Objective To develop a time saving and sensitive cell culture system based on hepatitis C virus chimera expressing enhanced green fluorescent protein(EGFP) and to facilitate the study on HCV pathogenesis and screening of anti-HCV drugs.Methods Enhanced green fluorescent protein reporter gene and a mutation V2440L that can yield higher virus titers were introduced into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome by using recombinant PCR.The viral RNA was transfected into Huh7.5 cells.Viral RNA in supernatant of HCV RNA-transfected cells was determined after transfection by RT-PCR.HCV replication and infection were determined by immunofluorescence assay.IFN-α was used to evaluate the feasibility of this system for anti-HCV drugs screening.Results The viral RNA replicated efficiently in transfected cells.These cells can produce HCV-EGFP reporter virus.Viral RNA levels in supernatant were 3.06× 105 copies/ml and 7.96×106 copies/ml at 72 h and 9 d after transfection,respectively.The virus titer reached to 104 FFU/ml 9 d after transfection.The expression of EGFP was inhibited by IFN-α in a dose dependent manner in Huh7.5 cells infected by HCV-EGFP reporter virus.Conclusion The recombinant HCV JFH1-EGFP reporter gene system is a time saving,cost effective and sensitive method for studying viral replication cycles and screening of anti-HCV drugs.

Objective To investigate the expression of microRNA-548ah (miR-548ah) and microRNA-4804 (miR-4804) in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis B virus infection and their clinical significance.Methods PBMCs were collected from 29 patients with chronic hepatitis B (CHB),30 hepatitis B virus carriers (HBVC),26 inactive HBsAg carriers (IASC) and 28 healthy controls in Taizhou People's Hospital during September 2012 and August 2013.Expressions of miR-548ah and miR-4804 in PBMCs were detected by fluorescence real-time quantitative RT-PCR (qRT-PCR).Receiver operating characteristic (ROC) curve was used to evaluate the expression of miR-548ah and miR-4804 in distinguishing immune tolerance phase and clearance phase of HBV infection.Pearson correlation analysis was performed to assess the correlations of miRNAs expression with clinical markers alanine aminotrans ferase (ALT) and HBV DNA loads.Results There were significant differences in expressions of miR-548ah and miR-4804 in PBMCs between CHB,HBVC,IASC groups and control group (F =28.16 and 83.17,P ＜ 0.01).Compared with control group,miR-548ah was up-regulated in CHB group(P ＜ 0.01),and down-regulated in HBVC and IASC groups (P ＜ 0.01) ; miR-4804 was up-regulated in CHB group (P ＜ 0.01),down-regulated in HBVC group (P ＜ 0.01),while there was no significant difference between IASC group and control group in miR-4804 expressions(P ＞ 0.05).The areas under ROC curve (AUCs) of miR-548ah and miR4804 in differentiation of immune tolerance and immune clearance were 0.966 and 0.997,and the sensitivities and specificities were 89.7％,96.6％ and 99.6％,100.0％,respectively.No significant correlation was found between the expression of miR-548ah,miR-4804 and ALT,HBV DNA loads (r=0.14,0.18,-0.20 and-0.19,P＞0.05).Conclusion miR-548ah and miR-4804 may be involved in the immunopathogenesis of CHB,and their expression levels in PBMCs are helpful in differentiation of immune tolerance and immune clearance in HBV infection.

ObjectiveTo develop a cell culture system with consistent expression of whole hepatitis C virus (HCV) gene and Renilla luciferase gene and to facilitate the study on HCV pathogenesis and the screening of new antiviral drugs.MethodsRenilla luciferase (RLuc) reporter gene and a mutation that could yield higher virus gene expression were introduced into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome by using recombinant PCR.The viral RNA was transfected into Huh7.5 cells.Naǐve Huh7.5 cells were infected by the supernatant from the viral RNA transfected cells.HCV replication and infection were determined by virus titration,Renilla luciferase assay,immunofluorescence assay and western blotting.IFN-α was used to evaluate the feasibility of this system for anti-HCV new drug screening.ResultsThe viral RNA replicated efficiently in transfected cells.These cells could produce high titer of HCV-Rluc reporter virus and the virus titer reached to 1.5 × 104 FFU/ml at day 15 of posttransfection.The activity of Renilla luciferase was inhibited by IFN-α in a dose dependent manner in Huh7.5 cells infected by HCV-Rluc reporter virus.ConclusionThe recombinant HCV-JFH1-Rluc reporter gene system is sensitive and efficient.It can be a useful tool for high throughput screening of anti-HCV drugs.

Object To study the phsiochemical properties of storax ? cyclodextrin (? CD) inclusion complex Methods The inclusion complex was identified by the methods of TLC, X ray powder diffractometry and IR The solubility and dissolution rate of cinnamic acid in inclusion complex were investigated by HPLC Results The TLC showed that the main composition of storax had no change before and after being included by ? CD The spectra of X ray powder diffractometry and IR of the inclusion complex were remarkably different from those of storax and storax ? CD mixture It was shown a great improvement of the solubility and dissolution rate of cinnamic acid in the inclusion complex in 0 1 mol/L HCl, pH 6 6 and pH 7 5 phosphate buffer solution Conclusion The storax ? CD inclusion complex exhibits some new physical characteristics and its physiochemical properties are greatly changed comparing with those of storax

Object To investigate the release of pH dependent gradient releasing heart protecting musk pellets (GRHPMP) in vitro. Methods The pH dependent GRHPMP was prepared by coating with hydroxy propylmethy cellulose, Eudragit ○R L 30D 55 and Eudragit ○R L100/S100, repectively. The release of borneol and total ginsenoside from GRHPMP were determined according to method described in Chinese Pharmacopoeia (2000 ed) at simulated gastrointestinal pH conditions. Results The f 2 value of release data of borneol and total ginsenoside was 79 6. Conclusion The result suggested that, in vitro, the liposoluble borneol and watersoluble total ginsenoside could release simultaneously at a sustained rate.

Object To investigate the pharmacokinetic parameters of Musk Protecting Heart Pellets with pH-dependent gradient-release (MPHP-pH) and Musk Protecting Heart Pills (MPHP). Methods The cardiac muscle nutritional blood flow in rat was measured as effective index. Results It was one-compartment model when the rat was ig MPHP. The minimal effecting dose was 0.54 mg/kg, the present half-life, the elimination half-life, medicinal effect and the peak time of effective action were 0.53, 1.21, 3.48 and 1.13 h, respectively. The absorption half-life, elimination half-life and peak time of effective dose were 0.23, 1.47 and 0.88 h, respectively. Statistical moment analysis showed that the mean residence time mean residence time (MRT) of effective action were 5.05 h for MPHP-pH and 2.33 h for MPHP, the MRT of effective dose were 7.70 h for MPHP-pH and 3.21 h for MPHP. The relative bioavailability of effective dose for MPHP-pH was 104.03%. Conclusion MPHP has the characteristics of fast absorption, fast eliminationand short effective action time. Whereas MPHP-pH has the characteristics of fast absorption, protonged and relaxed effective action compared with MPHP.

Objective:To investigate the nursing effect of irritant dermatitis around drains after debridement of infected pancreatic necrotic tissue.Methods:A total of 108 patients with post-infectious pancreatic necrosis tissue debridement drains were divided into the experimental group and the control group with 54 patients in each group according to the time of hospital admission. The control group was given routine care and the experimental group was given stepped skin care, including education and training, assessment, skin preparation, and skin protectants and the use of disposable urinary ostomy bags, and the effect was evaluated.Results:The incidence of irritant dermatitis was 20.4% (22/54) in the test group and 40.7% (11/54) in the control group, with a statistically significant difference between the 2 groups ( χ2 value was 5.280, P < 0.05); 4 cases (36.36%) were cured in the experimental group, 5 cases (45.45%) in class I, 2 cases (18.18%) in class Ⅱ, 0 cases in class Ⅲ, and 0,13 cases (59.09%), 8 cases (36.36%), 1 case (4.54%) in the control group, respectively. The difference between the two groups was statistically significant ( χ2 value was 8.353, P < 0.05); In the experimental group, the duration of dermatitis incidence and total nursing time during hospitalization were (18.00 ± 2.25)d and (12.69±1.96)h, respectively, compared with (12.13±5.84)d and (15.79±2.09)h in the control group, which were statistically significant between the two groups ( t value was 29.167, -2.791, all P<0.05); the total cost of care in the experimental group was (570.38 ± 88.01)yuan and (448.79±59.44)yuan in the control group, the difference between the two groups was not statistically significant ( t value was -0.372, P>0.05). Conclusion:The stepped skin care protocol can reduce the incidence of infected pancreatic necrosis after tissue debridement and improve caregivers′ work efficiency.

Objective To determine the predictive factors for antiviral therapy in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection [HBeAg(-) CHBI] patients with HBV DNA＜4.3 lg IU/mL.Methods A total of 179 HBeAg (-) CHBI patients were retrospective analyzed.Histology activity index (HAI) and fibrosis (S) were scored according to the Knodell HAI scoring system,and HAI＞3 and/or S≥3 was adopted as indications for treatment.Univariate and multiple regression analysis were used to assess factors associated with treatment indications.Receiver operating curves (ROC) and area under curve (AUC) were used to determine the predictive value of relevant factors.Results There were 81 cases with HAI＞3 (45.3％) and 72 with S≥3 (40.22％),and the proportion of patients with indications for treatment was 54.7 ％.Multiple regression analysis showed that age,γ-glutamyl transpeptadase (γ-GT),platelet (PLT) and albumin (Alb) were the predictive factors for the severity of liver damage and indication for treatment (all P＜0.05).The AUC for age,PLT,γ-GT and Alb were 0.655,0.657,0.726 and 0.65,respectively,and the corresponding Yoden index for age,PLT,γ-GT,and Alb were 0.297,0.426,0.03 and 0.012,respectively,the sensitivities of predicting HBeAg (-CHBI for treatment indications were 0.643,0.842,0.705 and 0.653,respectively.Conclusions This study shows that 54.7％ of HBeAg(-)CHBI patients with HBV DNA＜4.3 lg IU/ml have significant liver histological changes and require antiviral treatment.Older age,higher γ-GT,lower PLT and lower Alb levels are the predictive factors for treatment.

Objective:To explore the necessity of lowering the cut-off value of alanine aminotransferase (ALT) in identifying chronic HBV infection patients with significant liver histological changes.Methods:The clinical data of 123 chronic HBV infection patients with normal ALT according to domestic criteria who underwent liver biopsy in the Department of Hepatology of Taizhou People’s Hospital from June 2016 to March 2021 were analyzed retrospectively. According to the cut-off ALT values recommended by 2018 version of AASLD guidelines (male 35 U/L, female 25 U/L), the patients were divided into two groups: high normal value group (HNALT, ALT≥AASLD and

The presence of replication-competent HBV DNA in the liver and/or serum of HBsAg-negative individuals is a sufficient and necessary condition for the diagnosis of occult hepatitis B virus infection (OBI). In recent years, Chinese scholars have proposed what is considered a more "rigorous" definition, i.e., on this basis, HBV window period (WP) infection is excluded, which corresponds to a serum HBV DNA level of below the lower limit of detection or a low positive value (< 200 IU/mL). As the definition of WP for HBV infection remains unclear and its duration is highly variable, "HBV DNA < 200 IU/mL" is not the only criterion in OBI patients. Therefore, it is believed that there is still a lack of sufficient basis and operability for the definition of OBI based on "the exclusion of HBV WP infection" and "HBV DNA < 200 IU/mL" as "rigorous" conditions for the diagnosis of OBI.