glutathione-S -transferase, GST), uridine diphosphate glucuronosyltransferase (UGT) and albumin (Alb) were detected. Methods IHH cells were divided into observation group and control group. Cells in control group were cultured in medium containing 5% fetal bovine serum. Cells in observation group were cultured in medium containing 5% volume fraction of liver failure plasma. The growth of the two groups of cells was observed under a microscope. The proliferation of the two groups of cells on day 1, 3, 5, 7, and 9 was measured by CCK8 assay and the growth curve was drawn. Western blot was used to detect the expression of hepatic cell functional protein GS, GST , UGT, Alb and hepatocyte growth factor (HGF) protein levels were detected. The expression of HGF mRNA in the two groups of cells was detected by real-time fluorescence quantitative PCR. Results The morphology of the cells in two groups was normal and showed spindle-shaped adherent growth. The proliferative absorbance values (1.44 ± 0.08, 1.87 ± 0.11, 1.95 ± 0.06) on the 5th, 7th and 9th day in the observation group were significantly higher than those in the control group (1.26 ± 0.06 , 1.62 ± 0.08 and 1.78 ± 0.07 respectively) (P <0.05). The Alb expression level in the observation group (1.32 ± 0.07) was significantly higher than that in the control group (0.94 ± 0.05) (P <0.05) (P <0.05). The expression levels of HGF mRNA and protein in the observation group (7.940 ± 0.203, 1.17 ± 0.05) were significantly higher than those in the control group (1.020 ± 0.135, 0.97 ± 0.04, P <0.05) . Conclusions Plasma of patients with liver failure can promote the proliferation of IHH cells and up-regulate Alb expression of hepatocyte protein, and has no significant effect on the expression of GS, GST and UGT proteins. The mechanism of its promoting proliferation may be related to the up-regulation of HGF expression.