Early cardiac injury in patients with obstructive sleep apnea / 中国应用生理学杂志

OBJECTIVE@#To evaluate the early cardiac injury caused by obstructive sleep apnea (OSA) before the development of cardiovascular symptoms of OSA.@*METHODS@#Ninety-two patients without any known cardiovascular disorders who underwent polysomnography (PSG) were enrolled in the study. Subjects were divided into mild, moderate, and severe OSA groups by their apnea hypopnea index (AHI), and 25 healthy individuals were identified as controls. After PSG examination, fasting blood samples for the evaluation of N-terminal pro-brain natriuretic peptide (NT-proBNP) and heart-type fatty acid binding protein (h-FABP) were collected in the morning, and left ventricular(LV) functions were assessed by using echocardiographic methods. Thirty moderate and severe OSA patients were treated with continuous positive airway pressure respectively (CPAP).@*RESULTS@#The levels of h-FABP and NT-proBNP were obviously higher in all OSA groups than those in the control group (<0.01), and were positively correlated with AHI (<0.01). The Em/Am values of all OSA groups and E/A values of the moderate and severe OSA groups were significantly reduced (<0.01). The difference in Em/Am values among the groups was statistically significant (<0.01). Compared with those before treatment, h-FABP and NT-BNP levels in serum of OSA patients after CPAP treatment were significantly reduced (<0.01), and Em/Am and E/A values were significantly increased (<0.01).@*CONCLUSIONS@#Left ventricular diastolic dysfunction and early myocardial microtrauma are major manifestations of early heart damage in patients with OSA. CPAP therapy could significantly improve early cardiac damage in OSA patients.

Effects of trichostatin A on the interaction between HBx and histone deacetylase protein 1 / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1).</p><p><b>METHODS</b>Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo.</p><p><b>RESULTS</b>Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1.</p><p><b>CONCLUSIONS</b>HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.</p>

An analysis of the features of HBx protein distributed in liver cells and its expression in E. coli / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the features of HBx protein distributed in liver cells and its expression in E. coli.</p><p><b>METHODS</b>The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography.</p><p><b>RESULTS</b>The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded.</p><p><b>CONCLUSION</b>This study may provide a basis for further study on the biological function of HBx at the protein level.</p>

Cloning,Expression and Transcriptional Activity Assay of Human EYA Gene Family / 中国生物工程杂志

The complete coding sequences of Eya gene family was amplified by standard PCR fromhuman tissues or cells cDNA library.The product of PCR was cloned into the eukaryotic expression vector pcDNA3-FLAG,generating pcDNA3-FLAG-Eya1～4.Thenhuman embryo kidney 293T cells were transfected with the recombinant plasmids and the expression of Eya genes were identified by Western blot.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya cooperation with Six in 293T cells affected the Myogenin gene expression.The expression vectors of Eya genes were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya in coordination with Six in 293T cells stimulated the Myogenin gene expression.Eya proteins are transcriptional activator of Six and can improve the activity of myogenin promoter.

Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein.</p><p><b>METHODS</b>Two kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR.</p><p><b>RESULTS</b>The mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP.</p><p><b>CONCLUSION</b>Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.</p>

Development of hygromycin-resistant packaging cell line for hepatitis B virus-derived vectors / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles.</p><p><b>METHODS</b>Free of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.</p><p><b>RESULTS</b>Hygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium.</p><p><b>CONCLUSION</b>After the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.</p>

Replication and encapsidation of HBV mutants with the truncated C gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the replication and encapsidation of HBV mutants with the truncated C gene.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Approach to transforming hepatitis B virus as a gene therapeutic vector / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>It is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.</p>