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Analysis of prognostic factors for patients with acute-on-chronic liver failure.

Xiao-yan LIU ; Jin-hua HU ; Hui-fen WANG

Chinese Journal of Hepatology.2009;17(8):607-610.

OBJECTIVETo analyze the prognostic factors for patients with acute-on-chronic liver failure, and to build a scoring system for assessment of the prognosis of liver failure.

METHODS480 patients with acute-on-chronic liver failure in our hospital from January 2006 to June 2008 were enrolled in this study. The patients were divided into improved group and deteriorated group. The clinical data were analyzed by using chi square test, independent-Samples T Test and Binary logistic regression.

RESULTSThe factors that significantly affected the prognosis of Acute-on-chronic Liver Failure included age, hepatitis or liver cirrhosis, Staging, Hyponatremias, alpha-fetoprotein (AFP), the prothrombin time activity (PTA), total bilirubin (TBil), creatinine (Cr), albumin (ALB) and Hepatic encephalopathy, ascites, alimentary tract hemorrhage (P less than 0.05, P less than 0.01). PTA, Hyponatremias, hepatitis or liver cirrhosis, Hepatic encephalopathy and alimentary tract hemorrhage were independent risk factors of prognosis.

CONCLUSIONPTA, Hyponatremias, hepatitis or liver cirrhosis, Hepatic encephalopathy and alimentary tract hemorrhage are important to build a scoring system to assess the prognosis of Acute-on-chronic Liver Failure and may be useful to guide clinical treatment.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biomarkers ; blood ; Child ; Child, Preschool ; Chronic Disease ; Female ; Hepatic Encephalopathy ; complications ; Hepatitis, Viral, Human ; complications ; epidemiology ; Humans ; Hyponatremia ; complications ; Infant ; Liver Failure, Acute ; blood ; etiology ; pathology ; Logistic Models ; Male ; Middle Aged ; Prognosis ; Prothrombin Time ; Retrospective Studies ; Risk Assessment ; Risk Factors ; Severity of Illness Index ; Young Adult

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Real-time quantification of microRNAs in Huh7 cells by stem-loop reverse transcriptase polymerase chain reaction.

Bang-dong GONG ; Qing XIE ; Lin WANG ; Xiao-gang XIANG ; Lan-yi LIN ; Gang-de ZHAO ; Hui WANG ; Hong YU

Chinese Journal of Hepatology.2009;17(8):603-606.

OBJECTIVETo establish a convenient realtime PCR which can detect microRNAs in the human hepatoma cell line, Huh7 cells.

METHODSTotal RNAs in Huh7 cells were extracted. MicroRNA 122, 24 and 146a were assayed by microRNA array, and then verified by Northern blot. Stem-loop RT-PCR and poly(A)-tailed RT-PCR were used to detect the above microRNAs. Data were analyzed with Quantity One software and 7500 system software.

RESULTSMicroarray signal intensity of microRNA 122, 24 and 146a in Huh7 cells was 2201.49, 410.20 and 4.70, whose relative expression was confirmed as 0.0383, 0.0249, 0.0001 through Northern blot. While the poly(A)-tailed RT-PCR might only measure microRNA 122, Stem-loop RT-PCR could detect microRNA 122, 24 and 146a, whose average dCt was 2.5, 5.8 and 12.1 in accordance with microRNA array and Northern blot.

CONCLUSIONStem-loop RT-PCR can specifically and sensitively quantity microRNA levels, regardless of their abundance.

Base Sequence ; Blotting, Northern ; Carcinoma, Hepatocellular ; genetics ; metabolism ; Cell Line, Tumor ; DNA Primers ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; metabolism ; MicroRNAs ; analysis ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

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Analysis on the expression of the stem cells related genes in hepatocellular carcinoma cell lines.

Can ZOU ; Fang-jun YUAN ; Wen-bo ZHOU ; Ling HUANG ; Ju WANG ; You-shun ZHANG

Chinese Journal of Hepatology.2009;17(8):599-602.

OBJECTIVETo investigate the expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines of SMMC-7721, Bel-7402, HepG2, MHCC-97 and normal hepatocellular cell line of L02, and to compare the response of these cell lines to all-trans retinoic acid.

METHODSRT-PCR was used to detect expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines and normal hepatocellular cell line. Real time-PCR was used to quantify the expression of the genes.

RESULTSThere are different levels of expression of the stem cell-related gene in hepatocellular carcinoma cell lines and control cell line (P less than 0.05). There are significant differences in HepG2 and L-02 for the response to all-trans retinoic acid (P less than 0.05).

CONCLUSIONSThe stem cell-related genes are differentially expressed in different hepatocellular carcinoma cell lines.

Carcinoma, Hepatocellular ; metabolism ; pathology ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Nanog Homeobox Protein ; Octamer Transcription Factor-3 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; SOXB1 Transcription Factors ; genetics ; metabolism ; Signal Transduction ; Smoothened Receptor ; Stem Cells ; drug effects ; metabolism ; Tretinoin ; pharmacology ; Wnt Proteins ; genetics ; metabolism ; beta Catenin ; genetics ; metabolism

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Reversal of multidrug resistance in hepatocellular cell line HepG2R by mdr1-antisense RNA.

Ying MEI ; Yu-jun SHI ; Xiong DING ; Hua-gang JIAN ; Jian-ping GONG ; Chang-an LIU ; Xiao-guang CHEN

Chinese Journal of Hepatology.2009;17(8):594-598.

OBJECTIVETo investigate whether multidrug resistance gene 1(mdr1) could reverse multidrug resistance (MDR) in HepG2R cells.

METHODSAn adenovirus vector, Adeno-asmdr, containing the antisense RNA driven by AFP promoter, was construct. Adeno-EGFP was transfected into HepG2, an AFP producing cell line, L02, a normal human liver cell line, and HeLa, a human cervical cancer cell line, the EGFP transcription level was detected by RT-PCR. Adeno-asmdr was transfected into HepG2R cells, the expression of P-gp170 was detected by western blotting, apoptosis was detected using TUNEL and flow cytometry, cell cycle was analyzed by flow cytometry.

RESULTSEGFP was highly expressed in HepG2 cells, however, its expression in L02 or HeLa cells was very weak. Western blot showed that the P-gp170 was marked down-regulated 48h after transfection with Adeno-asmdr, and the expression of P-gp170 was detectable at least 7d post-transfection. Compared with control cells, Adeno-asmdr transfected HepG2R cells were more sensitive to different chemicals, as indicated by TUNEL staining and flow cytometry. Chemical treatment arrested the cells in S and G0/M phase.

CONCLUSIONThe recombinant adenoviral vector, Adeno-asmdr, can block the expression of mdr1, and reverse MDR in HepG2R cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Adenoviridae ; genetics ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; genetics ; metabolism ; Cell Cycle ; drug effects ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Gene Expression Regulation, Neoplastic ; HeLa Cells ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; genetics ; metabolism ; RNA, Antisense ; Transfection

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Expression, purification and polyclonal antibody preparation for a novel gene BC097361.

Qi WANG ; Yun-lei LIANG ; Hong-shan WEI ; Hui-chun XING ; Jun CHENG ; Meng-dong LAN ; Bin ZHANG

Chinese Journal of Hepatology.2009;17(8):589-593.

OBJECTIVETo express and purify of the BC097361 recombinant protein, and to prepare the BC097361 specific rabbit polyclonal antibody.

METHODSBC097361 cDNA was ligated into the prokaryotic expressive vector pET-32a (+), and the resulting plasmid was transformed into E.coli BL21 (DE3). The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE and western blotting. The expressed product was purified using Ni+ affinity column chromatography.Then the purified pET-32a (+) -BC097361 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA.

RESULTSThe BC097361 fusion protein was highly expressed.The protein was mainly in the inclusion body. ELISA indicated the titer of polyclonal antibody more than 1:320000. The high specificity was confirmed with Western blot.

CONCLUSIONSThe recombinant BC097361 fusion protein and the BC097361 specific polyclonal antibody will be valuable tools for the investigation on the biological function of BC097361.

Angiotensin II ; genetics ; Animals ; Antibodies ; immunology ; isolation & purification ; metabolism ; Antibody Specificity ; Blotting, Western ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Liver Cirrhosis ; genetics ; Male ; Plasmids ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Reverse Transcriptase Polymerase Chain Reaction

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Insertion of TC motif into hepatitis B virus core protein c/e1 site does not affect the expression of S and e antigen.

Yuan-yuan LIN ; Xiao-ming CHENG ; Yu-hu SONG ; Ying CHANG ; Jin-jian YAO ; Ju-sheng LIN

Chinese Journal of Hepatology.2009;17(8):585-588.

OBJECTIVETo investigate whether insertion of TC motif into hepatitis B virus (HBV) core protein c/e1 site affects the expression of S and e antigen.

METHODSDifferent oligonucleotides encoding TC motif were inserted into the c/e1 site of the core gene of a 1.3 copy wild-type HBV genome vector. HepG2 cells were divided into several groups of cells to transiently transfect with the wild-type and mutant HBV vectors, respectively. In each group, the expression level of core protein inside cells was detected by western blotting, and the levels of S and e antigen in culture medium were analyzed by ELISA assay.

RESULTSWestern blotting showed that these TC-tagged core proteins were expressed at similar level of wild-type one. ELISA assay indicated that the level of S and e antigen in culture medium of different groups were not significantly different.

CONCLUSIONInsertion of TC motif into HBV core protein c/e1 site does not interference with the expression of viral protein encoded by HBV genome.

Amino Acid Sequence ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; genetics ; metabolism ; Hep G2 Cells ; Hepatitis B Core Antigens ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; Humans ; Mutagenesis, Insertional ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Viral Core Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

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Long-term observation of hepatitis B virus (HBV) replication in new-born tree shrews inoculated with HBV.

Fang YANG ; Ji CAO ; Jing-jing ZHANG ; Qi WANG ; Jian-jia SU ; Chun YANG ; Chao OU ; Jun-lin SHI ; Duo-ping WANG ; Yuan LI

Chinese Journal of Hepatology.2009;17(8):580-584.

OBJECTIVETo observe the hepatitis B virus (HBV) replication in the tree shrews that were inoculated with HBV at neonatal period.

METHODSSix new-born tree shrews were inoculated with human HBV positive serum. Blood samples and liver biopsies were collected at different time points after inoculation. The HBV infection markers were tested by nested polymerase chain reaction (nPCR), fluorescence quantitative polymerase chain reaction (FQ-PCR), Southern blot, ELISA and immunohistochemistry staining. The liver tissues were observed under electron and light microscope.

RESULTS48 weeks after inoculation, HBV DNA and HBV cccDNA were detected in the serum and liver samples of three animals (number 1, 2 and 6) by nPCR. The copy-numbers of HBV DNA detected by FQ-PCR in their serum and liver samples were 103 and-104/ml respectively,and the total DNA in 1microg liver tissue was 107-108. Southern blot indicated that HBV replication intermediates such as HBV cccDNA and HBV ssDNA was detectable in liver tissues. HBsAg was detected by ELISA, and immunohistochemical staining showed a gradual increase of HBsAg-positive liver cells. High copy number of HBV DNA and suspected HBV EM particles could be detected in the liver samples from one of the three animals that have survived more than 2 years after inoculation. The other three animals showed low HBV DNA copy number, and the rest of the signs of HBV infection were negative or transiently positive.

CONCLUSIONSNeonatal tree shrews can be infected with human HBV. HBV can replicate inside the liver cells of tree shrew.

Animals ; Animals, Newborn ; Biopsy ; DNA, Circular ; analysis ; blood ; DNA, Viral ; analysis ; blood ; Disease Models, Animal ; Hepatitis B ; etiology ; pathology ; virology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; physiology ; Humans ; Immunohistochemistry ; Liver ; pathology ; virology ; Polymerase Chain Reaction ; methods ; Tupaiidae ; Virus Replication

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Human plasmacytoid dendritic cells from patients with chronic hepatitis B induce the generation of CD4+CD25+Treg in vitro.

Jun HONG ; Yan LI ; Zuo-jiong GONG

Chinese Journal of Hepatology.2009;17(8):574-579.

OBJECTIVETo compare the ability of plasmacytoid dendritic cells (pDCs) from chronic HBV patients and healthy controls to induce the the generation of CD4+CD25+Treg cells.

METHODSThe pDCs were isolated from PBMCs of 46 chronic HBV patients, 10 resolved HBV patients and 25 healthy controls by magnetic cell sorting. Purified CD4+CD45RA+ naive T cells were incubated with allogeneic pDCs from chronic HBV infected patients, resolved HBV patients or healthy controls. The cells were stimulated with HBcAg or tetanus toxin. The proportion of CD4+CD25+Treg in CD4+ T cells primed by pDCs was determined by flow cytometry, the expression of Fox p3 mRNA was detected by RT-PCR, IL-10 and TGFb1 expression was quantified using ELISA kits.

RESULTSCompared with pDC isolated from healthy controls and the resolved HBV patients, pDC from chronic HBV patients was more effective in suppression of CD4+ T cells proliferation and interferon production when CD25-depleted PBMCs were stimulated with HBcAg, (7999.36+/-374.74 vs 11 282.56+/-1174.46; 7999.36+/-374.74 vs 12 304.58+/-1462.81, P less than 0.05 ). Depletion of CD4+CD25+ Treg from CD4+ T cells primed by pDC led to the lose of capability to suppress HBV-specific T-cell responses. When CD25- depleted PBMCs were stimulated with purified tetanus toxin, there was no significantly difference in proliferation between CD25-depleted PBMC co-cultured with pDC-primed CD4+ T cells and CD25-depleted PBMC cultured without pDC-primed CD4+ T cells. A higher percentage of CD4+CD25+ Treg was detected within the population of CD4+ T cells primed by pDC from chronic HBV patients compared with healthy controls and resolved HBV patients (5.99%+/-1.85% vs 3.04%+/-0.79%; 5.99%+/-1.85% vs 3.01%+/-1.53%, P less than 0.05). Accordingly, CD25+Treg from pDC-primed CD4+ T cells displayed a higher Fox P3 mRNA level. The IL-10 and TGFb1 could be also detectable in the supernatants of pDC-primed CD4+ T cells.

CONCLUSIONpDCs from chronic hepatitis B induce the generation of a higher proportion of CD4+CD25+ Treg compared with pDCs from healthy controls.

Adult ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; methods ; Cytokines ; metabolism ; Dendritic Cells ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Hepatitis B Core Antigens ; pharmacology ; Hepatitis B, Chronic ; immunology ; metabolism ; pathology ; Humans ; Male ; Middle Aged ; Monocytes ; cytology ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; immunology ; metabolism ; Young Adult

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Pharmacoeconomic evaluation of telbivudine vs. lamivudine in treating the patients with HBeAg-positive and negative chronic hepatitis B.

Wen CHEN ; Jin-Lin HOU

Chinese Journal of Hepatology.2009;17(8):569-573.

OBJECTIVETo evaluate long-term cost effectiveness of telbivudine and lamivudine for the treatment of CHB.

METHODSCost effectiveness was conducted from social health insurance perspective. A Markov model was established based on disease progression pattern and the data from the 2 years GLOBE clinical trial. The information of annual medical expenditure and quality-of-life assessment for different CHB-related diseases was obtained from literature. Incremental cost per life year or quality-adjusted life year gained was measured.

RESULTSCompared with lamivudine, the incremental cost for 1 additional QALY gained with telbivudine in treating HBeAg-positive and -negative CHB were 5403 yuan and 28239 yuan in Beijing, as well 4916 yuan and 29618 yuan in Guangzhou, respectively. According to national economic burden of CHB-related diseases, the ICER with telbivudine vs lamivudine were 1282 yuan and 31565 yuan for HBeAg-positive and -negative CHB.

CONCLUSIONAccording to WHO recommendation for ICER threshold, telbivudine is cost effective in treating HBeAg-positive and -negative CHB, as compared to lamivudine.

Adult ; Antifungal Agents ; economics ; therapeutic use ; China ; epidemiology ; Cost-Benefit Analysis ; DNA, Viral ; blood ; Drug Costs ; statistics & numerical data ; Economics, Pharmaceutical ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; complications ; drug therapy ; economics ; epidemiology ; Humans ; Insurance, Long-Term Care ; Lamivudine ; economics ; therapeutic use ; Liver Cirrhosis ; etiology ; prevention & control ; Male ; Markov Chains ; Middle Aged ; Models, Economic ; Nucleosides ; economics ; therapeutic use ; Prescription Drugs ; economics ; Pyrimidinones ; economics ; therapeutic use ; Quality-Adjusted Life Years ; Thymidine ; analogs & derivatives

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Factors associated with response to lamivudine: retrospective study of 233 patients with chronic hepatitis B.

Hua-jian ZHOU ; Shao-guang LI ; Fan-yuan WEN ; Xiao-yun YANG ; Jing-li WU ; Bin TAN ; Juan FU

Chinese Journal of Hepatology.2009;17(8):564-568.

OBJECTIVETo identify factors associated with response to lamivudine in chronic hepatitis B patients.

METHODSClinical data of 233 chronic hepatitis B patients treated with lamivudine 100mg daily (91 patients were switched to Adefovir 10mg daily or Adefovir 10mg in combination with lamivudine 100mg daily) were retrospective. HBV DNA level and serum HBV markers were detected by polymerase chain reaction and enzyme-linked immunosorbent assay. Kaplan-Meier, long-rank, t test were conducted to evaluate the data.

RESULTS(1) The rates of HBV DNA loss, ALT normalization, viral breakthrough(VB), HBeAg loss and seroconversion were 63.4% , 83.8%, 30.9%, 30.9%, and 14.3%, respectively, in HBeAg(+) patients; and these were 84.6%, 81.3%, 14.3%, respectively in HBeAg(-) patients.(2) The rates of HBV DNA loss, HBeAg loss, HBeAg seroconversion, viral breakthrough (VB) were 55% and 66.7% (P more than 0.05), 55.0%, and 66.7% (P less than 0.05), 17.5% and 33.3% (P less than 0.05), 50% and 34.3% (P less than 0.05) in HBeAg(+) patients with baseline ALT less than 2.5 ULN and HBeAg(+) patients with baseline ALT is more than or equal to 2.5 ULN, respectively. (3) For HBeAg(+) patients, viral breakthrough rate was significantly lower in patients with baseline HBV DNA less than 10(6) copies/ml than that in patients with baseline HBV DNA more than 10(6) copies/ml patients (23.4% VS 46.3%, P less than 0.05) among HBeAg(+) patients. (4) The rate of HBV DNA loss, HBeAg loss, HBeAg seroconversion and viral breakthrough for the patients with IVR at week 24 were 76.3%, 72.3%, 40.8% and 28.9% compared to 47.6% (P less than 0.01), 46% (P less than 0.01), 12.7% (P less than 0.01) and 47.6% (P less than 0.05) for those without IVR. (4) For the 44 patients with viral breakthrough, 32 were switched to Adefovir monotherapy or adefovir in combination with lamivudine therapy, and 12 continued to receive lamivudine monotherapy. HBV DNA loss, HBeAg seroconversion were 40.6%, 21.9% for those switch/add group compare to 16.7%, 16.7% for the lamivudine monotherapy group. There were no significant differences in the background factors (sex, diagnosis, antiviral period, pre-tx ALT, pre-tx HBV DNA) between these two groups.

CONCLUSIONBoth the baseline ALT and HBV DNA are associated with the efficacy of lamivudine in chronic hepatitis B patients. Patients with baseline ALT is more than or equal to 2.5*ULN and (or) HBV DNA level of less than 1*10(6) copies/ml have better efficacy and lower rate of breakthrough rate. IVR at week 24 is an important predictive factor of a favorable response to lamivudine therapy. For the patients with viral breakthrough, those switched to/added on Adefovir have a favorable result.

Adenine ; analogs & derivatives ; pharmacology ; therapeutic use ; Adolescent ; Adult ; Alanine Transaminase ; blood ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA, Viral ; blood ; Drug Administration Schedule ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; drug effects ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Middle Aged ; Organophosphonates ; pharmacology ; therapeutic use ; Retrospective Studies ; Reverse Transcriptase Polymerase Chain Reaction ; Treatment Outcome ; Young Adult

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