Correlations of Serum Markers of Hepatitis B Virus,HBV DNA Load and Liver Function / 华中科技大学学报(医学版)

Objective To investigate the correlations of serum markers of hepatitis B virus,HBV DNA load and liver function indexes[alanine aminotransferase(ALT)and aspartate transaminase(AST)]in the peripheral blood.Methods Clinical data of 483 patients who were diagnosed with chronic hepatitis B and treated between March 2014 and March 2016 in Tongji hospital were retrospectively analyzed.The serum markers of hepatitis B virus were quantitatively detected by chemiluminescent microparticle immunoassay.Serum HBV DNA load was measured by real-time fluorescence quantitative PCR,and ALT and AST by continuous ultraviolet monitoring.Results There was no correlation between the HBsAg content and HBV DNA load or the rates of abnormal ALT(>41 U/L)and abnormal AST(>35 U/L)(P>0.05).The HBeAg content was not correlated with HBV DNA load and the rates of abnormal ALT(P>0.05),but weakly with the rate of abnormal AST(r=0.21,P0.05),it was weakly related to HBV DNA load and the rates of abnormal AST(r=0.16,P<0.05;r=0.19,P<0.01).The anti-HBc content had weak correlations with HBV DNA load,the rates of abnormal ALT and abnormal AST(r=0.25,P<0.01;r=0.29,P<0.01;r=0.29,P<0.01).The logarithm value of HBV DNA load was weakly positively correlated with ALT and AST(r=0.24;r=0.29).Conclusion Quantitative detection of both serum markers and the DNA of hepatitis B virus can complement each other,and when combined with detection of liver function indexes,it will help understand the damage of liver tissue.

Blood glucose abnormality in elderly patients with cardiocerebral vascular diseases / 中华全科医师杂志

One hundred and four patients aged of 65 years or above with cardiocerebral vascular diseases admitted from March to September 2008 were enrolled in the study. The fast plasma glucose (FPG) and 2 hours postprandial blood glucose (2 hPG) were tested. For patients with undiagnosed diabetes, if FPG≥7.0 mmol/L, 2 hPG≥11.1 mmol/L, their blood sugar index was checked again; if 2hPG was 7.8 mmol/L to 11.1 mmol/L, FPG from 6.1 mmol/L to 7.0 mmol/L, Oral glucose tolerance test (OGTT) was performed. Among the 104 patients, 30 cases of diabetes were diagnosed previously accounting for 28.8 %; 7 cases were newly diagnosed accounting for 6.8%; 21 cases were diagnosed as impaired glucose regulation accounting for 20.2%, with a total rate of sugar metabolism abnormality of 55.8%. The results indicate that blood glucose index should be regularly checked for elderly patients with cardiocerebral vascular diseases.

Genetic immunization with Hantavirus vaccine combining expression of G2 glycoprotein and fused interleukin-2.

In this research, we developed a novel chimeric HTNV-IL-2-G2 DNA vaccine plasmid by genetically linking IL-2 gene to the G2 segment DNA and tested whether it could be a candidate vaccine. Chimeric gene was first expressed in eukaryotic expression system pcDNA3.1 (+). The HTNV-IL-2-G2 expressed a 72 kDa fusion protein in COS-7 cells. Meanwhile, the fusion protein kept the activity of its parental proteins. Furthermore, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response.- The results showed that the chimeric gene could simultaneously evoke specific antibody against G2 glycoprotein and IL-2. And the immunized mice of every group elicited neutralizing antibodies with different titers. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to G2 and IL-2 were significantly higher than that of other groups. Our results suggest that IL-2-based HTNV G2 DNA can induce both humoral and cellular immune response specific for HTNV G2 and can be a candidate DNA vaccine for HTNV infection.

siRNA directed against c-Myc inhibits proliferation and downregulates human telomerase reverse transcriptase in human colon cancer Colo 320 cells.

The c-Myc and human telomerase reverse transcriptase gene (hTERT) gene are frequently deregulated and overexpressed in malignancy. hTERT activity is induced by c-Myc and strategies designed to inhibit c-Myc expression in cancer cells may have considerable therapeutic value. We designed and used a short hairpin RNA to inhibit c-Myc expression in Colo 320 cells and validated its effect on cell proliferation. In this study, four c-Myc-shRNA expression vectors were constructed and introduced into Colo 320 cells. The effects of c-Myc silencing on tumor cell growth was assessed by soft agar assay and DNA synthesis experiments. The expressions of c-Myc and hTERT were also assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it was found that expression of c-Myc and hTERT decreased in shRNA-transfected cells. The downregulation of c-Myc and hTERT inhibited cell growth, shortened telomere lengths, and suppressed telomerase activity. In conclusion, our findings demonstrate that shRNA of c-Myc can inhibit the DNA replication in Colo 320 cells effectively and reduce telomere length and telomerase activity, therefore, it could be used as a new potential anticancer tool for therapy of human colon cancer.

Depletion of c-Myc inhibits human colon cancer colo 320 cells' growth.

Human colon cancer is the leading cause of cancer death in both men and women worldwide. The c-Myc gene is frequently deregulated and overexpressed in this malignancy, and strategies designed to inhibit c-Myc expression in cancer cells may have considerable therapeutic value. We design and use short hairpin RNA (shRNA) to inhibit c-Myc expression in Colo 320 cells and validat its effect on cell proliferation. In this study, four c-Myc-shRNA expression vectors were constructed and introduced into Colo 320 cells, and the cell cycle and apoptotic cells were analyzed by flow cytometry. The effects of c-Myc silencing on tumor-cell growth was assessed by the soft agar assay and by DNA synthesis experiments. Expression of c-Myc was also assessed by real-time reverse transcription polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid-encoding shRNA, it was found that expression of c-Myc decreased in shRNA-transfected cells, and the downregulation of c-Myc inhibited cell growth and induced apoptosis in Colo 320 cells. c-Myc downregulation also increased cell population in the G0-G1 phase. In conclusion, our findings demonstrate that shRNA can inhibit the DNA replication and induce apoptosis in Colo 320 cells effectively and, therefore, could be used as a new potential anticancer tool for the therapy of human colon cancer.

Relationship between genomic types of Escherichia coli and clinical diseases / 华中科技大学学报（医学）（英德文版）

In this study, by analysis of genome structures of E. coli, the relationships between the genomic types of E. coli and the associated diseases were investigated. Samples of sputum, urine and other excretions from patients with different infective diseases were collected. And 62 E. coli strains were isolated from these samples. Intact bacterial genomic DNA was cleaved with I-CeuI, separated by pulsed field gel electrophoresis and then typed on the basis of cleavage map. The results showed that 7 I-CeuI sites were found in all the genome structures of the 62 E. coli, indicating that there were 7 rrn operons in the genomes. The size of genome ranged from 4500 kb to 5000 kb. According to the genome structures, 62 E. coli strains were divided into 30 genome types. It was concluded that genome structures of E. coli isolated from the patients with different infective diseases varied to some extent, suggesting that some genome types of E. coli were closely related to some infective diseases.

Neuroprotective effects of purslane herb aquenous extracts against D-galactose induced neurotoxicity.

In order to evaluate mechanisms of natural plant purslane herb aquenous extracts (PHAS) for neuroprotective, we assessed neuroprotective effects of PHAS at doses of 2.5, 5 and 10 mg/(kg day) on SD mice injected daily with D-gal (50 mg/(kg day)) by behavioral tests. PHAS-fed mice showed higher activity upon induction by new environmental stimuli, lower anxiety and higher novelty-seeking behavior in the open field tasks, and significantly improved learning and memory ability in step-through compared with D-gal-treated mice. We further examined the mechanisms involved in neuroprotective effects of PHAS on mouse brain. PHAS significantly increased superoxide dismutase (SOD) activity and decreased the malondialdehyde (MDA) level. Meanwhile, PHAS also could up-regulate telomere lengths and telomerase activity in PHAS-fed groups. Furthermore, we examined the expression of p21(waf1) and p53 mRNA and protein in mouse brain by western blot analysis and real-time RT-PCR. We found that p21(waf1)was down-regulated by PHAS without changing the expression of p53. The results of this study suggested that the PHAS might be a primary target of p21(waf1)and the neuroprotective effect of PHAS might be carried out through a p21(waf1)-dependent and p53-independent pathway.

Stable expression of Hantavirus H8205 strain G1/IL-2 gene and immune protection of the fusion gene / 华中科技大学学报（医学）（英德文版）

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20-1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene / 华中科技大学学报（医学）（英德文版）

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein.Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20-1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

Epidemiology of hepatitis B, C, D and G viruses and cytokine levels among intravenous drug users / 华中科技大学学报（医学）（英德文版）

To investigate the features of various hepatitis virus infection in intravenous drug users (IVDU), we conducted an epidemiological survey of hepatitis viruses including hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis G virus (HGV) in IVDU. The correlation of TH lymphocyte cytokine and hepatitis virus infection was examined. A study population of 406 IVDU consisted of 383 males and 23 females. HBV-DNA and HCV-RNA were detected by fluorescence quantitative polymerase chain reaction. HBsAg, HBeAg, anti-HBc, anti-HCV, HDV-Ag and anti-HGV were assayed by ELISA. The levels of cytokines of TH1 and TH2 were measured by ELISA. The similar indices taken from 102 healthy persons served as controls. The infection rate of each virus among IVDU was 36.45 % for HBV, 69.7 % for HCV, 2.22 % for HDV, and 1.97 % for HGV, respectively. The co-infection rate of HBV and HCV was detected in 113 of 406 (27.83 %). In contrast, among controls, the infection rate was 17.65 % for HBV and 0 % for the other hepatitis viruses. The levels of PHA-induced cytokines (IFN-gamma and IL-4) and the level of serum IL-2 were obviously decreased in IVDU. On the other hand, the level of serum IL-4 was increased. The IFN-gamma level was continuously decreased when the IVDU was infected with HBV/HCV. In conclusion, HBV and HCV infection were common in this population of IVDU and they had led to a high incidence of impaired TH1 cytokine levels.

Epidemiology of Hepatitis B, C, D and G Viruses and Cytokine Levels among Intravenous Drug Users / 华中科技大学学报（医学）（英德文版）

To investigate the features of various hepatitis virus infection in intravenous drug users (IVDU), we conducted an epidemiological survey of hepatitis viruses including hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis G virus (HGV) in IVDU. The correlation of TH lymphocyte cytokine and hepatitis virus infection was examined. A study population of 406 IVDU consisted of 383 males and 23 females. HBV-DNA and HCV-RNA were detected by fluorescence quantitative polymerase chain reaction. HBsAg, HBeAg, anti-HBc,anti-HCV, HDV-Ag and anti-HGV were assayed by ELISA. The levels of cytokines of TH 1 and TH2 were measured by ELISA. The similar indices taken from 102 healthy persons served as controls. The infection-rate of each virus among IVDU was 36.45 % for HBV, 69. 7 % for HCV,2.22 % for HDV, and 1. 97 % for HGV, respectively. The co-infection rate of HBV and HCV was detected in 113 of 406 (27. 83%). In contrast, among controls, the infection rate was 17.65 % for HBV and 0% for the other hepatitis viruses. The levels of PHA-induced cytokines (IFN-γ and IL-4) and the level of serum IL 2 were obviously decreased in IVDU. On the other hand, the level of serum IL-4 was increased. The IFN-γ level was continuously decreased when the IVDU was infected with HBV/HCV. In conclusion, HBV and HCV infection were common in this population ofIVDU and they had led to a high incidence of impaired TH 1 cytokine levels.

Cloning and regulatory expression of the transcriptional regulatory sequences in human breast cancer related DF3 antigen / 中国普通外科杂志

Objective To clone the transcriptional regulatory sequences (TRS) in human breast cancer related DF3 antigen, and to test the relationship between the activity of the TRS and the cell surface DF3 antigen. Methods Authors designed a pair of primers according to the registered 5′-flanking region of DF3 antigen. The 771 base pairs of DNA fragment were amplified from the genomic DNA of human MCF-7 breast carcinoma cells by PCR, and cloned to the pMD18-T vector. The results were tested by restrictive enzyme analysis and DNA sequencing. The DF3 TRS was cut by double enzyme: Mlu I、Hind III,and cloned to the pGL3 vector . The activity of the DF3 TRS was expressed by analyzing the relative luciferase activities. Results Restrictive endonuclease identification and DNA sequencing proved that the sequence authors got, was correct. The luciferase activity in MDA-MB-231 was hardly detected, whereas in MCF-7 the luciferase activity was about 200 times than in MDA-MB-231. Conclusions The DF3 TRS was cloned successfully. The DF3 activity has a distinct relationship with DF3 antigen. The study shows that DF3 TRS can be used in the gene therapy of breast carcinoma.