番鸭细小病毒MDPV-Q株VP2蛋白基因的克隆与序列测定

根据genebank中番鸭细小病毒 (MDPV)的基因序列 ,设计了一对引物LHMP7/LHMP8,同时在这 2条引物中分别加入 2种限制性核酸内切酶SacII和KpnI的酶切位点 ,使扩增后的DNA片段的两端 ,分别含有这 2种酶的酶切位点。应用PCR技术扩增了MDPV_Q株的VP2蛋白基因片段。将扩增后的VP2蛋白基因重组到pMD18_T质粒载体上 ,并对插入片段进行序列测定。测序结果表明 ,我国分离的MDPV_Q株基VP2蛋白基因序列与国外分离株的同源性达 97.9%。

Presence and integration of HBV DNA in mouse oocytes

AIM: Hepatitis B is a worldwide public health problem. To explore the feasibility of hepatitis B virus (HBV) vertical transmission via oocytes, the presence and integration of HBV DNA in mouse oocytes were studied. METHODS: Genomic DNA was isolated and metaphases were prepared, respectively from mouse oocytes cocultured with pBR322-HBV DNA plasmids. PCR, Southern blot, dot hybridization and fluorescence in situ hybridization (FISH) were performed to explore the existence and integration of HBV DNA in oocytes.RESULTS: PCR detected positive bands in the tested samples, and then Southern blot revealed clear hybridization signals in PCR products. Final washing solutions were collected for dot hybridization and no signal for HBV DNA was observed, which excluded the possibility that contamination of washing solutions gave rise to positive results of PCR and Southern blot. FISH demonstrated that 36 of 1 000 metaphases presented positive signals. CONCLUSION: HBV DNA sequences are able to pass through the zona and oolemma to enter into oocytes and tointegrate into their chromosomes. HBV DNA sequences might be brought into embryo via oocytes as vectors when they are fertilized with normal spermatozoa.

Chronic hepatitis B serum promotes apoptotic damage in human renal tubular cells

AIM： To investigate the effect of the serum of patients with chronic hepatitis B （CHB） on apoptosis of renal tubular epithelial cells in vitro and to study the role of hepatitis B virus （HBV） and transforming growth factor-β1 （TGF-β1） in the pathogenesis of hepatitis B virus associated glomerulonephritis （HBV-GN）. METHODS： The levels of serum TGF-β1 were measured by specific enzyme linked immunosorbent assay （ELISA） and HBV DNA was tested by polymerase chain reaction （PCR） in 44 patients with CHB ,and 20 healthy persons as the control. The normal human kidney proximal tubular cell （HK-2） was cultured together with the sera of healthy persons, CHB patients with HBV-DNA negative（20 cases） and HBV-DNA positive （24 cases） for up to 72 h. Apoptosis and Fas expression of the HK-2 were detected by flow cytometer. RESULTS： The apoptosis rate and Fas expression of HK-2 cells were significantly higher in HBV DNA positive serum group 19.01±5.85% and 17.58±8.35%, HBV DNA negative serum group 8.12±2.80% and 6.96 ± 2.76% than those in control group 4.25±0.65% and 2.33 ± 1.09%, respectively （P 〈 0.01）. The apoptosis rate and Fas expression of HK-2 in HBV DNA positive serum group was significantly higher than those in HBV DNA negative serum （P 〈 0.01）. Apoptosis rate of HK-2 cells in HBV DNA positive serum group was positively correlated with the level of HBV-DNA （r = 0.657）. The level of serum TGF-β1 in CHB group was 163.05 ± 91.35 μg/L, signifi- cantly higher as compared with 81.40 ± 40.75 μg/L in the control group （P 〈 0.01）.CONCLUSION： The serum of patients with chronic hepatitis B promotes apoptotic damage in human renal tubular cells by triggering a pathway of Fas up-regulation. HBV and TGF-β1 may play important roles in the mechanism of hepatitis B virus associated glomerulonephritis.

Killing of p53-deficient hepatoma cells by parvovirus H-1 and chemotherapeutics requires promyelocytic leukemia protein

AIM: To evaluate the synergistic targeting and killing of human hepatocellular carcinoma (HCC) cells lacking p53 by the oncolytic autonomous parvovirus (PV) H-1 and chemotherapeutic agents and its dependence on functional promyelocytic leukemia protein (PML). METHODS: The role of p53 and PML in regulating cy-totoxicity and gene transfer mediated by wild-type (wt) PV H-1 were explored in two pairs of isogenic human hepatoma cell lines with different p53 status. Further-more,H-1 PV infection was combined with cytostatic drug treatment. RESULTS: While the HCC cells with different p53 status studied were all susceptible to H-1 PV-induced apoptosis,the cytotoxicity of H-1 PV was morepronounced in p53-negative than in p53-positive cells. Apoptosis rates in p53-negative cell lines treated by genotoxic drugs were further enhanced by a treatment with H-1 PV. In flow cytometric analyses,H-1 PV infection resulted in a reduction of the mitochondrial transmembrane potential. In addition,H-1 PV cells showed a significant increase in PML expression. Knocking down PML expression resulted in a striking reduction of the level of H-1 PV infected tumor cell death. CONCLUSION: H-1 PV is a suitable agent to circumvent the resistance of p53-negative HCC cells to genotoxic agents,and it enhances the apoptotic process which is dependent on functional PML. Thus,H-1 PV and its oncolytic vector derivatives may be considered as therapeutic options for HCC,particularly for p53-negative tumors.

Cytomegalovirus enterocolitis in a patient with diffuse large B-cell lymphoma after chemotherapy with rituximab

Rituximab has been associated with the development of cytomegalovirus enterocolitis in immunosuppressed patients. A 51-year-old patient with diffuse large B-cell lymphoma who received a conditioning chemotherapy regimen (RCVP and RICE) consisting of rituximab before bone marrow transplantation went on to develop cy- tomegalovirus enterocolitis. This supports evidence from previously described cases that rituximab may be associ- ated with cytomegalovirus enterocolitis.

Immunogenicity of the Spike Glycoprotein of Bat SARS-like Coronavirus

A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γand IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.

Relationship between hepatitis B virus infection and hepatic metastasis in non-small cell lung cancer

Objective: The purpose of the study was to explore the relationship between hepatitis B virus(HBV) infection and hepatic metastasis in non-small cell lung cancer(NSCLC). Methods: Four hundred and eighty cases of NSCLC were retrospectively analyzed from January 2003 to January 2010, and the prevalence of hepatic metastasis of NSCLC in patients with and without hepatitis B virus infection were compared. Results: In the HBV carriers' group, the prevalence of synchronous hepatic metastasis and metachronous hepatic metastasis were 13.2% and 5.9%, respectively. Meanwhile in the non-HBV group, those were 21.6% and 9.5% respectively. A significant difference between the two groups was found(P < 0.05). Conclusion: The prevalence of synchronous hepatic metastasis and metachronous hepatic metastasis in non-small cell lung cancer with HBV infection are lower than those in non-HBV infection group. Hepatic metastasis is infrequent in HBV infected cases of NSCLC.

The Effects of Allitridin on the Expression of Transcription Factor T-bet/GATA-3 in Mice Infected by Murine Cytomegalovirus

The aim of this study is to investigate the effects of allitridin on the expression of transcription factor T-bet/GATA-3 in mice infected by murine cytomegalovirus. BALB/c mice model system of murine cytomegalovirus (MCMV) infection was established. In which 20 model mice were allocated randomly into allitridin treated group ( n =10) and infected control group ( n =10). Allitridin (25 mg·kg -1 ·d -1 ) was used in treated group at the 24 h by intraperitoneal route ( once/d ×14 d), and the same volume of saline solution was injected control mice. Normal control mice ( n =10), were only given with the same volume of 0.89% sodium chloride, without infection with MCMV. The expression levels of transcription factor T-bet/GATA-3 mRNA were measured by RT-PCR, and the expression levels of T helper 1(Th1) cytokine IFN-γ and Th2 cytokine IL-10 in supernatant of spleen cell culture were measured by ELISA. Experimental results showed MCMV infection could markedly down-modulate the expression of IFN-γ and T-bet, and significantly up-modulate the expression of IL-10 and GATA-3 mRNA. Allitridin could induce increased expression of transcription factor T-bet mRNA and Th1 cytokine IFN-γ significantly ( P <0.01), and decreased expression of transcription factor GATA-3 mRNA and Th2 cytokine IL-10 markedly ( P <0.01). It is concluded that MCMV infection leads to disequilibrium of Th1/Th2 cytokine expression: the level of Th1 cytokine IFN-γ decreases significantly and Th2 cytokine IL-10 overexpresses markedly. Allitridin can up-regulate the expression of T-bet and IFN-γ, and inhibit the expression of GATA-3 mRNA and IL-10 in MCMV infected mice, indicating a Th1 dominant state which should enhance the specific cellular immune reactions against CMV and be helpful for clearance of the cytomegalovirus in host.

PrP 106-126 Altered PrP mRNA Gene Expression in Mouse Microglia BV-2 Cells

Prion diseases are infectious and fatal neurodegenerative diseases.The pathogenic agent is an abnormal prion protein aggregate.Microglial activation in the centre nervous system is a characteristic feature of prion disease.In this study,we examined the effect of PrP 106-126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR.PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106-126,with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly.Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106-126,and indicate that microglial cells might play a critical role in prion pathogenesis.

Xeno-repopulation of Fah^(-/-)Nod/Scid mice livers by human hepatocytes

Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics,infection of hepatitis viruses,and the efficacy of hepatitis vaccines.Significant levels of liver xeno-repopulation have been reported in Fah-/-Rag2-/-Il2rg-/-mice.However,the high mortality and low breeding rate of this model may hinder its application.A new model,termed Fah-/-Nod/Scid mice,which combines the advantages of liver repopulation in Fah-/-mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding.Fah-/-Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities.FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah-/-Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes;the proportion of engrafted human hepatocytes reached 33.6%.The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis,the expression of characteristic proteins,and the biochemical analysis of liver function.These results show that Fah-/-Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.

Cyclovirobuxine D inhibits dengue virus replication by impeding the complete autophagy in a cholesterol-dependent manner

Dengue virus(DENV)is the most common mosquito-borne flavivirus,and it affects millions of people globally every year.Currently,there are no approved drugs for the treatment of dengue infection.By screening a natural product library,we identified a novel compound,cyclovirobuxine D(Cvb D),that displays anti-DENV activity.Cvb D inhibits DENV replication in vitro in a dose-dependent manner and protects suckling mice against lethal DENV infection.Mechanistically,Cvb D regulates the expression of genes related to the cellular cholesterol pathway.As a result,Cvb D increases cellular cholesterol synthesis and accumulation,activates mTOR,and inhibits viral-dependent autophagy.Cvb D does not suppress autophagy initiation but impedes the nuclear translocation of the lysosome transcription factor TFEB.In addition,Cvb D restricts the replication of other positive-strand RNA viruses such as Zika virus and Coxsackievirus B3.We speculate that Cvb D could be a broad-spectrum antiviral drug candidate for use against positive-strand RNA viruses that require autophagy for optimal replication.

Human endogenous retrovirus W env increases nitric oxide production and enhances the migration ability of microglia by regulating the expression of inducible nitric oxide synthase

Human endogenous retrovirus W env(HERV-W env) plays a critical role in many neuropsychological diseases such as schizophrenia and multiple sclerosis(MS). These diseases are accompanied by immunological reactions in the central nervous system(CNS). Microglia are important immunocytes in brain inflammation that can produce a gasotransmitter – nitric oxide(NO). NO not only plays a role in the function of neuronal cells but also participates in the pathogenesis of various neuropsychological diseases. In this study, we reported increased NO production in CHME-5 microglia cells after they were transfected with HERV-W env. Moreover, HERV-W env increased the expression and function of human inducible nitric oxide synthase(hi NOS) and enhanced the promoter activity of hi NOS. Microglial migration was also enhanced. These data revealed that HERV-W env might contribute to increase NO production and microglial migration ability in neuropsychological disorders by regulating the expression of inducible NOS. Results from this study might lead to the identification of novel targets for the treatment of neuropsychological diseases, including neuroinflammatory diseases, stroke, and neurodegenerative diseases.