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.

The present therapy for primary hepatocellular carcinoma (HCC)consists of surgery as well as local radiotherapy and chemother-apy.However,the majority of patients are susceptible to recurrence after comprehensive treatment,and the overall treatment outcome is not ideal due to the lack of effective drugs and strategies.Increasing evidence has demonstrated that the immune system is closely related to the development,progression,metastasis,and recurrence of HCC.Thus,immune therapy,especially cellular immunotherapy,could regulate immune function and induce specific antitumor immunity to achieve the goal of controlling HCC and reducing its recurrence and metastasis, which has become an essential part in the comprehensive treatment of HCC.The findings in preclinical and clinical studies on cellular immu-notherapy for HCC data are reviewed,and the current problems are discussed.

Objective To construct the eukaryotic expression vector coding HCV gene E2 fused with His-Tag, and to express fused protein in CHO cells for investigating the function of HCV envelope protein E2. Methods The gene encoding HCV envelope protein E2 was amplified from pBRTM/HCV1-3011, a plasmid containing the cDNA of HCV's ORF, by polymerase chain reaction (PCR) method and cloned into the vector pET28(a) containing His-Tag to obtain the fused HCV envelope protein E2 gene fused with His-Tag. The fused gene was cloned into pcDNA3.1 to construct the recombinant plasmid pcDNA3.1-His-E2, which will express the E2 protein, fused with His tag. This recombinant plasmid was transfected into CHO cells by Lipofactamine 2000 reagent. The fused protein was identified by indirect immunofluorescence (IIF) and Western-blot (WB) methods. Result The positive results were obtained when the fused protein of HCV E2 with His-Tag were identified by IIF and WB methods. Conclusion The eukaryotic expression vector pcDNA3.1-His-E2 was constructed successfully and the fused proteins were expressed in cells.

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.

Seventy-three patients with type 2 diabetes mellitus were divided into atheroselerosis(AS) group and non-AS group according to the intima-media thickness(IMT)of the carotid artery.The plasma visfatin level in AS group was higher than that in non-As group[(44.95±10.14 vs 34.52±9.08)μg/L,P<0.05],and both of them were higher than that of the control [(24.46±7.18)μg/L,both P<0.05 ].The visfatin level Was positively correlated with IMT,waist-to-hip ratio,visceral fat thickness,fasting insulin,and HOMA insulin resistance index.Age,duration of diabetes,HbA_(1C),and visfatin level were the major risk factors for IMT of the carotid artery.

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.

The research on prophylaxis and treatment of hepatitis C has been performed for more than 30 years, with outstanding achievements. The discovery and confirmation of hepatitis C virus (HCV) was a milestone for how humans discovered new life. Emphasis on HCV molecular biology, infection immunity, and pathogenesis is the basic rule for scientific research on infectious diseases. PEG-IFN combined with ribavirin as the standardized antiviral treatment has been a great success; this combination therapy achieves a sustained viral response more than 80% in Chinese people, which is a typical example for successful clinical application of cytokines. Direct-acting antiviral agent(DAA) or combined application has made it possible to cure all patients infected with hepatitis C, which is the most successful example for reference. Persistent viral infection and maintenance of immune homeostasis under certain conditions are the results of the interaction between the host and the virus, and the development of vaccines will be continued.

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.