Objective:To investigate the anti-aging and antioxidant effect of the heat shock transcription factor 1 (HSF1) on human retinal pigment epithelial cells.Methods:Two HSF1-deficient ARPE cells (ARPE/Hsf1 -/-) were constructed by using the clustered regularly interspaced short palindromic repeat and associated protein 9 (CRISPR/Cas9) gene editing system and named H8, H9 konckout cell strains.Experiments were operated on the 3 cell strains: wild-type, H8 and H9 cells.The content of reactive oxygen species in ARPE-19 cell was measured by DHE probe staining combined with flow cytometry technology, and the cell cycle was measured by flow cytometry technology.The cell viability at different time points was measured using cell counting kit-8 (CCK-8).Crystal violet staining assay was used to measure the relative ratio of cell survival.SA-β-gal staining assay was used to detect the ratio of ARPE-19 senescent cells.The expressions of HSP70, HSP27, clusterin (CLU), p53, p21 and interleukin (IL)-1β proteins were measured by Western blot technology.The expressions of p53, p21, IL-6, IL-8, IL-1β and monocyte chemoattractant protein 1 (MCP1) mRNA were measured by quantitative real-time PCR technology.Relative expression of heat shock response protein under different heat shock treatment conditions and HSP90 inhibitor IPI504, relative survival with different concentrations of H 2O 2, relative expression of p21 protein after treatment with or without ROS scavenger N-acetylcysteine (NAC) were compared in each cell strain. Results:Gene sequencing showed that H8 and H9 cell strains successfully carried mutated genes.Western blot experiment results showed that H8 and H9 cell strains did not express HSF1 protein, and HSF1 was successfully knocked out in ARPE-19 cells.Compared with wild-type cell, the expression levels of HSP70, HSP27 and CLU proteins in H8 and H9 cell strains significantly decreased, with statistically significant differences (all at P<0.05), and no significant difference was found in the relative HSP90 protein expression level ( F=0.29, P>0.05).Under different heat shock stimulation and IPI504 induction, the HSP70, HSP27, and CLU protein levels significantly increased in wild-type cells compared with before treatment, and the HSP70, HSP27, and CLU protein levels were significantly lower in H8 and H9 cell strains than in corresponding treated wild-type cells (all at P<0.05).Compared with wild-type cell strains, cell viability significantly decreased in H8 and H9 cell strains at 24, 48, 72, and 96 hours (all at P<0.05).Compared with wild-type cell strains, the percentage of cells in G1 phase was significantly higher and the mRNA and protein levels of the cell cycle inhibitors p53 and p21 significantly increased in H8 and H9 strains, showing statistically significant differences (all at P<0.05), and the ratio of positive cells for SA-β-gal staining significantly increased, showing statistically significant differences (all at P<0.001).The relative expression of aging-related inflammatory factors IL-6, IL-8, IL-1β, and MCP1 mRNA decreased, and the differences were statistically significant (all at P<0.001).In addition, compared with wild-type cell strains, the content of reactive oxygen species (ROS) was higher in H8 and H9 cell strains, and the differences were statistically significant (all at P<0.001).The expression of p21 protein in H8 and H9 cell strains wtih NAC treatment decreased significantly compared with non-NAC treatment cells (both at P<0.05).Compared with wild-type cell strains, H8 and H9 cell viability decreased at 200, 400, 600, and 800 μmol/L H 2O 2 treatment conditions, and the differences were statistically significant (all at P<0.05). Conclusions:Knockdown of HSF1 can downregulate the expression of heat shock proteins, activate the ROS/P53/P21 pathway, induce senescence in RPE cells, and increase the sensitivity of RPE to oxidative stress stimuli.HSF1 may have anti-senescence and anti-oxidant regulatory effects in RPE cells.

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.

Nurses are subjected to high amount of stress in the medical setting,and work-related stress often leads to mental problems.This study aims to investigate the mental health status of nurses exposed to blood through needlestick injuries.A total of 302 nurses working in the hospital of Guangdong,China,participated in this study.Out of the 302 nurses,140 did not experience any needlestick injuries during the previous week,whereas 162 nurses experienced needlestick injuries.The General Health Questionnaire (GHQ)-28 Standardized Questionnaire,which uses physical,anxiety,social function,and depression subscales,was used in this study.No significant difference between nurses exposed to blood and nurses not exposed to blood was found in terms of gender,age,length of employment,and civil status (P ＞ 0.05).Results from the GHQ-28 Standardized Questionnaire showed that 75.9％ (123/162) of nurses exposed to blood were suspected to suffer from mental disorders,whereas 40％ (56/140) of nurses not exposed to blood were suspected to suffer from mental disorders.The mean mental health scores of nurses exposed to blood and those not exposed were 8.73 ± 7.32 and 5.69 ± 5.70,respectively.From these results,we can conclude that blood exposure from needlestick injuries leads to higher prevalence of depression,anxiety,and stress symptoms in nurses.This finding highlights the importance of providing efficient,adequate,and appropriate support services after nurses are exposed to blood from needlestick injuries.

Objective To assess the efficacy and safety of peginterferon ( PegIFN) α-2b in treatment of HBeAg-positive chronic hepatitis B ( CHB).Methods Thirty two patients with HBeAg-positive CHB admitted in Peking University Shenzhen Hospital during November 2013 and January 2014 were recruited in the study.Patients were center randomly assigned into two groups : 22 patients in test group were treated with 180 μg PegIFN α-2b, 1 /w for 48 wk; 10 patients in control group were treated with 180 μg PegIFN α-2a (Pegasys), 1 /w for 48wk.All patients were followed up for 24wk after treatment.Virology markers, HBV DNA levels and liver functions were monitored regularly , and adverse events were observed . Fisher’s exact test was used to compare the efficacy and safety between two groups .Results There were no statistically significant differences between the control group and test group in ALT normalization rates , HBV DNA negative rates and HBeAg serological conversion rates both at the end of treatment and at the end of 24-wk follow-up (all P >0.05).Both groups had similar adverse effect incidence rates (P >0.05), but retina disease occurred in 7 cases of test group, which was not observed in control group .Conclusion Compared with PegIFN α-2a, PegIFN α-2b has similar efficacy and safety for patients with HBeAg -positive CHB.

ObjectiveTo investigate the clinical effect of Dahuang Zhechong capsules combined with entecavir in the treatment of chronic hepatitis B (CHB) patients with liver fibrosis. MethodsA total of 100 CHB patients with liver fibrosis who visited or were hospitalized in Shenzhen Hospital of Peking University from October 2014 to January 2016 were enrolled and randomly divided into Western medicine group and combined treatment group, with 50 patients in each group. The patients in the Western medicine group were given entecavir, and those in the combined treatment group were given Dahuang Zhechong capsules in addition to entecavir. The course of the treatment was 48 weeks. The changes in liver function, HBV DNA load, four serum parameters of liver fibrosis, liver stiffness, and aspartate aminotransferase-to-platelet ratio index (APRI)/FibroIndex were observed in both groups. The t-test was used for comparison of continuous data between groups, and the chi-square test was used for comparison of categorical data between groups. ResultsBoth groups showed significant reductions in serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and HBV DNA load after 48 weeks of treatment (all P＜0.05). After treatment, the four serum parameters of liver fibrosis all returned to normal after treatment, and the serum levels of hyaluronic acid, type Ⅲ precollagen, and type IV collagen showed significant differences between the two groups (all P＜005), and the portal vein diameter, thickness of the spleen, liver stiffness and APRI/FibroIndex also showed significant differences between the two groups (both P＜0.05). The combined treatment group had a significantly higher overall response rate than the Western medicine group (920% vs 720%, χ2=6.775, P=0009). ConclusionDahuang Zhechong capsules combined with entecavir have a better effect in the treatment of CHB patients with liver fibrosis compared with entecavir alone.

Objective To investigate the effect of recombinant human interferon α-2b on influenza virus in vitro.Methods Influenza A virus subtype H1N1 and influenza B/Y virus were inoculated into Vero cells and different concentrations of interferon α-2b and oseltamivir were added.Numbers of virus plaques were observed and calculated,and quantitative RT-PCR were used to assess the inhibitory effect of interferon α-2b and oseltamivir in vitro.The nuclear export of viral ribonucleoprotein (RNP) complexes were monitored under fluorescence microscope.Results Virus plaque test showed that influenza A viruses subtype H1N1 were significantly inhibited when 10 μg/μL interferon α-2b and 10 μg/μL oseltamivir were added,and the numbers of plaques were 7.5 × 108 and 15 × 108 PFU/mL,respectively;the inhibitory effect of oseltamivir was better than that of interferon α-2b.Influenza B/Y viruses were also inhibited when 10 μg/μL interferon α-2b and 10 μg/μL oseltamivir were added,and the numbers of plaques were 1.1 × 108 and 1.5 × 108 PFU/mL,respectively.Quantitative RT-PCR results showed that the cycle threshold (CT) values of influenza A virus subtype H1N1 and influenza B/Y virus were much higher when 10 μmol/L interferon α-2b and 10 μmol/L oseltamivir were added.CT values of influenza A virus subtype H1N1 were 16,26 and 35 before and after inferferon α-2b and oseltamivir were added.CT values of influenza B/Y virus were 18,27 and 31 before and after interferon α-2b and oseltamivir were added.Reduction in the nuclear export of viral RNP in influenza A virus subtype H1N1-infected Vero cells was also observed when 10 μmol/L interferon α-2b were added.Conclusion Interferon α-2b has significantly inhibitory effect on both influenza A virus subtype H1N1 and influenza B/Y virus in vitro.

ObjectiveTo investigate the expression and clinical significance of heat shock transcription factor 1 (HSF1) protein in human hepatocellular carcinoma (HCC) tissues,and deduce the probable molecular mechanism of HSF1 in the development and advancement of HCC.MethodsSixty-seven samples of HCC tissue and 21 samples of normal liver tissue were obtained from March 2006 to March 2007 at the Xijing Hospital.The expressions of HSF1 protein and heat shock protein 70 (HSP70) were detected by using immunohistochemistry.The probable molecular mechanism of HSF1 in the development and advancement of HCC was deduced according to the relationship between the expressions of HSF1 protein and HSP70.Positive rates of HSF1 protein in different tissues and the relationship between HSF1 protein expression in the HCC tissues and clinical pathological factors were analyzed by the chi-square test and by calculating Fisher exact probability,respectively.The correlation between the expressions of HSF1 protein and HSP70 in the HCC tissue was analyzed by the Spearman correlation coefficient.The survival curve was drawn by the Kaplan-Meier method,and the survival rate was analyzed by the Log-rank test.ResultsThe positive rates of HSF1 protein expression was 69％ (46/67) in the HCC tissue,which was significantly higher than 29％ (6/21) in the normal liver tissue ( x2 =10.628,P ＜ 0.05 ),The positive rates of HSP70 expression in the HCC tissue was 57％ (38/67),which was significantly higher than 24％ (5/21) in the normal liver tissue ( x2 =6.929,P ＜ 0.05 ).The expression of HSF1 protein in the HCC tissue was positively correlated with that of HSP70 (r=0.319,P ＜0.05).The high expression of HSF1 protein was correlated with the integrity of capsule of HCC,tumor differentiation and TNM stage (x2 =5.935,9.762,5.159,11.267,P＜0.05 ),while the high expression of HSF1 protein was not correlated with the gender,age,levels of hepatitis B surface antigen and alpha fetoprotein,and portal vein tumor thrombus ( x2 =0.822,0.172,2.059,P ＞0.05 ).The survival time was (21.4 ± 1.9 )months for patients with positive HSF1 protein expression and (29.8 ± 2.7 ) months for patients with negative HSF1 protein expression.There was a significant difference in the survival time between patients with positive and negative HSF1 protein expression ( x2 =4.276,P ＜ 0.05 ).Conclusions HSF1 is correlated with the development,advancement,invasion,metastasis and malignant prognosis of HCC.HSF1 takes effects by regulating the expression of HSP70,and it has a good perspective of clinical application for the diagnosis and treatment of HCC.

Objective To compare the clinical efficacy and safety of pegylated interferon α-2a (Peg IFN α-2a) or adefovir dipivoxil(ADV) monotherapy and their combination therapy in HBeAg positive chronic hepatitis B (CHB) patients. Methods An open randomized controlled multicenter clinical trial was performed. One hundred and twenty cases with CHB were divided into 3 groups: Peg IFN α-2a monotherapy (group A), ADV monotherapy (group B) and Peg IFN α-2a plus ADV combination therapy (group C). The virological response (VR), serological response (HBeAg, HBsAg clearance and seroconversion), biochemical response (BR) and sustained response (SR) were tested at week 24 and 48 of therapy and week 48 of follow-up after end of treatment (EOT) for'evaluation of therapeutic effects, safety and drug resistance. The efficacy was compared using X2 test. Results At week 48 of treatment, the VR (HBV DNA ≤500 copy/mL) rates were 36. 8%(14/38), 37. 5%(15/40) and 62. 9% (22/35), respectively in groups A, B and C; that in group C was higher than those in groups A and B (X2 = 4. 933, 4. 801, respectively; both P < 0. 05); HBeAg seroconversion rates in three groups were 44. 7% (17/38), 17. 5% (7/40) and 51. 4% (18/35), respectively. At week 48 of follow-up,SR rates in three groups were 34. 2%(13/38), 15. 0%(6/40) and 48. 6% (17/35), respectively; those in groups C and A were higher than that in group B (X2 = 9. 894,P<0. 01;X2 =3. 903, P<0. 05, respectively). Conclusions VRs at week 24 and 48 of Peg IFN α-2a plus ADV combination therapy are better than Peg IFN α-2a or ADV monotherapy. SRs at week 48 of follow-up after Peg IFN α-2a monotherapy and combination therapy are both better than ADV monotherapy.

Objective To investigate the role of CD4 ~+ CD25~+ regulatory T lymphocytes (Treg)in modulating the cellular immune response and pathogenesis of murine pulmonary tuberculosis.Methods Inactivation of Treg was achieved by intraperitoneal injection anti-CD25 (clone PC61,50 μ/mouse) in PC61 group, and rat-IgG (50 μ/mouse) was injected intraperitoneally in control group. All the mice were inoculated intravenously with H37Rv 0. 1 mL (1 × 10~6 CFU) 3 days after Treg inactivation. The effects of Treg inactivation in different tissues were analyzed by flow cytometry. The cellular immune response, pulmonary histopathology and bacterial load were determined in vitro at different time points. The data were compared using homogeneity of variance F test and non-paired t test. Results In spleen, the percentages of Treg/CD4 T lymphocytes in PC61 group and control group were (21. 13± 3. 58)% and (30. 42± 4. 20)%, respectively at day 10 of inoculation (t = 2. 38, P < 0. 05), and those were (16. 12 ± 1. 26)% and ( 17. 34± 1. 62)%,respectively at day 30 of inoculation (t = 0. 84,P>0. 05). The percentages of Foxp3~+/CD4~+ T lymphocytes in PC61 group and control group were (32. 07 ± 3. 95)% and (60. 55 ± 5. 48)%,respectively at day 10 of inoculation (t = 5. 96, P<0. 05). Similar results were achieved in the peripheral blood. Bacillus calmette-guerin (BCG)-specific 1L-17 (ng/L) secreted by murine spleen cells in PC61 group and control group at day 10, 30 and 60 of inoculation were 5. 1± 0.9 vs 0, 43. 1± 10.0 vs5. 9± 2. 8 and 124.8 ± 5.8 vs 102. 5±8. 1, respectively (t = 7. 90, t=5. 10,t = 3. 19; all P<0.05); those of BCG-specific IFN-γ (ng/L) were 28. 4 ± 8. 2 vs 4. 0±1. 3, 685. 9± 128. 6 vs418. 7±20.4 and 310.9±119. 7 vs 32. 8±7. 5, respectively(tO = 4. 21,t = 8. 43, t = 3. 27; all P<0.05);those of TNF-a (ng/L) were 38. 6±5.0 vs 16. 3±4. 0, 112. 9 ±12. 3 vs 71. 5±12. 6 and 86. 2±8. 2vs0, respectively(t = 4. 95, t=3. 33,t/=14.8; all P<0. 05). The lung bacterial load at day 10 of inoculation in PC61 group was lower than that in control group (t = 4. 63, P < 0. 01), but the differences were not significant thereafter. The changes of lung histopathology at late stage of infection (day 120) in PC61 group were less severe than those in control group. Conclusions Murine Tregs increase dramatically after Mycobacterium tuberculosis infection. Treg could inhibit the specific cellular immunity against Mycobacterium tuberculosis, and therefore, may facilitate the persistent infection of Mycobacterium tuberculosis and development of tuberculosis.

Objective To establish a fluorescent polymerase chain reaction (PCR) method for rapid, sensitive and specific determination of -88/-123 polymorphisms in Myxovirus resistance protein A (MxA) gene promoter so as to provide molecular biology tool for optimized interferon-a treatment in chronic hepatitis B patients. Methods Hepatitis B virus (HBV) genotyping,serum HBV DNA level,and- 88/- 123 polymorphisms in MxA gene promoter of patients who had been treated with interferon-α were detected. The statistical analysis was done by using SPSS software to understand the relationship between MxA gene polymorphisms and interferon-α treatment. Afterwards, an optimal fluorescent PCR system was established to determine -88/-123 polymorphisms in MxA gene promoter. The sensitivity and the specificity of this system were confirmed by DNA sequencing. P-value of chi square test, odds ratios of regression analysis and 95% confidence intervals were employed. Results Patients with- 88 G/T and - 123 C/A in the interferon-stimulated response element in MxA gene promoter were interferon-α sensitive, while patients with - 88 GIG and - 123 C/C were not interferon-α sensitive. The coincidence rate of this system was 99.65% in comparison with DNA sequencing.Conclusion MxA gene polymorphisms could be rapidly and sensitively determined by this fluorescent PCR system.