Objective To establish the HepG2 cell lines which can stably express and replicate hepatitis 13 virus (HBV).Methods One point two X unit length of HBV genome was cloned intn SalⅠsite of the eukaryotic expression vector pREP10 to construct the recombinant plasmid pREP-HBV. Human hepatoblastoma cell HepG2 was transfected with pREP-HBV by Lipofectamine 2000 and seh,cted by bygromycin at the concentration of 250?g/mL.HBsAg and HBeAg were monitored by enzyme linked immunosorbent assay (ELISA)kits.H13V particles presemed in supernatant were ex- amincd by electronic microscopy.DNA isolated from intracellular HBV core particles was analyzed by Southbern blot using HBV-specific probe.Results The recombinant vector pREP HBV containing 1.2?unit length of HBV DNA was constructed successfully.After transfection of pREP-HBV to HepG2 cells and consistently cultured in hygromycin selective medium.5 drug-resistant cell lines, RHBV1-RHBV5.were established,and all of them could stably express HBsAgand HBeAg.South ern blot analysis revealed that HBV could replicate in all cell lines,as confirmed by the presence of replicateintermediatc DNA in intracellular HBV core particles.Clustered 42 nm Dane particles as well as 22-26 nm spherical H13sAg particles in condensed cuhure supernatant were visualized by elec tronic microsopic analysis.Conclusion HepG2 ceil lines in which HBV can replicate and express specific antigens are successfully established.Up to now,the cells have been passaged every three days for 50 times.

BACKGROUND:For pediatric patients with aplastic anemia in China, it is difficult to find human leucocyte antigen-matched sibling donors that are mostly replaced by parental donors.
OBJECTIVE:To retrospectively analyze the clinical efficacy and safety of parental haploidentical peripheral blood hematopoietic stem cel transplantation in children with relapsed and refractory severe aplastic anemia.
METHODS:Seventeen children with relapsed and refractory severe aplastic anemia who had no matched sibling or unrelated donor and failed to respond to immunosuppressive therapy were subjected to parental haploidentical peripheral blood hematopoietic stem cel transplantation. A conditioning regimen of fludarabine+cyclophosphamide+rabbit anti-human thymocyte immunoglobulin antibody and the triple therapy of methotrexate, cyclosporine A and mycophenolate mofetil were applied to prevent graft-versus-host disease.
RESULTS AND CONCLUSION: (1) Of the 17 children, 16 cases (94%) reached hematopoietic reconstitution, and the median time of neutrophils≥ 0.5×109/L and platelets≥ 20×109/L was 13 (11-15) days and 17 (12-28) days, respectively. (2) Incidence of acute graft-versus-host disease was 47% (8 of 17 cases), including 29% (5/17) of grades I-II and 18% (3/17) of grades III-IV. Incidence of chronic graft-versus-host disease was 41% (7/17). (3) With a median folow-up duration of 268 (43-753) days, the overal survival rate was 70.6% (12/17). Five dead cases (29%) belonged to transplantation-related death, including one case of fungal skin infections, one case of graft-versus-host disease, three cases of severe lung infection. No relapse case was reported. These findings indicate that if there are no matched sibling or unrelated donors and the immunosuppression effect is poor, parental haploidentical peripheral blood hematopoietic stem cel transplantation is a safe and effective salvage treatment for children with relapsed and refractory severe aplastic anemia.

ObjectiveTo evaluate the feasibility of determining the chemical composition of kidney stones using gemstone spectral imaging ( GSI ).Methods One hundred and sixty eight extracted human kidney stones immersed in a 10 cm deep water tank underwent CT (Discovery CT750 HD) scans with GSI mode and conventional polychromatic imaging ( CPI,120 kVp) mode.All GSI data were transferred to Workstation AW 4.4 to acquire monochromatic images of 50 keY,effective atomic number (Zeff) mapping images,water (calcium)-based images and calcium (Water)-based images with GSI Viewer.CT numbers of stones were measured and compared at 50 keV monochromatic images and 120 kVp polychromatic images,the mean Zeff,calcium density and water density were measured at Zeff mapping images,Calcium (Water) -based images and Water (Calcium)-based images,respectively.The mean Zeff,spectral HU curve slope and calcium water ratio (CWR) were compared with ANOVA and Wilcoxon test.The composition of kidney stones was determined by infrared spectrometer after CT examination.According to the result of stone composition determined by infrared spectroscopy,108 pure kidney stones were divided into five groups:Uric acid stones ( UA,n = 13 ),struvite stones ( STR,n = 24),cystine stones ( CYS,n = 14),calcium phosphate stones ( CaP,n = 18),and calcium oxalate stones ( COX,n = 39).ResultsThe mean Zeff,CWR,the mean CT numbers at 50 keV images,120 kVp images and spectral HU curve slope of each group were listed as the following:UA [ 7.4 ± 0.4,0.0085 ± 0.0021,( 503 ± 168 ) HU,(495 ± 106 ) HU and - 0.77 ] ; STR [ 11.8 ± 0.9,0.1743 ± 0.0677,( 1056 ± 290 ) HU,( 799 ± 165 ) HU and 18.72 ] ; CYS [ 11.2 ± 0.6,0.1253 ± 0.0297,( 740 ± 172 ) HU,( 565 ± 129 ) HU and 12.79 ] ; CaP [ 16.0 ± 0.4,0.6781 ± 0.0952,( 2567 ±178 ) HU,( 1602 ± 200 ) HU and 37.14 ] ; COX [ 15.4 ± 0.4,0.5683 ± 0.0759,( 2267 ± 385 ) HU,( 1489 ±284) HU and 36.36 ],there were significant differences among groups ( P ＜ 0.01 ).The differences in the mean Zeff,CRW,spectral HU curve slope were statistically significant among the five groups ( P ＜ 0.05 ).Conclusion Spectral CT imaging provides a new method to characterize the kidney stones with the information orovided by mean Zeff,CRW and the CT numbers at 50 keV.

In recent years, there has been increasing evidence that nonalcoholic fatty liver disease (NAFLD) is a manifestation of a systemic metabolic disease in liver. However, limitations in the definition and diagnostic criteria of NAFLD could not fully and accurately describe the metabolic disorder. A consensus statement proposed renaming NAFLD to metabolic associated fatty liver disease (MAFLD). Compared with NAFLD, MAFLD links fatty liver disease with metabolic factors such as overweight or obesity and diabetes, and the diagnosis of the disease no longer excludes the presence of metabolic abnormalities in patients with alcohol consumption or other chronic liver diseases. The authors focus on definition changes of fatty liver disease, discuss the epidemiological changes from NAFLD to MAFLD and the relationship of MAFLD with liver fibrosis and extrahepatic diseases, and also the significance of MAFLD for public health.

BACKGROUND:Careful regulation of bone immune response during repair of bone scaffold is important for bone regeneration. OBJECTIVE:To review the influence of bone immune response on bone repair and the design of bone tissue engineering scaffold with regulating bone immune function and its application in bone repair. METHODS:Relevant articles published from 1973 to 2023 were retrieved from Science Direct,PubMed,Web of Science,and CNKI databases.English search terms were"osteoimmunology,macrophages,bone repair materials,bone scaffold,bone defects,bone regeneration".Chinese search terms were"bone immunity,macrophages,bone repair material,bone stent,bone defect,bone regeneration".Totally 80 articles of the latest research progress in this field were summarized and analyzed. RESULTS AND CONCLUSION:(1)A detailed review was conducted on the important time points in the origin and development process of bone immunity,and it was explained that macrophages,as important members of the bone immune regulatory system,can be divided into two phenotypes:M1(pro-inflammatory)and M2(anti-inflammatory),and play a key role in different stages of bone regeneration.During the inflammatory phase,M1 type macrophages can activate osteoclasts,initiate tissue repair processes,and participate in the reconstruction of bone microvascular networks.On the other hand,during the bone tissue regeneration process in the later stages of inflammation,sustained high expression of M1 type macrophages can hinder the formation of new bones.During the repair phase,M2 macrophages can secrete osteogenic cytokines,stimulate osteogenic differentiation and mineralization of bone marrow mesenchymal stem cells,and promote bone formation.On the other hand,long-term activation of M2 macrophages can increase the secretion of fibrogenic molecules,leading to excessive formation of scar tissue and delaying the healing process.Therefore,regulating macrophages to undergo phenotype transformation at appropriate stages and constructing an immune microenvironment beneficial for osteogenesis has great significance for bone regeneration.(2)In the process of designing bone scaffolds with bone immune regulation characteristics,the physical and chemical properties such as scaffold roughness,pore structure,stiffness,hydrophilicity,surface charge,and surface functional groups can be changed to affect non-specific protein and cell adhesion,thereby affecting the interaction between bone scaffolds and the immune system.By designing surface functional coatings of bioactive substances such as hydroxyapatite,bioactive glass,metal ions,extracellular matrix,drugs,cytokines,and exosomes,the immune microenvironment can be actively regulated by releasing bioactive substances after implantation into the body,affecting macrophage polarization and crosstalk between macrophages and bone cells,and promoting more M2 polarization of macrophages,so as to build a bone immune microenvironment that is conducive to bone regeneration.(3)Based on the research and development of bone tissue engineering scaffolds,in addition to focusing on the direct regulatory factors of stem cell osteogenic differentiation,this article also proposes that attention should be paid to the management of the immune microenvironment of stem cell differentiation.By regulating the appropriate bone immune microenvironment,more stem cell osteogenic differentiation can be induced;the osteogenic efficiency of the scaffold can be enhanced,and the concept of"bone immune regulatory characteristics"can be condensed;deeply elucidated the multi-directional regulatory role of the bone immune microenvironment and introduced the existing strategies for changing the physicochemical properties and surface functional coating of scaffolds to endow them with bone immune regulatory potential,providing new ideas for guiding the development of a new generation of bone tissue engineering scaffolds with bone immune regulatory characteristics.However,the bone immune microenvironment is a dynamic equilibrium state,and most of the existing regulatory strategies do not consider the dynamic matching of regulation.Therefore,the research and development of intelligent bone immune regulatory scaffolds with efficient and targeted regulation of the immune microenvironment will be a key focus of attention for scholars in future.

Objective:To compare and analyze the clinical characteristics between acute fatty liver of pregnancy (AFLP) and the hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome.Methods:This is a retrospective cohort study. The clinical data of 13 cases with AFLP and 34 cases with HELLP syndrome were collected from three tertiary referral centers in Yunnan (the First Affiliated Hospital of Kunming Medical University, the Second Affiliated Hospital of Kunming Medical University, and Yan'an Hospital of Kunming City) from January 2016 to December 2021. The patients were diagnosed to AFLP and HELLP syndrome according to the Swansea criteria and the Tennessee classification system. The general characteristics, clinical features, laboratory results within 24 hours after admission, complications, maternal and neonatal outcomes were compared to analysis the differences between the two groups.Results:① Maternal characteristics: compared with HELLP syndrome group, AFLP group had lower body mass index (BMI) and blood pressure at admission (both P < 0.01). ②Clinical features: the most common symptoms in AFLP patients were skin jaundice, abdominal pain, nausea and vomiting, edema. The main manifestations of patients with HELLP syndrome were albuminuria, hypertension, edema, headache. Some patients had multiple symptoms concurrently. ③ Laboratory results: compared with HELLP syndrome group, the levels of platelet count (PLT), total bilirubin (TBil), direct bilirubin (DBil), γ-glutamyl transferase (γ-GGT), alkaline phosphatase (ALP), total bile acid (TBA), serum creatinine (SCr) and international standardized ratio (INR) in AFLP group were significantly increased within 24 hours after admission [PLT (×10 9/L): 107.69±51.13 vs.76.71±43.25, TBil (μmol/L): 121.60 (83.20, 170.00) vs.15.25 (7.22, 29.05), DBil (μmol/L): 86.50 (58.60, 104.00) vs. 4.30 (2.22, 10.10), γ-GGT (U/L): 87.00 (37.00, 127.00) vs. 41.00 (19.00, 64.42), ALP (U/L): 199.10 (109.00, 349.20) vs. 125.50 (90.50, 155.25), TBA (μmol/L): 51.50 (16.20, 117.40) vs. 4.15 (2.02, 6.95), SCr (μmol/L): 155.80 (129.00, 237.00) vs. 79.00 (65.43, 113.70), INR: 1.28 (1.17, 1.63) vs. 0.94 (0.88, 1.08), all P < 0.05], prothrombin time (PT) was significantly prolonged [seconds: 16.10 (14.50, 19.20) vs. 12.40 (11.43, 13.40), P < 0.05]. The level of blood glucose (GLU), fibrinogen (FIB) and the activity of antithrombin Ⅲ (ATⅢ) decreased significantly [GLU (mmol/L): 5.18±1.33 vs. 6.33±1.19, FIB (g/L): 1.96±1.46 vs. 3.81±1.58, ATⅢ (%): 40.61±25.84 vs. 66.39±24.11, all P < 0.05]; ④ Complications: compared with HELLP syndrome group, the incidence of patients with hypoglycemia [30.77% (4/13) vs. 0% (0/34)], acute liver failure [53.85% (7/13) vs. 5.88% (2/34)], acute renal insufficiency [69.23% (9/13) vs. 8.82% (3/34)], coagulopathy [76.92% (10/13) vs. 38.24% (13/34)], disseminated intravascular coagulation (DIC) [53.85% (7/13) vs. 5.88% (2/34)], and multiple organ dysfunction syndrome (MODS) [53.85% (7/13) vs. 5.88% (2/34)] were significantly higher in AFLP group (all P < 0.05). ⑤ Maternal and neonatal outcome: all patients delivered after admission. The total length of hospital and intensive care unit stay were significantly longer in the AFLP group than in the HELLP syndrome group [days: 17.00 (11.00, 25.00) vs. 9.00 (7.00, 12.00), 12.00 (4.00, 22.00) vs. 3.91 (0, 7.00), both P < 0.01]. Two AFLP patients died, including one due to intracranial venous thrombosis and one due to multiple organ failure and cardiopulmonary arrest. There were no deaths in the HELLP syndrome group. Conclusions:There are significant differences in maternal characteristics, laboratory results and complications between AFLP and HELLP syndrome. TBil, γ-GGT, SCr, FIB, INR and ATⅢ activity may help to distinguish the two diseases.

Yuebawei refers to 8 kinds of genuine medicinal materials in south China area，namely Citri Grandis Exocarpium， Citrus reticulata， Amomum villosum， Pogostemon cablin， Morinda officinalis， Aquilaria sinensis， Citrus medica and Polygonum multiflorum. The results of this review show that， the pharmacological effects of Yuebawei are very extensive. It has significant effects in relieving cough and phlegm， anti-bacterial and anti-inflammatory， enhancing immunity， anti-oxidation， antidepressant， immune regulation and regulating gastrointestinal function. Its development and application prospects are broad， but most of the existing pharmacological studies stay on pharmacodynamic studies. In the aspect of the safety research of Yuebawei， there are few studies on the safety of other medicinal materials， except for the liver toxicity of P. multiflorum； at the same time， there are few comparative studies on the therapeutic advantages of Yuebawei and other local medicinal materials in clinical application. It is necessary to strengthen the research on the material basis of pharmacological action mechanism， and safety and efficacy observation of clinical medication for Yuebawei， so as to provide scientific basis for the development of new drugs and clinical promotion and application of Yuebawei.