OBJECTIVETo evaluate the replication and encapsidation of HBV mutants with the truncated C gene.

METHODSThe HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome.

RESULTSThe C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay.

CONCLUSIONThe C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.

Cell Line, Tumor ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; physiology ; Humans ; Mutation ; Plasmids ; genetics ; Transfection ; Virus Replication

BACKGROUNDTo reveal the characteristics of genotype and phenotype of HIV strains in blood and some tissues of AIDS patients.

METHODSThe virus was isolated from peripheral blood mononuclear cell (PBMC),cerebrospinal fluid (CSF)and lymph nodes of 3 AIDS patients by coculture with PBMC stimulated by PHA for 72 hours from uninfected donor. The cytopathic effect of the HIV isolates was determined in cultured MT2 cell line. The env gene sequences form proviral DNA were analyzed by GCG software.

RESULTSIn one patient,there were differences between the strains from blood and different tissues both in genotype and phenotype. The biological phenotypes of two strains from CSF were non syncytium (NSI) type, their env sequences were similar to standard CNS tropic strain (SF162).

CONCLUSIONSThe viral heterogeneity exists in different body compartments within an infected individual. The neurotropic isolate which is similar to international standard strain exists in some AIDS patients in China.

Acquired Immunodeficiency Syndrome ; virology ; Adult ; Coculture Techniques ; Female ; Genetic Heterogeneity ; Genotype ; HIV ; isolation & purification ; Humans ; Leukocytes, Mononuclear ; virology ; Lymph Nodes ; virology ; Male ; Phenotype

Animals ; COS Cells ; Female ; Genetic Therapy ; Interleukin-12 ; genetics ; Liver Neoplasms, Experimental ; immunology ; therapy ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy.

METHODSA fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot.

RESULTSThe HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon.

CONCLUSIONIt is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.

Cell Transformation, Viral ; Cells, Cultured ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; physiology ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; cytology ; virology ; Humans ; Liver ; cytology ; virology ; Recombinant Proteins ; genetics ; Transfection ; Virus Replication

OBJECTIVETo explore the effect and mechanism on HBV replication in C gene truncated mutant.

METHODSProtein expression of C gene truncated vector and wild C gene vector were assay by SDS-PAGE Western blot. Constructed C gene truncated expression vector was cotransfected with wild HBV genome; virus load was detected by PCR in the culture medium and the cell. The formation of core particle was assay by Native western blot.

RESULTSThe recombinant vectors can efficiently express. Virus load of the cotransfected group by pcDNA3-deltaC and adwR9 was lower than that of control group in the culture medium and the cell. Protein band of the co-expressed group by pcDNA3-deltaC and pcDNA3-C showed slightly weaker than that of the co-expressed group by pcDNA3 and pcDNA3-C.

CONCLUSIONC gene truncated mutant could interfere with the formation of core particle and reduce of HBV replication

Cell Line ; Genetic Therapy ; Hepatitis B ; therapy ; Humans ; Mutation ; Transfection ; Viral Core Proteins ; genetics ; Virus Replication

Objective To diagnose the process of hepatic injury,a method of quantitating the concentration of sH2a subunit from asialoglycoprotein receptor (ASGPR) in serum by enzyme-linked immunosorbent assay was established and clinical evaluated.Methods 210 serum samples were collected in Suzhou Kowloon Hospital.Among them,70 subjects with cirrhosis,viral hepatitis and fatty liver disease were as hepatic injury group and 140 subjects of healthy and high fat,hemolysis,jaundice and with autoimmune disease were as control group.The serum sH2a of two group were measured by ELISA kit.The results of sH2a ELISA were analyzed by four table chi-square test and SPSS20.0 software.Results sH2a protein level in liver injury group and control group were 105.92+ 53.41 ng/ml and 69.25+27.45 ng/ml,respectively.The difference between the control group and the liver injury group was statistically significant (F=14.375,t=5.397,P=0.000).The sensitivity and specificity of the sH2a ELISA kit were 68.57％ (95％CI:56.37～79.15％) and 82.86％ (95％CI:75.58％～88.70％),and the total compliance rate was 78.10％ (95％CI:71.88％～83.49％) with KAPPA coefficient:0.510 6 (95％ CI:0.3877～0.6336).Conclusion SH2a serum ELISA kit with positive and negative coincidence rate between SH2a serum level and meet the clinical requirement,which could be used as new marker for diagnosis of heptieal injury related diseases.

The application of mental image in clinical practice of TCM reflects the thinking process of syndrome differentiation and treatment. The process, which is undertaken on the“mind”, one of forms of representation of mental image, can be summarized in understanding of image, collection of image, storage and decision of image, examination and analysis of image, confirmation of image.

Qingfei Paidu decoction has a remarkable clinical effect, which has been recommended by the Diagnosis and Treatment Plan of Novel Coronavirus Pneumonia (Trial Sixth Edition) of the National Health Commission and National Administration of Traditional Chinese Medicine. In this paper, the authors intend to explore the pathogenesis of cold pestilence of coronavirus disease-2019 (COVID-19) and composition mechanism of Qingfei Paidu decoction, and believe that this formula can be used to relieve internal and external pressure and regulate triple energizer with the functions of promoting lung Qi, dispelling evil and detoxification, moistening and dampening, and purging heat by removing water. This formula is suitable for the pathogenesis of COVID-19, including cold, dryness and dampness, and can effectively treat symptoms. Therefore, it should be widely used throughout the country to contain the spread of the epidemic.

AR (androgen receptor) and CCAT2 are two prostate cancer (PCa)-related genes whereas their relationship is not yet reported. AR is the classical major functional gene in PCa progression. CCAT2, a non-coding gene, was identified based on big-data GWAS (Genome-Wide Association Studies) in the year of 2013. Androgen deprivation therapy (ADT) is usually used to treat PCa in the early stage. After persistent androgen deprivation, PCa would generally lead to castration resistant prostate cancer (CRPC), whereas the mechanism is yet unclear. Here we explore the function of AR and CCAT2 in PCa progression, especially their relation in androgen sensitive and insensitive cell model LNCap and DU145. We found a loop between AR and CCAT2 transcription by over-expression and knock-down strategies. In DU145 cells, G-CCAT2 activated AR mRNA level 2. 6 times, while T-CCAT2 inhibited it to 0. 2 times (P<0. 05). In LNCaP cells, G-CCAT2 could activate AR mRNA levels 1. 5 times, and TCCAT2 had no significant effect (P<0. 05). Under overexpression of AR in DU145 cells, the expression of CCAT2 increased 2. 9 times (P < 0. 05). The abundance of CCAT2 decreased to 0. 48 (P < 0. 05) in LNCaP cells by AR knock-down. Reporter gene analysis showed that CCAT2 could function on the AR promoter. We then performed CCK8 assays and AR protein level detection as supplement for the new gene CCAT2 studies. Finally we primarily studied some target genes that are related to AR and CCAT2 . The results showed that the G-CCAT2 transcript could activate AR expression in LNCap cells while UCCAT2 had no significant effect. In DU145 cells, G-CCAT2 exhibited a more relative stronger activation effect on AR, and U-CCAT2 could inhibit AR transcription. AR activates the transcriptional activity of CCAT2 in both cell lines, suggesting a feedback regulation between them. Our data showed that there would be a feedback loop between CCAT2 and AR, which may indicate a new method for PCa treatment.

Objective To study the effect of passive motion from lower extremity on electromyography (EMG) activity of major muscles when the back under head down tilting state. Methods Thirty healthy subjects were selected. The EMGs of erector spinal and trapezius muscles were recorded and analyzed when human body was under head-down tilting at angel of 0°, 10°, 20° and 27° in static mode, respectively, in sway mode (along the axis of Z with frequencies of 120 and 140/min， respectively) and in vibration mode (along the axis of Y with frequency of 680/min) with the help of multifunctional test bed. Results In the static mode, the median frequency (MF) decreased with the increase of head-down titling angle; in the sway mode, the MF at each of 4 head-down tilting angles was smaller than that in the static mode. When the frequency was 120/min, the MF decreased with head-down tilting angle increasing, but when the frequency reached 140/min, the variation of MF became irregular. In the vibration mode, the MF increased with the increase of head-down tilting angle and was larger than that in the static mode. Conclusions In the static mode, the muscle fatigue increased with the head down tilting angle increasing; in the vibration mode, smaller head-down tilting angle should be selected to achieve better treatment effect; but in the sway mode, larger head-down tilting angle would be better, and the sway frequency should be set lower than 120/min to avoid potential damage due to excessive muscle fatigue.