As an important immune organ,the liver has a unique immune microenvironment,and is one of the important places for innate immunity and adaptive immunity.The liver is closely connected with the immune system through the gut-liver axis and is also the target or-gan of immune injury.However,the defensive role of the liver for Hepadnaviridae remains unclear.Cell therapy for liver diseases includes input of liver cells or stem cells for functional replacement,therapy by immune cells for antiviral and antitumor purposes,and genetic therapy for hereditary hepatopathy with cells as the carrier.The features of mesenchymal stem cells,multi-directional differentiation potential and immunomodulatory property,become the hot spots of cell therapy for liver diseases,and efficient in vitro amplification of cells makes it possi-ble for the use of NK cells in the treatment of hepatocellular carcinoma.

Homeostasis between the host and viruses is naturally maintained.On the one hand,the immune system activates the immune re-sponse to kill or eliminate viruses;on the other hand,the immune system controls the immune response to maintain immune homeostasis. The cause of persistent infections with hepatitis viruses such as HBV and HCV is that viral molecules damage the immune system of the host and their variants escape immune clearance.Long-term coexistence of the host and viruses is the process involving various immune cells and molecules and is the result of homeostasis maintenance in antiviral immune response.The immune homeostasis maintained during persis-tent infections with hepatitis viruses is analyzed by the cellular and molecular mechanisms.

Objective To study the inhibition of HCV IRES mediated HCV core protein expression in cells by inhibitor RNA. Methods Plasmid pcRz-IRNA, a eukaryotic expression vector with IRNA and two self cleavage ribozyme overhang at both sides respectively, was constructed and co-transfected with pcHCVcluc (containing HCV NCR, core and Luc genome) into the HHCC cell line (Human Hepatocellular Carcinoma cell line). Immunoflurescence tests were applied to detect the co-transfected cells, which were thereafter analysed with confocal microscope quantitatively. Luciferase activity was valued using Luc Assay System (Promega). Results The cotransfected cells expressed HCV core protein, and the fluorecein in which was reduced significantly in comparison with control. Conclusions IRNA can inhibit the expression of HCV IRES mediated core protein in the cotransfected cells.

Objective To detect circulating CD4 + CD25 + regulatory T cells (Treg) and Toll-like receptor(TLR)2 and TLR4 expression on the peripheral blood mononuclear cells (PBMCs) of patients with HBV-related liver cirrhosis (LC), and to explore the correlation between them. Methods PBMCs isolated from 30 LC patients, 21 chronic hepatitis B (CHB) patients and 16 normal controls(NC) were stained with fluorescent labeling anti-TLR2-PE, anti-TLR4-APC, anti-CD14-FITC monoclonal antibodies and anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE. Samples were detected by flow cytometry. Statistic analysis be-tween groups was performed by Kruskal-Wallis H test. Spearman rank correlation was used to analyze the correlation of Treg and TLR2, TLR4. Results The expression of TLR2 and TLR4 were significantly up-reg-ulated in patients with LC than those in the controls (TLR2 : 200.3 ± 96.8 vs 94.1 ± 17.6, P < 0.05 ; TLR4:32.1 ±7.2 vs 17.8 ±3.9, P<0.05). The expression of TLR4 was significantly increased in pa-tients with LC than those in patients with CHB (TLR4 : 32. 1 ± 7.2 vs 25.2 ± 8.3, P < 0.05), but there were no differences of TLR2 expression between LC and CHB(200.3 ± 96.8 vs 214.0 ± 72.6, P > 0.05). Treg/CD4+ T cells were 5.07% ±1.43%, 5.88% ±1.66%, 4.21% ±1.24% in patients with LC, CHB and NC, respectively. Treg/CD4+ T cells were significantly increased in patients with CHB than those in pa-tients with NC(P<0. 05) and LC(P <0.05), but there were no differences between LC and NC(P > 0.05). TLR4 expression and Treg were positive correlation (r = 0. 469, P = 0. 032) and TLB2 expression were negative correlation in patients with LC (r = -0.428, P = 0.021). Conclusion The expression of TLR2 and TLR4 were up-regulated on PBMCs in patients with LC. It seems to be expression of TLR2 and TLR4 in-volved in the pathogenesis of LC.

Objective To observe the morphological characteristics of HCV particles and intracel-lular ultrastructure changes in Huh7. 5 cells which was infected with chimeric HCV via transmission electron microscopy. Methods Plasmid J6/JFH encoding the full length HCV chimeric genome was transcribed to HCV RNA in vitro and the RNA was transfected into Huh7.5 cells by electroporation. Quantitative real-time PCR(qRT-PCR) was used to assay HCV copies of the supernatant of transfected cells. Indirect immunofluo-rescence was used to detect the expression of HCV proteins. The cell culture superoatant were used to infect narve Huh7.5 cells, transmission electron microscopy was used to observe morphological characteristics of vi-rus particles and intracellular ultrastructure changes in infected Huh7. 5 cells. Results qRT-PCR showed high level virus copies in supernatant of transfected cells collected in different times, indirect immuno-fluo-rescencc proved high expression of HCV NS5A proteins in the transfected cells. Large numbers of enveloped or unenveloped virus-like particles (VLPs) were observed in infected Huh7. 5 cells via transmission electronmicroscopy. We also found hyperplasia of some membrane-enclosed organelles in the cytoplasm. Several fea-tures characterizing flavivirus infected cells and a cytoplasmic inclusion of unknown origin were observed. Conclusion The chimeric HCV from in vitro cell culture system is proved to be intact virus particles which can efficiently infect Huh7.5 cells.

Clinical medicine is a comprehensive discipline integrating natural science and hu-manities and social science. Lemology is closely related with basic medicine and medical microbiology and medical immunology are the basis of lemology. Therefore, in the process of cultivating postgradu-ates of lemology, we should not only should attach importance to the cultivation of basic medical knowl-edge and clinical professional quality, but also pay more attention to the development of the intelligence factors and non-intelligence factors. Meanwhile education on humanity, social sciences and relevant laws and regulations should be enhanced to cultivate doctors' professional quality. Reverse thinking and lateral thinking in the clinical diagnosis should be strengthened to achieve the training objectives of cultivating international medical talents.

Objective To observe the protective effects of Toll-like receptor(TLR)-4 siRNA against acute liver injury in mice induced by lipopolysaccharide(LPS)and D-galactosamine(D-GalN).Methods One hundred and fifty C57BL/6 male mice were divided into 5 groups: phosphate buffered solution(PBS)pretreatment group,negative control plasmid pretreatment group,TS4 pretreatment group,TS6 pretreatment group and TS7 pretreatment group.Acute liver injury was induced in mice by intraperitoneal coinjection of LPS(10 ng/g)and D-GalN(1 mg/g).In vivo delivery of siRNA was performed via the tail vein by hydrodynamic injections(50 μg siRNA dissolved in 1 mL PBS)24 h and 48 h before coinjection of LPS and D-GalN. Expression of TLR-4 in liver tissues was measured by immunohistochemistry.The changes of TLR-4,tumor necrosis factor(TNF)-α and macrophage nflammatory protein(MIP)-2 mRNA levels in liver tissues were determined by reverse transcriptasepolymerase chain reaction(RT-PCR)analysis.MIP-2 and TNF-α concentrations in the sera of mice were determined by enzyme-linked immunosorbent assay(ELISA). Levels of alanine transaminase (ALT) and aspartate transaminase(AST) in serum were measured by standard autoanalyzer techniques. Liver pathological changes were observed by haematoxylin-eosin staining, while cell apoptosis levels in liver were determined by terminal deoxynucleotidyl-mediated-dUTP nick end labeling (TUNEL)assay. The difference of survival rates in 5 groups was analyzed by Fisher's exact probability test.ResultsPretreatment with TLR-4 siRNA down-regulated the TLR-4 mRNA and protein expressions,and significantly decreased the mortality and liver injury caused by coinjection of LPS and D-GalN in C57BL/6 mice.TLR-4 siRNA significantly down-regulated the TNF-α and MIP-2 mRNA expression and cytokine levels as determined by RT-PCR and ELISA,respectively. TLR-4 siRNA abrogated hepatocyte necrosis and inflammatory infiltration and also remarkably reduced serum concentrations of transaminases. The percentage of TUNEL-positive hepatocytes was significantly reduced in TLR-4 siRNA pretreatment group(TS4 pretreatment group: 0.065±0.015 vs PBS pretreatment group; 0.346±0.062,P＜0.05).ConclusionIt suggest that inhibition of TLR-4 expression by TLR-4 siRNA may provide potential application value for preventing liver injury.

Tenofovir alafenamide (TAF) is a novel nucleoside reverse transcriptase inhibitor used in the treatment of human immunodeficiency virus (HIV) infection and chronic hepatitis B virus (HBV) infection. Compared with tenofovir disoproxil fumarate, TAF has better plasma stability and stronger liver-targeting ability and can significantly reduce the adverse events of renal injury and reduced bone mineral density. This article summarizes the research advances in the pharmacological characteristics, metabolic pathways, drug interactions, drug resistance, and renal safety of TAF and its role in patients with chronic HBV infection.

Hepatitis B virus(HBV)infection is the main cause of chronic liver diseases in China. The clearance of HBV is important for patients with chronic hepatitis B.Current antiviral therapy includes interferon and nucleotide analogues.The adverse reactions of interferon are common which restrict its application.Nucleotide analogues need long-term maintenance treatment.Kupffer cells (KCs) are the main innate immune cells in the liver.Studies have shown that KCs play an important role in hepatitis B virus infection.It can inhibit virus replication effectively , but may also result in immune dysfunction , causing HBV persistence.Understanding the mechanism related to the role of KCs in hepatitis B virus infection may lead to a new approach of antiviral therapy.

Almost all pathogens have DNA or RNA genomes.Theoretically, genomes of pathogens can be effectively detected by sequencing technology , it would be of value for etiological diagnosis of infectious diseases.The next generation sequencing ( NGS) emerged in 2005, the advantages of low cost , high efficiency and high accuracy make it a favorable technical platform for clinical identification of microorganisms.As a new sequencing technique , metagenomic next generation sequencing ( mNGS) has great potential in improving the accuracy and efficiency of pathogenic diagnosis , especially in the areas where conventional diagnostic methods have limitations.This article introduces the common platforms and principle of the high throughput sequencing , reviews the clinical applications and the advantages of mNGS in identification of different pathogenic microorganisms , and reflects on its limitations and seeks possible solutions.