OBJECTIVETo establish a culture system of HBV positive serum infected Hep G2 cells in vitro.

METHODSHep G2 cells were seeded into six-well cluster dishes, at 1 x 10(-6) cells per well and incubated with 3 ml 10% fetal calf serum/ Dulbecco's modified Eagle's medium (10% FCS/DMEM) at 37 degrees in 5% CO2 air. At 24 h after plating, infection group Hep G2 cells were cultured with 0.5 ml HBV positive serum, in control group HBV negative serum was used, 24 h later the inoculums was removed. The cells were then extensively washed with 0.01 mol/L phosphate-buffered saline (PBS). After washing with PBS, 4 ml 2% FCS/DMEM were added to each well and the medium was collected every 12 h. ELISA method was used to detect HBsAg in culture medium. HBV DNA in cells and culture medium was detected by PCR.

RESULTSIn infection group, HBsAg could be detected from cell culture medium from 12 h (after PBS washed) to 84 h. HBV DNA could be detected by PCR in culture medium and cells.

CONCLUSIONInfection of Hep G2 cells by HBV positive serum is feasible.

Carcinoma, Hepatocellular ; pathology ; virology ; Cell Line, Tumor ; DNA, Viral ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; blood ; virology ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; genetics ; growth & development ; immunology ; Humans ; Liver Neoplasms ; pathology ; virology ; Polymerase Chain Reaction ; Serum ; virology

Objective:To investigate the application of transbronchial needle aspiration (TBNA) in the diagnosis of tuberculosis with mediastinal lymphadenopathy in children.Methods:A retrospective study was conducted on clinical data in 8 children of tuberculosis with mediastinal lymphadenopathy treated in the Center for Respiratory Intervention, Children′s Hospital Affiliated to Shandong University from March 2014 to July 2019.TBNA was performed after the mediastinal lymphadenopathy were diagnosed by chest enhanced CT and the final diagnosis was made.The diagnostic experience of TBNA was summarized.Results:Eight children with mediastinal lymphadenopathy included in this present study aged from 7 months to 8 years and 6 months (infants accounted for 75.0%), with a median age of 22.5 months.There were 3 males (37.5%) and 5 females (62.5%). The body mass was 8.5-39.0 kg, and the median body mass was 10.7 kg.The course of disease was 15-90 days, and the median number of days was 18.5 days.The clinical manifestations included cough in 8 cases, fever in 4 cases, wheezing in 1 case and laryngeal ringing in 1 case.Bronchoscopy and TBNA biopsy were performed.Cytology, etiology and pathology were examined after TBNA.A definite diagnosis could be made in 6 children, with a diagnosis rate of 75.0%.Among them, 4 cases were found with acid-fast bacilli in smear but pathological examination was negative; 1 case was pathologically conformed to the characteristics of tuberculosis infection but the smear was negative; the smear and pathology of 1 case were both suggestive of tuberculosis; 2 cases did not present etiological and histological evidence with TBNA.The diagnosis was made according to the positive acid-fast bacilli of alveolar lavage fluid smear.There were no complications during and after operation.Conclusions:TBNA is an important method to diagnose tuberculosis in children, which is effective, safe and has high clinical application value.

OBJECTIVETo establish a MDS mouse model with iron overload and to study the effect of iron overload on MDS.

METHODSThe exogenous mutant gene RUNX1-S291fs was inserted into the mice bone marrow mononuclear cell's genome in mice by retrovirus and transplanted into C57BL/6 mice irradiated by Co γ-ray. After 8 weeks,intraperitoneal injection of iron was performed to establish an MDS mouse model with iron overload. After 24 weeks of transplantation, the peripheral blood, bone marrow, femur, liver and spleen of mice were taken, then the morphological characteristics of peripheral blood and bone marrow cells were observed by Wright's staining; the liver, spleen and bone marrow were stained with Prussian blue to observe the iron deposition. The surface antigens of bone marrow cells were detected by flow cytometry. Bone marrow mononuclear cells and spleen tissue proteins were detected by Western blot to confirm the transfection of RUNX1-S291fs gene and expression of protein. The blood routine and transplanted cell chimeric rate of mice were monitored periodically.

RESULTSCompared with the empty plasmid control mice, levels of leukocyte and hemoglobin as well as platelet were decreased in RUNX1-S291fs mutant mice; the peripheral blood cells and bone marrow cells showed pathological hematopoiesis; the liver and spleen enlarged significantly; the tissue structure of femur, liver and spleen was abnormal; the expression of bone marrow cell surface antigens was abnormal. Bone marrow cells and spleen tissue expressed the RUNX1-S291fs protein. Compared with the controlled mice injected with normal saline, iron deposition occurred in the bone marrow, liver and spleen stained with Prussian blue in the mice injected with iron agent.

CONCLUSIONMice engineered to carry exogenous mutant gene RUNX1-S291fs and injected with iron showed pathologic features of MDS and iron overload, resulting in establishing MDS iron overloaded mouse model successfully, which lays a foundation for studying the effect of iron overload on MDS.

Animals ; Bone Marrow ; Disease Models, Animal ; Iron Overload ; Mice ; Mice, Inbred C57BL ; Spleen