Table of Content

30 April 2016, Volume 21 Issue 4

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Original Articles

Performance comparison between QIAGEN real-time PCR and COBAS TaqMan for serum HBV DNA quantitation in predicting the pathological state of liver tissue from chronic hepatitis B patients

ZHANG Zhan-qing, LU Wei, WANG Ping-an, WANG Yan-bing, ZHOU Xin-lan, DING Rong-rong, LI Xiu-fen, HUANG Dan

2016, 21(4):  241-247. 

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Objective To investigate and compare the performance of QIAGEN real-time PCR and COBAS TaqMan for serum hepatitis B virus (HBV) DNA quantitation in predicting the pathological state of liver tissue from chronic hepatitis B patients.Methods A total of 278 patients with chronic hepatitis B, including 162 HBeAg-positive and 116 HBeAg-negative ones, were enrolled in this study. Serum HBV DNA (QIAGEN) and HBV DNA (COBAS) were detected by real-time quantitative PCR and Roche TaqMan COBAS system, respectively. The Scheuer score system used for pathological diagnosis of liver tissue had 5 grades (G0-G4) and 5 stages (S0-S4), respectively. Results In HBeAg-positive patients, there were statistically significant differences of serum HBV DNA (QIAGEN) and HBV DNA (COBAS) between G2 and G3, S1 and S4, S2 and S4, S3 and S4 (all P<0.05), respectively; in HBeAg-negative patients, there were statistically significant differences between G1 and G2, G1 and G3, S1 and S2, S1 and S3, S1 and S4 (all P<0.05), respectively. Rates of disagreement between serum HBV DNA (QIAGEN) and HBV DNA (COBAS) quantitation of G1-2, G3, S1-3 and S4 in HBeAg-positive patients were 4.24% (5/118), 9.09% (4/44), 3.68%(5/136) and 7.69%(2/26), respectively; and the rates of G1, G2-3, S1 and S2-4 in HBeAg-negative patients were 6.02% (5/83), 3.03% (1/33), 3.29% (2/61) and 3.64% (2/55), respectively. Areas under the receiver operating characteristic (ROC) curves of serum HBV DNA (QIAGEN) and serum HBV DNA (COBAS) for predicting ≥G3 and S4 in HBeAg-positive patients were 0.645 and 0.695, 0.703 and 0.755, respectively; and the areas for predicting ≥G2 and ≥S2 in HBeAg-negative patients were 0.848 and 0.817, 0.756 and 0.756, respectively. Optimal cut-offs of serum HBV DNA (QIAGEN) and serum HBV DNA (COBAS) for predicting S4 in HBeAg-positive patients were ≤3.784×106 IU/mL and ≤6.668×10⁷ IU/mL, respectively; and the corresponding sensitivities and specificities were 0.654 and 1.000, 0.735 and 0.581, respectively; and the optimal cut-offs of serum HBV DNA (QIAGEN) and serum HBV DNA (COBAS) for predicting ≥G2 in HBeAg-negative patients were ≥5.821×10³ IU/mL and 9.311×10³ IU/mL, respectively; and the corresponding sensitivities and specificities were 0.970 and 0.909, 0.614 and 0.651, respectively. Conclusion The most efficiency of serum HBV DNA for predicting the pathological state of liver tissue is characterized by predicting ≥G2 in HBeAg negative patients, and there is a high consistance of the performance between serum HBV DNA (QIAGEN) and serum HBV DNA (COBAS) for predicting ≥G2 in HBeAg negative patients.

Serum C-terminal agrin fragment level in AKI patients with ACLF and its clinical diagnostic value

YOU Guo-qiong, WANG Li, DUAN Meng

2016, 21(4):  248-252. 

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Objective To investigate serum C-terminal agrin fragment (CAF) level and its clinical diagnostic value in acute kidney injury (AKI) patients with acute-on-chronic liver failure (ACLF).Methods A total of 347 patients with hepatitis B virus (HBV) related ACLF from April 2012 to February 2015 in our center were enrolled retrospectively, and then divided into AKI group (n=165) and non-AKI group (n=182). The general information and routine laboratory parameters were recorded, and the serum CAF level was detected. The correlation between CAF and parameters was analyzed, and diagnostic efficiency of CAF for AKI in patients with ACLF was evaluated by receiver operating characteristic (ROC) curve. Results The level of CAF in AKI group (1243.3(678.4-436.5) pg/mL) was significantly higher than that in non-AKI group (250.2 (2105.9 ~ 135.7) pg/mL, P<0.001), and increased with different stage of AKI, in order from AKI-1 to AKI-2 to AKI-3. In addition, the CAF levels were significantly negatively correlated with mean arterial pressure (MAP) (R=-0.242, P=0.004) and glomerular filtration rate (R=-0.480, P<0.001), while positively correlated with alanine aminotransferase level (R=0.222, P=0.004), white blood cell count (R=0.212, P=0.006), serum creatinine (sCr) level (R=0.272, P<0.001) and model for end stage liver disease score (R=0.392, P<0.001). Area under the curve (AUC) of CAF used for the diagnosis of AKI in patients with ACLF was 0.859 (95%CI: 0.819-0.898), and the AUC of CAF with sCr was 0.923 (95%CI: 0.894-0.951).Conclusion CAF can reflect the severity of renal damage of AKI in patients with ACLF, and it can be combined with sCr to improve the diagnosis efficiency of AKI.

Expression and significance of DHPS,an activating factor of eIF5A,in nonalcoholic fatty liver disease

MIAO Qi, BIAN Zhao-lian, WANG Zhao-yue, PENG Yan-shen, MA Xiong

2016, 21(4):  253-258. 

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Objective To investigate the expression and mechanism of deoxyhypusine synthase (DHPS), an activating factor of eIF5A, in high-fat (HF) diet-fed mice and nonalcoholic fatty liver disease (NAFLD) patients.Methods Immunohistochemistry, western blot and real-time polymerase chain reaction (PCR) were performed to identify the DHPS expression in both HF mice and normal diet-fed (ND) mice. Hematoxylin-eosin (H&E) and oil red staining were conducted between DHPS overexpression group and green fluorescent protein (GFP) control group to compare the degree of liver steatosis and inflammation. In addition, PCR arrays were made to find potential genes related to DHPS. Immunohistochemistry and immunofluorescence were conducted to identify the expression of DHPS in NAFLD patients and HL-7702 cell line modeled with OP 2:1. Results HF mice highly expressed DHPS (1.80 vs 0.69, P<0.05), especially in liver (13.21 vs 1.00, P<0.01) and adipose (7.97 vs 1.00, P<0.01) tissue. Mice with overexpression DHPS endured more severe hepatic steatosis compared with mice treated with GFP (2.78 vs 2.20, P<0.05), which might be correlated with Ascbg2, Irs1, iNos and IL-6. Expression of DHPS was more common in NAFLD patients than that in normal controls (1.29 vs 0.50, P<0.05). In histology, the number of DHPS-positive cells was correlated with the degree of steatosis (r=0.5927,P=0.018). HL-7702 treated with OP 2:1 showed higher expression of DHPS.Conclusion Expression of DHPS is significantly increased in HF mice, liver tissue in patients with NAFLD and HL-7702 cell line treated with OP, which suggests an important role of DHPS in the pathogenesis of NAFLD.

The role of interleukin-21 in Th17/Treg imbalance in patients with chronic hepatitis B

GENG Jian, LIU Rui-xia, GAO Xi-yang, LI Yan, PAN Xiu-cheng, FU Juan-juan, LI Li

2016, 21(4):  259-262. 

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Objective To investigate the role of interleukin-21 in T helper cell 17 (Th17)/regulatory T cells (Treg) imbalance in chronic hepatitis B (CHB) patients, and its mechanism.Methods Frequencies of Th17 cells and Treg in peripheral blood mononuclear cells (PBMCs) of 76 CHB patients and 20 health controls were detected by flow cytometry, and serum levels of interleukin (IL)-21, IL-17, transforming growth factor (TGF)-β and IL-10 were measured by enzyme-linked immune-sorbent assay (ELISA). Under stimulation of IL-21 or IL-2 in vitro, levels of RORγt and Foxp3 mRNA in PBMCs were detected by real time reverse transcription polymerase chain reaction (RT-PCR), and levels of IL-17, IL-10, TGF-β in supernatants were determined by ELISA assay, respectively. Results The level of IL-21 was positively associated with the ratio of Th17 to Treg, which was significantly enhanced in CHB patients when compared with that in health controls (P<0.05). In IL-21 stimulated PBMCs of CHB patients, the expression of RORγt mRNA was significantly increased (P=0.016), while the expression of Foxp3 mRNA was obviously decreased (P=0.018), comparing with those in IL-2 stimulation group. Additionally, level of IL-17 in the corresponding supernatants was notably enhanced (P=0.049), while levels of IL-10 and TGF-β were decreased significantly (P=0.017, P=0.004), respectively.Conclusion IL-21 could enhance the ratio of Th17 to Treg through increasing the expression of RORγt mRNA and decreasing the expression of Foxp3 mRNA in patients with CHB.

Inhibition of zinc-2 glycoprotein in a rat model of hepatocellular carcinoma

LIAO Hong-yu, YU Jing-xia, LIU Ting, LU Lun-gen, XU Ming-yi

2016, 21(4):  263-266. 

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Objective To investigate the expression of zinc-α2-glycoprotein (AZGP1) in a rat model of hepatocellular carcinoma (HCC), and its relationship with HCC progression.Methods Cirrhosis and HCC in rat models were established by diethylnitrosamine (DEN) water feeding, and subcutaneous tumor and orthotopic liver transplantation models in nude mice were constructed by injection of HepG2 cells with AZGP1 gene over-expressed interferenced vector. The expressions of AZGP1 and transforming growth factor β1 (TGFβ1) were detected by immunohistochemistry staining, westernblot (WB) and polymerase chain reaction (PCR), respectively, then the relationship among AZGP1, tumor size and metastasis rate was evaluated. Results The level of AZGP1 mRNA (0.20±0.02, p=0.003) was lower in HCC tissues than that in normal or cirrhosis tissues. Hepatic expression of AZGP1 proteins was absent or low-expressed, which was confirmed by quantitative immunohistochemical and WB analysis. Hepatic TGFβ1 gene and protein in cirrhotic and HCC tissues were significantly increased. The size of subcutaneous tumors had no difference between AZGP1 over-expressed HepG2 cells treated group (HepG2-AZGP1 group) and the control group (HepG2-GFP group). The incidence of lung metastasis was 57% in HepG2-AZGP1 group and 14% in HepG2-GFP group at week 6 after transplantation of subcutaneous tumors in nude mice. Compared with those in HepG2-GFP group, the numbers and sizes of tumor nodules in liver and lung were significantly decreased and the distinct atypia degrees of cancer cells were significantly lower in HepG2-AZGP1 group.Conclusion During the progression from cirrhosis to HCC, AZGP1 loss occurred accompanied with derepression of TGFβ1, which was proposed that function recovery of AZGP1 might be a new promising approach to reversing HCC.

The expression profiles and significance of IL-33 and its receptor ST2 in a murine model of D-GalN/LPS -induced acute liver failure

JIANG Shao-wen, LIN Lan-yi, XIANG Xiao-gang, LU Jie, WANG Fan, MO Rui-dong, LIU Yu-han, CAI Wei, WANG Hui, XIE Qing

2016, 21(4):  267-272. 

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Objective To investigate the expression profiles and implication of IL-33 and its receptor ST2 in a murine model of acute liver failure (ALF) induced by D-GalN/LPS.Methods The ALF murine model was set up by intraperitoneal injection of D-GalN (900 mg/kg)/LPS(10 ug/kg), and confirmed by histopathology and biochemistry. Dynamic expression profiles of IL-33 and its receptor ST2 in ALF murine model were investigated by quantitative polymerase chain reaction (q-PCR), western-blot, enzyme-linked immune-sorbent assay (ELISA) and immunohistochemistry at different time points, respectively. Results The murine model of ALF was successfully established by intraperitoneal injection of D-GalN (900 mg/kg)/LPS(10 ug/kg). The mRNA level of intra-hepatic IL-33 continuously increased with the progression of ALF, and reached the peak at 7 h after D-GalN/LPS challenge. Compared to baseline, intra-hepatic ST2L protein level was markedly up-regulated at 3 h, which was before the occurrence of obvious hepatocytes damage. However, it declined later on and fall to the lowest at 7 h. In addition, it was observed that IL-33 protein level in serum was elevated sustainedly over time and reached the highest level at 7 h, which was consistent with its dynamic mRNA changes. In contrast, the serum level of sST2 had no significant differences between 0 h and 3 h at the early stage of liver damage, but showed an obvious rise to climax at 5 h in the medium-term of liver damage, and then was followed by a drastic decline. The immunohistochemistry results confirmed that intra-hepatic IL-33 was mainly located in the nucleus of endothelial cells and sinusoidal cells in ALF mouse liver.Conclusion The dynamic changes of IL-33 and its receptor ST2 in ALF mice are closely linked with the progression of acute liver failure, suggesting that IL-33/ST2 axis is indeed involved in the process of ALF. This might provide a novel potential target for ALF treatment.