Synchronization isolation method for multiple types of cells from mouse liver / 中华肝脏病杂志

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Effects of hepatitis B virus on Th17, Treg and Th17/Treg ratio in different alanine aminetransferase stages / 北京大学学报(医学版)

OBJECTIVE@#To evaluate the effects of hepatitis B virus (HBV) on helper T lymphocytes 17 (Th17), regulatory T lymphocyte (Treg) and Th17/Treg ratio in chronic hepatitis B patients in different alanine aminetransferase (ALT) stages.@*METHODS@#In the study, 336 chronic hepatitis B patients in the first hospital of Lanzhou University were analyzed. The hepatitis B antigen antibody parameters were measured by chemiluminescence immunoassay analyzer, the liver function parameters were measured by automatic biochemical analyzer, the HBV loads were measured by quantitative PCR, Th17, Treg and Th17/Treg ratios were detected by flow cytometry. Among them, 111 cases (ALT < 40 U/L) of ALT were normal hepatitis B, 108 cases of chronic hepatitis B with ALT above normal upper limit and < 2 times higher (40 U/L≤ALT < 80 U/L), and 117 cases of chronic hepatitis B with ALT above 2 times normal upper limit (80 U/L≤ALT). According to the viral load, they were divided into low replication group with HBV DNA < 4.0 lg copies/mL, medium replication group with 4.0 lg copies/mL≤HBV DNA < 6.0 lg copies/mL and high replication group with HBV DNA ≥ 6.0 lg copies / mL. Dunnett T3 variance analysis were used to analyze the effects of HBV on Th17, Treg and Th17/Treg ratio in the chronic hepatitis B patients in different ALT stages. The changes of virological and immunological indexes before and after treatment were observed for 24 weeks of antiviral therapy in the hepatitis B patients with ALT≥double upper limit of normal group.@*RESULTS@#In the ALT normal group, different virus load HBV had minor effects on Th17, Treg and Th17/Treg ratio. In the ALT≥2 times upper limit of normal group, with the virus load increased, Th17 (3.18%±0.79% in low replication group, 3.78%±0.92% in medium replication group and 4.57%±1.15% in high replication group), Treg cells (5.52%±1.58% in low replication group, 5.89%±1.84% in medium replication group and 6.37%±2.35% in high replication group) and their ratio Th17/Treg (0.57±0.25 in low replication group, 0.65±0.29 in medium replication group and 0.73±0.36 in high replication group) were significantly increased (P < 0.05). After entecavir treatment 24 weeks, the patient' s HBV-DNA decreased significantly, Th17 (3.89%±1.02% vs. 2.06%±0.46%), Treg (6.02%±2.03% vs. 5.06%±1.25%), Th17/Treg ratio (0.65±0.28 vs. 0.41±0.14) decreased significantly (P < 0.05).@*CONCLUSION@#Investigation on the effects of HBV on Th17 and Treg cells and their ratios in different ALT states can clarify the effects of HBV on the body from the immunological perspective and can further understand the ALT grouping for antiviral treatment theoretical significance, which is helpful for clinical treatment.

Distribution of HCV genotypes in Chinese Han population with chronic hepatitis C / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the distribution of HCV genotypes in Chinese Han population with chronic hepatitis C (CHC).</p><p><b>METHODS</b>This randomized multicenter study included 1 014 CHC patients from 28 hospitals in different regions of China. SPSS 20.0 was applied to analyze the relationship among region, HCV genotype, gender and the replication level of HCV-RNA.</p><p><b>RESULTS</b>HCV 1 genotype (56.80%) was the most common genotype. The majority of CHC patients were of genotype 1, 2, 3, 6 in the order of frequency, except those in southwestern, southern and central China. HCV 1, 2, 3, 6 genotypes were most common among male patients in southern China; among female patients in northern China; among male patients in northern and northwestern China and among male patients in northwestern China, respectively (all P <0.05). There was no statistical significance between different genders in other regions. The high viral load was more common than the low viral load among HCV 1, 2, 3, 6 genotype-infected patients.</p><p><b>CONCLUSION</b>There are different distributions of HCV genotypes among the different regions. In addition, HCV genotypes are correlated with gender and HCV-RNA load.</p>

Clinical efficacy of various antiviral-based strategies to treat chronic hepatitis patients with positivity for hepatitis B e antigen and rtN236T mutation / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To compare the efficacy and safety of the common antivirals, including adefovir dipivoxil (ADV), pegylated-interferon alpha-2a (peg-IFN) and lamivudine (LAM), used as combination therapies to treat chronic hepatitis B (CHB) patients with positivity for the hepatitis B e antigen (HBeAg) and hepatitis B virus (HBV) harboring the ADV-resistance mutation, rtN236T, and to explore the factors associated with curative outcome.</p><p><b>METHODS</b>Sixty-five adult CHB patients (age range: 20-60 years) who were unresponsive to ADV therapy (HBeAg-positive; HBV DNA >or= 10(5) copies/ml), LAM-naive, and tested positive for the rtN236T HBV mutation were enrolled in the study and randomly divided into two treatment groups: Group A (n = 33), who were administered ADV (10 mg/day, orally) plus peg-IFN (180 microg/week, subcutaneous injection) for 48 weeks; and Group B (n = 32 patients), who received the ADV plus LAM (100 mg/day, orally) for 48 weeks followed by continued LAM treatment for an additional 24 weeks. Pre- (baseline), during and post-treatment measurements of HBV viral loads and hepatitis B markers were made by quantitative PCR and electrochemiluminescence assays, respectively. All patients underwent liver biopsies to determine the histological activity index (HAI) and treatment response regarding inflammation and fibrosis stage. The rates of virological response (VR), HBeAg-negativity, HBeAg seroconversion, and alanine aminotransferase (ALT) normalization were calculated, and the significance of differences between groups were assessed by Student's t-test and Chi2 test.</p><p><b>RESULTS</b>There were no significant differences between the two groups in regards to sex, age, or baseline levels of HBV DNA, ALT, and total bilirubin (P > 0.05). At weeks 24 and 48 of treatment and 24 after treatment end, group A showed significantly higher (vs. group B, P < 0.05) rates of reduced HBV DNA viral loads (81.8%, 90.9%, and 75.8% vs. 53.1%, 56.2%, and 59.4%), VR (48.5%, 60.6%, and 42.4% vs. 31.3%, 34.4%, and 31.3%), HBeAg-negativity (39.4%, 60.6%, and 54.5% vs. 12.5%, 37.5%, and 37.5%), HBeAg seroconversion (27.3%, 54.5%, and 48.5% vs. 6.3%, 15.6%, and 18.8%), and ALT normalization (72.7%, 84.8%, and 78.8% vs. 46.9%, 56.3%, and 46.9%). After 48 weeks of treatment, group A showed significantly improved HAI (vs. group B, P < 0.05). With the exception of treatment-related increased creatinine (P < 0.05), group A showed significantly higher rates of adverse reactions; although, none was serious enough to threaten patient safety or necessitate early termination of the treatment regimen. Twenty-four weeks after treatment completion, five patients had HBV viral loads of >or= 2log10 copies/ml and four had < or= 500 copies/ml, and ALT was normalized in 28 patients. The four patients in group A with HBV DNA < or= 500 copies/ml and elevated ALT during treatment did not show HBeAg seroconversion.</p><p><b>CONCLUSION</b>Peg-IFN plus ADV combination therapy produced better outcomes than the ADV plus LAM combination therapy in regards to HBV viral loads, VR rate, HBeAg-negative rate, HBeAg seroconversion rate, ALT normalization rate, and HAI, but was associated with a higher rate of adverse reactions (none of which were severe). Lack of HBeAg seroconversion was associated with higher virus load and ALT levels.</p>

Inhibition effect of small interfering RNA targeting connective tissue growth factor on liver fibrosis in rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To design and synthesize small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) and to investigate its effect on liver fibrosis.</p><p><b>METHODS</b>The interference sequence of CTGF was designed and synthesized. Rat hepatic fibrosis model was induced by intraperitoneal injection of 40 % CCl4(3 ml/kg). Thirty male rats were randomly divided into 5 groups: in normal control and model groups rats received tail vein injection of normal saline every 3 days for 8 consecutive weeks; in preventive group rats received tail vein injection of CTGF siRNA (0.1 mg/kg) every 3 days for 8 weeks; in 2-w treatment group CTGF siRNA was given for 6 weeks starting from two weeks after CCl4 injection; in 4-w treatment group CTGF siRNA was given for 4 weeks starting 4 weeks after CCl4 injection. The serum and hepatic tissue samples were harvested 3 days after the last CCl4 injection. Hepatic fibrosis indices were measured. Expression of CTGF mRNA and protein in the liver was evaluated by RT-PCR and Western blot, respectively. Fibrosis in rat liver was analyzed by Masson staining.</p><p><b>RESULTS</b>Compared with model group (0.544 0.019), the expression of CTGF mRNA and protein in liver of both preventive(0.105 ± 0.003) and 2-w treatment groups (0.190 ± 0.006) were markedly down-regulated (P<0.05). Inflammation, necrosis and fibrosis in hepatic tissue were significantly attenuated. In addition, the serum ration of liver fibrosis indices was greatly reduced(P<0.05). Compared with preventive and 2-w treatment groups, the expression of CTGF mRNA and protein in liver in 4 weeks of treatment group were up-regulated (P<0.05); inflammation, necrosis and fibrosis in hepatic tissue were relative increased; and the serum concentrations of liver fibrosis indices were relatively higher (P<0.05).</p><p><b>CONCLUSION</b>The highly effective CTGF siRNA has been successfully synthesized, which can inhibit CTGF expression in liver, prevent hepatic fibrosis and its progress in rats.</p>