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To detect the levels of miR-146a and miR-155 in different samples from chronic hepatitis B (CHB), reveal whether there is a correlation between the 2 miRNAs in different samples, and to provide a theoretical basis for sample choice of miRNA research in liver. Methods: Real-time PCR was conducted to examine the expression of miR-146a and miR-155 in the plasma, peripheral blood mononuclear cell (PBMC), and liver tissues from 41 CHB patients who underwent nucleoside analogues antiviral therapy for 104 weeks. Correlations between the levels of miR-146a and miR-155 among the 3 samples were analyzed. Results: The expressions of miR-146a and miR-155 in the plasma, PBMC and liver tissues were significantly down-regulated at the 104th week than those at the baseline (all P0.05). Conclusion: Compared with PBMC, miR-146a and miR-155 from plasma can better reflect the expression in the liver tissues, suggesting that plasma can be applied in the mechanism research on miR-146a and miR-155 in the liver diseases instead of liver tissues.
الموضوعات
Humans , Hepatitis B, Chronic , Genetics , Leukocytes, Mononuclear , MicroRNAs , Genetics , Real-Time Polymerase Chain Reactionالملخص
Objective:To investigate expression profiles of the plasma exosomal miRNAs of the chronic hepatitis B (CHB) patients with persistently normal alamine aminotransferase (PNALT) for the first time and try to find exosomal miRNAs which could reflect liver inflammation better.Methods:Five CHB patients with liver tissue inflammation grade ≥A2 of PNALT and 5 CHB patients with liver tissue inflammation grade ＜A2 of PNALT were enrolled and their blood samples were collected.The exosomes were extracted from these blood samples and measured by electron microscope to determine the extraction effect.The exosomal miRNAs were extracted and sent for high throughput sequencing,and the expression of exosomal miRNAs in the 2 groups of patients was analyzed.Results:Under the electron microscope,exosomes were small membranous vesicles with 30-100 nm in diameter.The peak value of particle size ranged from 10 to 100 nm.High throughput sequencing showed that there were 591 differentially expressed exosomal miRNAs between the 2 groups.Compared with the control group,18 exosomal miRNAs were up-regulated and 6 exosomal miRNAs were down-regulated in PNALT patients with the liver tissue inflammation grade ≥ A2.Conclusion:Exosomal miRNAs in the CHB patients with PNALT who have the different grades of liver inflammation are differently expressed.Some of the differently expressed exosomal miRNAs are expected to be sensitive biomarkers for timely assessment of liver inflammation in the CHB patients with PNALT.
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Objective To evaluate the accuracy and feasibility of time-resolved immunofluorometric assay (TRI FA) for detection of HBsAg based on Abbott automated chemiluminescence immunoassay(CMIA),so as to carry out this project in primary hospitals,and provide reference for individual antiviral strategy and prediction of therapeutic effect.Methods Serum of 157 patients infected with hepatitis B virus were detected with CMIA and TRIFA,specimens with HBsAg titers exceeding the detection limit were firstly diluted,then performed quantitative analysis.HBsAg levels were divided into 4 groups:≤100 IU/mL,101-1 000 IU/mL,1 001-20 000 IU/mL,and ＞ 20 000 IU/mL,quantitative correlation between two methods was analyzed.Results The linear regression equation of two methods was Y=2.323X-896.3,correlation coefficent r=0.943,P＜0.001.CMIA was as a reference,4 groups were divided for analysis,results showed that when detected specimens was at low concentration of HBsAg,TRIFA value was low compared with CMIA method,while detected specimens was at high concentration of HB sAg,CMIA value was high,two reagents had good consistency in the detection of different concentrations of HBsAg(both P＜0.05),when concentration was at 1 001-20 000 IU/mL,consistency was the best.Conclusion The accuracy of two reagents in the quantitative detection of HBsAg is similar,and the best correlation of detection value is 1 000-20 000 IU/mL.TRIFA assay has wide application for its low-cost and easy to be operated,which is especially suitable for primary hospitals.
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Objective@#To investigate the clinical effect and safety of long-acting pegylated interferon-α-2b (Peg-IFN-α-2b) (Y shape, 40 kD) injection (180 μg/week) in the treatment of HBeAg-positive chronic hepatitis B (CHB) patients, with standard-dose Peg-IFN-α-2a as positive control.@*Methods@#This study was a multicenter, randomized, open-label, and positive-controlled phase III clinical trial. Eligible HBeAg-positive CHB patients were screened out and randomized to Peg-IFN-α-2b (Y shape, 40 kD) trial group and Peg-IFN-α-2a control group at a ratio of 2:1. The course of treatment was 48 weeks and the patients were followed up for 24 weeks after drug withdrawal. Plasma samples were collected at screening, baseline, and 12, 24, 36, 48, 60, and 72 weeks for centralized detection. COBAS® Ampliprep/COBAS® TaqMan® HBV Test was used to measure HBV DNA level by quantitative real-time PCR. Electrochemiluminescence immunoassay with Elecsys kit was used to measure HBV markers (HBsAg, anti-HBs, HBeAg, anti-HBe). Adverse events were recorded in detail. The primary outcome measure was HBeAg seroconversion rate after the 24-week follow-up, and non-inferiority was also tested. The difference in HBeAg seroconversion rate after treatment between the trial group and the control group and two-sided confidence interval (CI) were calculated, and non-inferiority was demonstrated if the lower limit of 95% CI was > -10%. The t-test, chi-square test, or rank sum test was used according to the types and features of data.@*Results@#A total of 855 HBeAg-positive CHB patients were enrolled and 820 of them received treatment (538 in the trial group and 282 in the control group). The data of the full analysis set showed that HBeAg seroconversion rate at week 72 was 27.32% in the trial group and 22.70% in the control group with a rate difference of 4.63% (95% CI -1.54% to 10.80%, P = 0.1493). The data of the per-protocol set showed that HBeAg seroconversion rate at week 72 was 30.75% in the trial group and 27.14% in the control group with a rate difference of 3.61% (95% CI -3.87% to 11.09%, P = 0.3436). 95% CI met the non-inferiority criteria, and the trial group was non-inferior to the control group. The two groups had similar incidence rates of adverse events, serious adverse events, and common adverse events.@*Conclusion@#In Peg-IFN-α regimen for HBeAg-positive CHB patients, the new drug Peg-IFN-α-2b (Y shape, 40 kD) has comparable effect and safety to the control drug Peg-IFN-α-2a.
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Objective:To observe the sensitivity of transcription mediated amplification (TMA),and to compare its performance with real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in detecting human immunodeficiency virus RNA (HIV RNA).Methods:TMA system was established with TaqMan probes,specific primers,moloney murine leukemia virus (MMLV) reverse transcriptase,T7 RNA polymerase,and reaction substrates.The sensitivity of TMA was evaluated by amplifying a group of 10-fold diluted HIV RNA standards which were transcribed in vitro.A total of 60 plasma of HIV infected patients were measured by TMA and Cobas Amplicor HIV-1 Monitor test to observe the positive rate.The correlation and concordance of the above two technologies were investigated by linear regression and BlandAltman analysis.Results:TMA system was established successfully and HIV RNA transcribed standards at concentration of equal or more than 10 copies/mL could be detected by TMA technology.Among 60 samples of plasma from HIV infected patients,46 were positively detected and 12 were negatively amplified by both TMA and Cobas reagents;2 samples were positively tested by Cobas reagent but negatively tested by TMA system.The concordance rate of the two methods was 97.1％ and the difference of positive detection rate between the two methods was not statistically significant (P＞0.05).Linear regression was used for 46 samples which were positively detected by both TMA and Cobas reagents and showed an excellent correlation between the two reagents (r=0.997,P＜0.001).Bland-Altma analysis revealed that the mean different value ofHIV RNA levels for denary logarithm was 0.02.Forty-four samples were included in 95％ of credibility interval of concordance.Conclusion:TMA system has the potential of high sensitivity.TMA and real-time RT-PCR keep an excellent correlation and consistency in detecting HIV RNA.
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<p><b>OBJECTIVE</b>To evaluate the effect of different immune status on the incidence of hepatic lesions in patients with hepatitis B virus (HBV) infection undergoing anti-tuberculosis therapy.</p><p><b>METHODS</b>The PubMed (1966-2013), Embase (1966-2013), Wanfang (1998-2013), Chinese National Knowledge Infrastructure (CNKI; 1997-2013), and Chinese Biomedical (CBMdisc; 1860-2013) literature databases were searched for case-control studies of hepatic lesions in patients undergoing anti-tuberculosis therapy with or without concomitant HBV infection. The HBV patients were divided into subgroups according to hepatitis B e antigen (HBeAg) positivity or negativity, all members of the control group were HBsAg⁻. The data from all 7 studies included in the meta-analysis were extracted and analysed using RevMan5.2 soft-ware.</p><p><b>RESULTS</b>Patients with HBV infection who were undergoing anti-tuberculosis therapy had a higher risk factor than the control patients (OR =5.81, 95% CI =[4.26, 7.39]). The HBV patients with HBeAg positivity who were undergoing anti-tuberculosis therapy had a high risk factor than the HBV patients with HBeAg negativity (OR =2.56, 95% CI=[1.90, 3.44]).</p><p><b>CONCLUSION</b>HBV infection is a risk factor for hepatic lesions when undergoing anti-tuberculosis therapy, and HBeAg-positive status may put a patient at higher risk.</p>
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Humans , Antitubercular Agents , Therapeutic Uses , Hepatitis B , Pathology , Hepatitis B e Antigens , Blood , Liver , Pathology , Tuberculosis , Drug Therapy , Pathologyالملخص
Objective:To observe the stability and sensitivity of transcription mediated ampliifcation (TMA) system, and to compare it with real-time reverse transcription polymerase chain reaction (RT-PCR) in amplifying serum HCV RNA in HCV infected patients. Methods: TMA system was established by moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase and 2 speciifc primers ifrstly,and then its stability and repeatability were compared at different storage temperatures by the correlation change of HCV RNA amplification curve. The sensitivity difference between TMA and RT-PCR was evaluated by amplifying a group of 10-fold diluted HCV RNA samples which were transcribed in vitro. A total of 101 serums of chronic HCV infected patients were measured by TMA system and RT-PCR to observe the positive rate and their correlation. Linear correlation and linear regression were used to observe the correlation of the two methods. Results:TMA system was successfully established. TMA system was not stable when stored at 20℃ (placed for 24 hours only), but it was stable for 6 days when stored at 4℃ or within 6 months when stored at-20 ℃. Compared with RT-PCR whose reagent was made by Hunan Sansure Biotechology Corporation, TMA system showed 20 positive samples and 11 negative samples in a total of 31 samples. So was the RT-PCR kit of the Sansure Biotechology Corporation, and the concordance rate of the two methods was 100%. Advanced quantitative study of the 20 positive samples found that the two methods had good correlation and consistency (r=0.91,P0.05). Advanced quantitative study of 29 positive samples found that the two methods had good correlation and consistency (r=0.96,P<0.01). Conclusion:The stability and repeatability of TMA system are good within 6 months when stored at-20 ℃ storage temperature. Both TMA and RT-PCR HCV RNA can detect serum HCV RNA well, and the two methods have good correlation and consistency.
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BACKGROUND/AIMS: This study aimed to investigate the microRNA (miRNA) expression profiles in peripheral blood mononuclear cell (PBMC) of hepatitis B virus (HBV)-infected patients with different clinical manifestations and to analyze the function of miR-197. METHODS: PBMC miRNA expression profiles in 51 healthy controls, 70 chronic asymptomatic carriers, 107 chronic hepatitis B patients, and 76 HBV-related acute on chronic liver failure patients were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). miR-197 mimic and inhibitor were transfected in THP-1 cells. qRT-PCR and ELISA for interleukin (IL)-18 mRNA and protein levels were performed, respectively. RESULTS: The microarray analysis revealed that 17 PBMC miRNA expression profiles (12 miRNAs downregulated and five miRNAs upregulated) differed significantly in HBV-induced liver disease patients presenting with various symptoms. The qRT-PCR results suggested that the PBMC miR-197 levels regularly decreased as the severity of liver disease symptoms became aggravated. IL-18, a key regulator in inflammation and immunity, was inversely correlated with miR-197 levels. Bioinformatic analysis indicated that IL-18 was a target of miR-197. Exogenous expression of miR-197 could significantly repress IL-18 expression at both the mRNA and protein levels in THP-1 cells. CONCLUSIONS: We concluded that multiple PBMC miRNAs had differential expression profiles during HBV infection and that miR-197 may play an important role in the reactivation of liver inflammation by targeting IL-18.
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Humans , End Stage Liver Disease , Enzyme-Linked Immunosorbent Assay , Hepatitis , Hepatitis B , Hepatitis B virus , Hepatitis B, Chronic , Hydrazines , Inflammation , Interleukin-18 , Interleukins , Liver , Liver Diseases , Liver Failure , Microarray Analysis , MicroRNAs , Real-Time Polymerase Chain Reaction , RNA, Messengerالملخص
OBJECTIVE@#To evaluate the mid-term prognostic value of procalcitonin (PCT), endotoxin and common inflammatory markers combining the model for end-stage liver disease (MELD) score in patients with chronic severe hepatitis.@*METHODS@#A total of 124 chronic severe hepatitis patients were enrolled, who were hospitalized in the Department of Infectious Diseases, Xiangya Hospital, Central South University from May 2011 to December 2011. Indexes of inflammation, liver and kidney function tests and MELD were determined within 24 h after the admission, and blood samples were collected for measurement of endotoxin , procalcitonin (PCT), and C-reactin protein (CRP). The outcome was confirmed after discharge follow-up at the end of the 3rd month. According to the outcome, the 124 patients were divided into a survival group (n=58) and a death group(n=66).@*RESULTS@#1) Of the 124 patients, 66 died and 58 survived, with statistical difference in age, MELD score, white blood cell (WBC), polymorphonuclear (PMN), CRP and PCT by single factor analysis between the 2 groups(P<0.05). Binary logistic regression analysis indicated that age, MELD scores and PCT were highly correlated with the outcome (OR=1.07, 1.42 and 1.02 respectively, P<0.05), which could be used to predict the 3 month mid-term mortality of chronic severe hepatitis. 2)There was significant correlation between the MELD scores and the mid-term mortality. Age was positively correlated with the MELD score, and Pearson's correlation coefficient was 0.21 (P<0.05). PCT was also positively correlated with the MELD, and Spearman's correlation coefficient was 0.54 (P<0.01). 3)According to the receiver operation characteristic (ROC) curve analysis , the area under the curve (AUC) of MELD score and PCT were 0.91 and 0.77 respectively, higher than those of other indexes (P<0.01). When the MELD score was up to 30.09 or higher, the predicted mortality risk among these tested patients was the highest(82.26%). The mortality risk predicted by PCT combining MELD score and PCT alone was lower than by MELD score alone (75.00%), but the specificity of MELD score combining PCT was 100%, and the positive prediction value was 1.00.@*CONCLUSION@#Endotoxin and common inflammatory markers (WBC, PMN, and CRP) are not reliable indicators to predict the prognosis in patients with chronic-severe hepatitis. MELD score is significantly correlated with the outcome of mid-term chronic severe hepatitis, PCT and age are both positively correlated with the MELD score. PCT and age combining MELD score can be used to predict the 3 month mid-term mortality of chronic severe hepatitis. MELD score has better prognostic value than PCT. MELD score combining PCT can improve the specificity of prediction.
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Adult , Female , Humans , Male , Middle Aged , Age Factors , C-Reactive Protein , Metabolism , Calcitonin , Blood , Calcitonin Gene-Related Peptide , End Stage Liver Disease , Diagnosis , Mortality , Endotoxins , Blood , Hepatitis, Chronic , Diagnosis , Mortality , Predictive Value of Tests , Prognosis , Protein Precursors , Blood , ROC Curve , Severity of Illness Index , Survival Analysisالملخص
OBJECTIVE@#To investigate the impact of coagulative parameters on different anticoagulation systems in molecular adsorbent recirculating system (MARS) in subjects with liver failure, and to evaluate the safety of different anticoagulation methods .@*METHODS@#A prospective experimental observation was designed. According to anticoagulation Methods , 174 MARS treatment sessions for 146 patients with liver failure and prothrombin time activity percentage (PTA) ≤ 40% were randomly divided into 2 groups: 92 MARS treatment sessions in the heparin-free group and 82 in the low-dose heparin group. Time points of 0, 0.5, 1, 2, 3, 4, 5 and 6 h were selected to observe the coagulation changes of prothrombin time (PT), PTA, thrombin time (TT), activated partial thromboplastin time (APTT) and international normalized ratio (INR) dynamically. Adverse events such as line / filter coagulation, rupture and bleeding were also investigated and compared due to frequency and severity between the 2 groups.@*RESULTS@#There was no difference in PT, PTA, INR between the 2 groups, but significant differences were observed in APTT and TT and fibrinogen (Fbg). APTT and TT levels in the low-dose heparin group was increased rapidly after the first given dose of anticoagulant heparin and reached the peak within 30 min.The levels at each time point was statistically different between the 2 groups (P<0.05). A significant difference in the Fbg level was obtained between the 2 groups. In the low-dose heparin group it was stabilized and increased slightly at the end of the treatment. While in the heparin-free group it was decreased gradually and reached a ravine at the end of the treatment. A curve was observed after 2.5 h treatment between the 2 groups (P=0.001). There were 2 cases of severe bleeding after MARS was finished in the heparin group, and 1 was terminated because of degree III clotting in the heparin-free group.@*CONCLUSION@#Fibrinogen should be adsorbed while the blood touches the MARS circuit path and anticoagulants can prevent it. Comprehensive analysis of blood platelet count (BPC), fibrin degradation products (FDP), D-dimer and clinical symptoms is critical and required to determine the coagulation status to select an anticoagulation system before MARS. The use of low dose heparin in MARS improves the disorder of hypercoagulable state during the high coaguation period, while heparin-free during low coagulation period can effectively prevent the occurrence of bleeding and improve the mechanism of blood coagulation by reducing heparin-like substance in the blood.
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Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Adsorption , Anticoagulants , Disseminated Intravascular Coagulation , Heparin , Liver Failure , Therapeutics , Liver, Artificial , Prothrombin Time , Sorption Detoxification , Methodsالملخص
To investigate whether there is mutation in DC-SIGN promoter region in patients with chronic hepatitis B (CHB) and healthy persons previously infected with hepatitis B virus (HBV) and to explore the relationship between the mutation in dendritic cell-specific intercellular adhension molecule-3-grabbing nonintegrin (DC-SIGN) promoter region and HBV.Methods The studied population was composed of two cohorts:47 CHB patients and 20 healthy persons previously infected with HBV.The mutation in DC-SIGN promoter region was detected with PCR,single-stranded conformational polymorphism and heteroduplex analysis,cloning,sequencing and aligning the published DC-SIGN promoter sequence.Results The characteristic mutation within DCSIGN promoter region in HBV infected individuals was observed.In the DC-SIGN promoter region,4 hot spot mutations located in positions - 139,- 142,- 222,and - 336 were observed in the CHB patients,but only 1 spot mutation located in position - 139 was observed in the healthy persons previously infected with HBV.The -336C which was absent in the healthy persons previously infected with HBV was shown in 11 CHB patients (23.40％).The - 139T was far more frequent in the healthy persons previously infected with HBV ( 100％ ) than in the CHB patients (34.04％).Conclusion In the DC-SIGN promoter region,-336C may be a genetic risk factor for developing CHB,but -139T may be associated with protection against HBV.
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To determine CD4 + CD25 + T regulatory cells (Tregs),forkhead box P3 (FoxP3) mRNA expression and levels of cytokines secreted by peripheral blood mononuclear cells (PBMCs) in individuals responsive or unresponsive to hepatitis B (HB) vaccination,and to explore the relationships between immune response and immune regulatory cells or cytokines.Methods Based on the antibody against hepatitis B surface antigen (HBsAg) after HB vaccination,the CD4 + CD25 + Tregs frequencies in PBMCs from 18 responders,22 nonresponders and 10 non-immunized healthy controls were analyzed by flow cytometry.The expression of FoxP3 mRNA in PBMCs with or without stimulation of phytohemagglutinin ( PHA ) and HBsAg was analyzed by real-time quantitative PCR.Levels of IL-4,IL-12,IL-18,and IFN-γ secreted by PBMCs after PHA and HBsAg stimulation were analyzed by enzyme-linked immunosorbent assay.Results The ratio of CD4 + CD25 + Tregs to CD4 + T cells in the nonresponders was markedly higher than that in the responders (P ＜0.05),but lower than that in the controls (P ＜0.01 ).FoxP3 was differentially expressed among the responders,nonresponders,and controls in PBMCs before and after PHA and HBsAg stimulation,and nonresponders had the highest FoxP3 mRNA expression (P ＜ 0.05 or P ＜ 0.01 ).The content of IFN-γ by PBMCs after PHA and HBsAg stimulation was markedly lower in the nonresponders as compared with the controls and responders (P ＜ 0.05).However,there were no significant differences in the levels of IL-18,IL-4,and IL-12 from PBMCs after PHA and HBsAg stimulation between the responders and controls as well as the nonresponders (P ＞ 0.05 ).Conclusion CD4 + CD25 + FoxP3 + Tregs may be involved in the negative regulation of responses to hepatitis B vaccination.Immunologic non-responses to hepatitis B vaccination may be related to IFN-γhyposecretion in PBMCs.
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Objective To investigate clinical features and antifungal therapeutic effect of chronic severe hepatitis (CSH) patients with invasive fungal infection (IFI), and to improve the diagnosis and treatment.Methods Clinical manifestation, blood routine, imageology and mycetology characteristic, antifungal treatment perscription and therapeutic effect of 79 CSH patients with IFI were retrospectively analyzed. Antifungal therapeutic effect was compared between fluconazole and voriconazole. Results Thirteen (16.5%) patients received glucocorticoid or other immunodepressants for a relatively long time, 40 (50.6%) patients had invasive operation, and 61 (77.2 %) patients were administered 1-6 kinds of broad-spectrum antibiotics. Seventy-three patients had fever. Leucocytes and neutrophilic granulocyte increased in 96.2% of the patients. Lung (31.6%), intestinal tract (26.2%) and oral cavity (14%) infections were common. Fungus was found in 70.9% of the patients. Candida albicans (40.9%) and aspergillus (21.1%) were often seen. Halo signs and crescent signs on lung CT were relatively specific in 40% of the patients with fungal pneumonia. Voriconazole was more effective than fluconazole(71.4% vs. 39.0%, P<0.05). Twelve patients with lung aspergillus infection were administered voriconazole, 8 (66.7%) patients of whom was effective, and the other 4 (33.3%) patients died. Conclusion There are high risk factors in major CSH patients with IFI. The most common clinical manifestations of CSH patients with IFI are fever, leukocytosis, lung and intestinal tract infection. Candida albicans and aspergillus infection are common. Voriconazole is more effective than fluconazole, and can increase the survival rate of CSH patients with IFI.
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Objective To determine whether HBx gene can directly induce hepatocellular carcinoma in vivo, and to explore the mechanism of transplantation tumor in nude mice.Methods pCMVX/QSG7701 cell lines were vaccinated into subcutaneous tissue of nude mice. pRcCMV2/QSG7701 and QSG7701 cell lines were used as controls. The sections of transplantation tumor were observed microscopically by HE staining. RT-PCR was used to detect the expression of mutant p53 and c-Myc mRNA in transplant tumor and an other 3 cell lines. Results The transplant tumor occurred within the subcutaneous tissue of the nude mice inoculated with pCMVX/QSG7701 cell lines at 2nd week after the vaccination. No metastatic tumor was found in other organs. Transplant tumor was not formed in all the controls. HE staining confirmed that the transplant tumor was hepatocellular carcinoma. The mutant p53 mRNA and c-Myc mRNA expression level of transplant tumor and pCMVX/QSG7701 cells was significantly higher than that of pRcCMV2/QSG7701 and QSG7701 cells, respectively (P＜0.01).Conclusion HBx gene can up-regulate the expression of mutant p53 and c-Myc genes, and directly induce hepatocellular carcinoma in vivo.
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f fibrosis-related factors in LX-2cells were significantly increased after co-cultured with QSG7701-HBx cells, which proved that HBx could induce fibrogenesis in vitro.
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Objective To observe the effects of hepatitis B virus(HBV)X gene muhifunctional protein(HBx)on the biological characteristics of QSG7701 and the transformational effects on QSG7701 cell.Methods QSG7701 ceils were stably transformed by recombinant plasmid pCMV/X and eukaryotic expressed plasmid pRc/CMV2 by liposome-based assay,respectively.Non-transfeeted QSG7701 cells were employed as controls.The expressions of HBx,c-Myc and Bel-2 proteins in QSG7701 cells were detected by Western blot.MTT colorimetric analysis,flow cytometry and soft agar clone-forming assay were performed tO detect the biolo~dcal activity of cells.Results HBx Drotein was highly expressed in pCMV X/QSG7701 cells.The expression level of c-Myc protein in the pCMV X/QSG7701 cells was much higher than those in the other tWO groups of cells.The expression of Bcl2 protein was detected in the three groups of cells,but the expression levels were similar.Percentage of S stage cells in pCMV X/QSG7701 ceils was significantly higher than those in pRcCMV2/QSG7701 and non-transfected QSG7701 cells E(28.80±2.32)%,(15.5±2.64)%and(21.5±3.66)%,LSD 0.05=3.95%,LSD 0.01=5.47%,P<0.01].While percentage of G1 stage cells in pCMV X/QSG7701 cells was significantly lower than those in pRcCMV2/QSG7701 and non-transfected QSG7701 cells[(62.30±3.85)%,(78.70±4.12)%and(78.10q±4.45)%,LSD 0.05=5.63%,LSD 0.01-7.79 %,P<0.01].The apoptosis rate of pCMV X/QSG7701 cells was much higher than those in pRcCMV2/QSG770t and non-transfected QSG7701 cells[(14.90±1.01)%,(8.91±0.48)%and (4.03±0.47)%,LSD 0.05=O.94%,LSD 0.01=1.31%,P<0.01].The population doubling time of pCMV X/QSG7701 cells was shorter than those in pRcCMV2/QSG and non-trarmfected cells(14 h,29 h,38 h,respectively).The cloning ratio in soft agar of pCMV X/QSG7701 cells WaS significantly higher than those of pRcCMV2/QSG and non-transfected QSG7701 cells[(19.83±1.96)%,(1.76±0.03)%and (1.33±0.18)%,LSD 0.05=1.53%,LSD 0.01=2.11%,P<0.01].Conclusion HBx may transform human non-tumor hepatoeyte QSG7701,which makes the cell growth malignizeA.
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Objective To investigate the inhibitive activities of hepatitis B immunoglobulin(HBIG)poly(butylcynaoacrylate)nanoparticles(HBIG-PBCA-NP)to hepatitis B surface antigen(HBsAg)and hepatitis B virus(HBV)DNA secretions using HBV infected cell model in vitro.Methods HepG 2.2.15 cells were cultured with media containing HBIG-PBCA-NP or HBIG for several days,or cultured with HBIG-PBC-NP and HBIG for 2 days and without HBIG-PBCA-NP and HBIG from day 3.The supernatants at different time points were collected for quantitative detection of HBsAg and HBV DNA.The comparisons between groups were done by variance analysis.Resalts Secretions of HBsAg and HBV DNA in supernatants of HepG2.2.15 cultured with 0.1-10.0 IU/mL of HBIG-PBCA-NP and HBIG were inhibited significantly compared with control group.HBsAg titers and HBV DNA levels in supernatants of HBIG-PBCA-NP group and HBIG group cultured with media without HBIG-PBCA-NP and HBIG kept decreasing at day 5 and 7,then rebounded at day 9 and 11.HBsAg titera in supernatants of 0.1,1.0,5.0 IU/mL HBIG-PBCA-NP group were all significantly different from those in HBIG group at day 9[(31.31±1.98)μg/L vs(40.62±2.99)μg/L,(23.79±1.31)μg/L vs(36.51±2.12)μg/L,(19.91±1.74)μg/L vs(33.03±1.65)μg/L;F=412.24,P＜0.01].Couclusion HBIG-PBCA-NP can inhibit secretions of HgsAg and HBV DNA in vitro,which is more effective than HBIG.
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Objective To develop the technique to detect total core antigen of HCV(Total HCV-cAg) by Enzyme-Linked Immu-nosorbent Assay (ELISA) and apply it for clinical diagnosis. Methods 201 serum samples with anti-HCV antibody were detected total HCV-cAg after pre - treating the samples, then the sensitivity of results were compared with HCV RNA tests. Among them, 176 cases was determined by FQ-PCR, and 25 cases by RT-PCR for HCV-RNA. Results HCV RNA was found in sera from 88 of 201 samples (43.8%). Total HCV-cAg was positive in 71 (35.3%) of 201 samples . There was no significant difference between the detection rate of HCV RNA by PCR and total HCV-cAg by ELISA. Conclusion Detection of total core antigen of HCV is suitable to be used as to diagnose HCV in clinic.
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Objective To investigate the possibility of anti-liver fibrosis of 13-estradiol nanoparticle prepared by interfacial polymeri-zation method with butylcyanoacylate as carrier material (E2-PBCA-NP) and its effect on the expression of transforming growth factor β1 and connective tissue growth factor in pig serum induced animal fibrotic model. Methods Male Sprague-Dawley (SD) rats were random divided into five groups. Except normal control group, other four groups were all given intraperitoneal injection with pig serum. Therapeutic drugs were administered to rats from the ninth week after injection of pig serum. All rats were killed at the end of the twelfth week. Several experi-ments were done as below, the tissues of liver were observed by Masson staining, and the mRNA of TGF-β1 and CTGF of liver samples were detected by RT-PCR. Meanwhile, the expression of TGF-β1 and CTGF protein were detected by immunohistochemistry. Results It showed that both E2 and E2-PBCA-NP treatment groups had lower stage of liver fibrosis, according to the observation of pathology by Masson staining (P < 0.05). The anti-liver fibrosis effect of E2-PBCA-NP treatment group was better than that of E2 treatment group (P < 0.05). The mRNA and protein level of TGF-β1 and CTGF were markedly reduced by E2 and E2-PBCA-NP treatment, compared with liver fibrotic model groups (P <0.01). There was no statistical difference between E2-PBCA-NP and E2 treatment (P >0. 05), while no significant change was observed in blank nano -particle group (P > 0.05). Conclusion Both E2-PBCA-NP and E2 had anti-liver fibrosis activity. E2-PB-CA-NP has stronger anti - liver fibrosis activity than E2, which could be resulted from the inhibition of TGF-β1 and CTGF expression.
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Objective To investigate the clinical significance of serum amyloid A protein in patients with chronic hepatitis C (HCV) infection. Methods Serum amyloid A (SAA) levels were detected by ELISA in 131 patients with HCV infection and 20 normal controls. The expression of SAA-mRNA in peripheral blood mononuclear cells (PBMC) was detected by RT-PCR from some blood samples of HCV patients and normal controls. Results The SAA levels in the patients with chronic HCV infection were markedly higher than those in normal controls (t = 17. 14, P < 0. 01 ). The expression of SAA-mRNA detected by RT-PCR was closely correlated with concentrations of SAA measured by ELISA ( r = 0.86, P <0.01 ). No correlation was found between SAA expression and serum HCV RNA titers, as well as between SAA and serum ALT in patients with chronic HCV infection. Conclusion SAA levels are increased in patients with chronic HCV infection, which is not correlated with HCV RNA titers and serum ALT levels.