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.

Change in the activity of glucosylceramide synthase in human hepatoma cell line with hepatitis B virus infection in vitro / 临床肝胆病杂志

ObjectiveTo investigate the change in the activity of glucosylceramide synthase, the key enzyme in glycosphingolipid metabolism and synthesis, in Huh7 cells infected by hepatitis B virus (HBV) in vitro. MethodsBlood samples were collected from nine previously untreated patients with acute hepatitis B who attended Department of Infectious Diseases, The First Hospital of Lanzhou University, from June to August, 2019, and the blood samples collected from seven healthy individuals who underwent physical examination were established as control. Huh7 cells were inoculated with the high-copy HBV particles (＞9.9×107 IU/ml) in the serum of patients with HBV infection (infection group), and Huh7 cells co-cultured with the serum of healthy individuals were established as control group. The expression levels of HBsAg and HBV DNA in the cytoplasm of HBV-infected Huh7 cells were measured, and the correlation between GCS activity and virus was analyzed. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups, and a Pearson correlation analysis was performed. ResultsCompared with the control group, the infection group had a significant reduction in the number of cells, an increase in cell volume, and cell membrane fragmentation. The infection group had a significant increase in the expression of HBsAg in cytoplasm at 4 hours, 8 hours, 2 days, and 5 days after infection (P＜0.05); the expression level of HBV DNA tended to increase significantly from 4 hours after infection to 8 hours, 2 days, and 5 days after infection (16.67±11.55 IU/ml vs 112.01±25.94 IU/ml/328.01±10350 IU/ml/101.60±49.84 IU/ml, P＜0.001), with the highest level at 2 days after infection. During HBV infection, the activity of GCS gradually increased with the increase in viral replication from 4 hours after infection (126.21±9.59 IU/ml) and reached a peak at 2 days after infection (226.53±36.27 IU/ml), with a significant difference between the infection group and the control group at 2 days after infection (226.53±36.27 IU/ml vs 136.50±1544 IU/ml, t=3.956, P=0.016 7). The activity of GCS was positively correlated with HBV DNA level (r=0.576 8, P=0047 1). ConclusionHuh7 cells are successfully infected with the high-copy HBV particles in the serum of patients with HBV infection, which mimics the characteristics of HBV infection in vitro to a certain degree. The activity of GCS may be associated with HBV infection, suggesting that glycosphingolipid synthesis and metabolism may be closely associated with HBV.

Value of New Screening Scoring System of Gastric Cancer for Detecting Early Gastric Cancer Patients in Local Region / 胃肠病学

Background: The prognosis of early gastric cancer is significantly better than that of advanced gastric cancer. Because of the lacking of rational screening means, the detection rate of early gastric cancer is low. Serum pepsinogen Ⅰ (PGⅠ), PGⅡ, gastrin-17 (G-17) can be used for the screening of early gastric cancer. Aims: To investigate the value of new screening scoring system of gastric cancer for detecting patients with early gastric cancer in local region. Methods: A total of 393 patients from April 2017 to December 2018 were enrolled. According to pathological findings, patients were divided into control group, gastric ulcer group, dysplasia group and early gastric cancer group. Levels of PGⅠ, PGⅡ, G-17, Hp antibody were detected, and PGR was calculated. ROC curve was used to analyze the value of parameters for detecting early gastric cancer. Detection rates of early gastric cancer via serum ABC method, new ABC method and new screening scoring system were compared. Results: Compared with control group, serum PGⅠ and PGⅡ in gastric ulcer group were significantly increased, PGR was significantly decreased (P12.80 pmol/L and ≤6.90, respectively, the combination of these three indices for screening early gastric cancer had a sensitivity of 90.6%, specificity of 91.1%, and AUC of 0.965. The detection rate of early gastric cancer via new screening scoring system was significantly increased than that of serum ABC method, however, no significant difference was found between new screening scoring system and new ABC method (P>0.05). Conclusions: Decreased serum PGⅠ, PGR and increased G-17 may suggest the occurrence of early gastric cancer. New screening scoring system of gastric cancer is an efficient method for screening early gastric cancer.