Enhancement of Virus Replication in An Influenza A Virus NS1-Expresssing 293 Cell Line / 生物医学与环境科学（英文）

The nonstructural protein 1 (NS1) of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon (IFN)-mediated antiviral response. We engineered an NS1-expressing 293 (293-NS1) cell line with no response to IFN stimulation. Compared with the parental 293 cells, the IFN-nonresponsive 293-NS1 cells improved the growth capacity of various viruses, but the introduction of NS1 barely enhanced the propagation of Tahyna virus, a negative-strand RNA virus. In particular, fastidious enteric adenovirus that replicates poorly in 293 cells may grow more efficiently in 293-NS1 cells; thus, IFN-nonresponsive 293-NS1 cells might be of great value in diagnostic laboratories for the cultivation and isolation of human enteric adenoviruses.

Morphogenetic study of human adenovirus type 41 in 293TE cells / 病毒学报

To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.

Characterization of fibrillous inclusion body (FIB) of human adenovirus type 41 (Ad41) using immunoelectron microscopy / 病毒学报

To investigate the components of fibrillous inclusion body (FIB), which was formed in packaging cells during the replication of human adenovirus type 41 (Ad41), Ad41 long fiber knob (LFK) and short fiber knob (SFK) proteins were expressed in prokaryote respectively and then used to immunize BALI mice for preparation of anti-LFK serum and anti-SFK sera. The activity and specificity of anti-LFK and an ti-SFK sera were confirmed with Western blot, indirect immunofluorescence assay (IFA) and immunonegative staining, suggesting these sera could be applied in immuno-colloidal gold labelling electron microscopy (EM). 293TE cells were infected with wild-type Ad41. Ultrathin sections of infected cells were made, and labelled with immuno-colloidal gold technique using anti-Ad41 sera, anti-LFK sera, anti-SFK sera, or anti-fiber monoclonal antibody 4D2, respectively. The labelled sections were observed under EM, and the results demonstrated that both Ad41 long fiber protein and short fiber protein were included FIB.

The genetic stability of recombinant adenovirus expressing human rotavirus VP6 gene which used Ad41 as vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the genetic stability of non-replicating recombinant adenovirus which used Ad41 as vector and could express VP6 gene of group A rotavirus during continous passage, in order to develop the vaccine of rotavirus.</p><p><b>METHODS</b>The recombinant adenovirus rvAd41-VP6 (o) was prepared by our laboratory early, it then was continuously propagated on 293TE7 cells for 14 passages. After that samples of the infected cells were collected at every 2 passages for the detection of the integration of the VP6 gene by PCR, and the expression of the target protein was detected by Western Blot analysis.</p><p><b>RESULTS</b>Analysis by PCR revealed that, there was stable integration of specific VP6 gene in the rvAd41-VP6 (o), Western Blot analysis confirmed that rvAd41-VP6 (o) could stably expressed the group-specific antigen structural protein VP6 (o), and it had preferable genetic stability.</p><p><b>CONCLUSION</b>The recombinant adenovirus rvAd41-VP6 (o) which could stably express the VP6 (o) gene had favorable biological property in vitro, and it has provided a basis for further research of animal immunization.</p>

Establishment of internally controlled real-time PCR for the detection of human parvovirus B19 DNA in serum / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum.</p><p><b>METHODS</b>Two DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA.</p><p><b>RESULTS</b>The standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively.</p><p><b>CONCLUSION</b>The Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.</p>

Influence of rhG-CSF on activity of sphingosine kinase in monocytes / 中国实验血液学杂志

The aim of this research was to understand the influence of rhG-CSF on the sphingosine kinase (SphK) activity of monocytes. The peripheral blood monocytes were collected from 6 peripheral blood progenitor cell donors on the fifth day of mobilization with rhG-CSF and from 5 blood donors' buffy coats. The mRNA expressions of monocyte G-CSF receptor and SphK were tested with RT-PCR. The changes of SphK activity of monocytes were assayed after being treated with rhG-CSF. The results showed that the two kinds monocytes collected from both blood donors and peripheral blood progenitor cell donors mobilized with rhG-CSF expressed mRNA of G-CSF receptor and SphK. The SphK activity of monocytes collected from blood donors was not changed significantly after being treated with rhG-CSF (P > 0.05). The SphK activity of monocytes collected from peripheral blood progenitor cell donors transiently increased by (39.6 - 87.2)% after being treated by means of rhG-CSF (P < 0.05) without obviously dose-dependent effect. It is concluded that the SphK activity of monocytes collected from peripheral blood progenitor cell donors can be activated by rhG-CSF.

Construction of 293pT2-P210 cell line enables expression of bcr/abl to be regulated by Tet-off inducing-expression-system / 中国实验血液学杂志

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210(bcr/abl) fusion protein, which is generated by the reciprocal translocation t (9; 22), inhibits apoptosis and increase proliferation. P210(bcr/abl) plays a central role in the pathophysiology of CML. The purpose of this study was to construct a cell line model that bcr/abl expression can be regulated by Tet-off inducing-expression-system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to construct the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasmid and the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC was selected by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. The results showed that individual subclones expressing bcr/abl after withdrawing doxycycline were 293pT2-P210 cell line. In conclusion, selected 293pT2-P210 cells are cells that bcr/abl expression can be regulated by Tet-off inducing-expression-system. They are suitable thoroughly to study the function of bcr/abl fusion gene and its signal regulation mechanism.

Improved replication of enteric adenovirus type 41 in Hep2 cell line expressing E1B55K / 病毒学报

Adenovirus type 40 and 41 (Ad40, Ad41), which belong to human adenovirus subgroup F, are called fastidious adenoviruses due to their property of poor growth in cultured cell lines in vitro The effect of expression of exogenous E1B55K in Hep2 on Ad41 replication in this cell line was investigated. E1B55K gene was amplified by PCR with DNA extracted from Ad41-positive feces supernatant as template. Eukaryotic expression plasmid (pcDNA3) carrying E1B55K was constructed, purified, and transferred into Hep2 cell. Expression of E1B55K in G418-resistant clones was assayed by RT-PCR, and one clone named as Hep2-E1B4#4 could produce more Ad41 progenies when compared with other clones by the method of inducing complete cytopathic effect (CPE) in 293 cells. Infection of equivalent Ad41 caused more significant cytopathic effect (CPE) in Hep2-E1B#4 than that in the control cells of Hep2 or Hep2-DNA3, also suggesting enhanced viral replication in Hep2-E1B#4. The titer of Ad41 was further determined by method of immunocytochemical staining, and semi-quantity PCR was employed to compare the copy number of Ad41 genome DNA. The results showed that the yield of Ad41 in Hep2-E1B#4 was more than 9 times of that in control cells when equal amount of seed viruses were incubated, and the copy number of Ad41 genome increased 4 times in the raw extract from the infected Hep2-E1B#4 when compared with that from control cells. In conclusion, E1B55K gene transfer improved the ability of Hep2 in packaging Ad41, and the Hep2-E1B#4 cell line, which expressed E1B55K constitutively, would be helpful in isolation, cultivation and amplification of Ad41.

Impact of rhG-CSF on sphingosine 1-phosphate concentration in blood plasma of donors / 中国实验血液学杂志

Sphingosine 1-phosphate (S1P), which can be impacted by different growth factors through sphingosine kinase (SphK), is a bioactive lipid produced by metabolism of sphingolipid with various biological responses. Recombinant human granulocyte-colony-stimulating factor (rhG-CSF) have been used widely in peripheral blood stem/progenitor cell mobilization. This study was aimed to investigate the effects of rhG-CSF on S1P concentration in plasma of donors. The peripheral blood mononuclear cells and blood plasma were collected from 8 peripheral blood progenitor cell donors before mobilization and on the fifth day of mobilization with rhG-CSF. The SphK mRNA expression of blood mononuclear cells were detected by RT-PCR. The changes of S1P concentration were assayed with SphK enzyme catalyzed reaction. The results showed that both kinds blood mononuclear cells collected before and after rhG-CSF mobilization expressed SphK mRNA. The S1P concentration is low in donor's plasma both before and after mobilization with rhG-CSF, and there was no markedly change of S1P concentration in plasma before and after mobilization (P > 0.05). In conclusion, mobilization with rhG-CSF does not impact S1P concentration in donors' plasma.

The impact of rhG-CSF mobilization on migration and adhesive function of CD4+ T cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the impact of mobilization with recombinant human granulocyte colony stimulated factor (rhG-CSF) on the migration and adhesive function and their related signal mechanism mediated by the CXCR4 and lymphocyte function antigen-1 (LFA-1) molecules on the surfaces of CD4(+) T cells.</p><p><b>METHODS</b>Before and at day 5 on rhG-CSF mobilization, the expression rates of CXCR4 and LFA-1 (CD11a) on CD4(+) T cells in the peripheral blood were detected by tricolor fluorescence labeling, and the migration and adhesive activities of CD4(+) T cells to stroma cell-derived factor 1 alpha (SDF-1 alpha) and intercellular adhesion molecule-1 (ICAM-1) were also tested.</p><p><b>RESULTS</b>The expression of CXCR4 on CD4(+) T lymphocytes was (84.58 +/- 20.31)% before mobilization and (81.23 +/- 22.46)% at day 5 on mobilization. The expression of LFA-1 on CD4(+) T lymphocytes before and at day 5 on mobilization was 100%. There was no significant difference in the expression CXCR-4 and LFA-1 on CD4(+) T lymphocytes whether mobilization (P > 0.05). SDF-1 alpha induced 4 hours' CD4(+) T cells migration didn't change markedly before and after mobilization \[(28.5 +/- 10.3)% vs (31.2 +/- 8.9)%\] (P > 0.05). The adhesive activity of CD4(+) T cells to ICAM-1 was decreased from (85.59 +/- 14.21)% to (61.45 +/- 15.07)% after mobilization (P < 0.05).</p><p><b>CONCLUSIONS</b>The expression of CXCR4 and LFA-1 on CD4(+) T lymphocytes didn't change markedly during rhG-CSF mobilization, but the adhesive activity of CD4(+) T cells to ICAM-1 was frustrated after that.</p>

Impact of mobilization with rhG-CSF on the proliferation and cytotoxicity of donor's T cells / 中国实验血液学杂志

The study was to understand the impact on the proliferation and cytotoxicity of donor's T cells during mobilization with rhG-CSF. The peripheral blood mononuclear cells (PBMNC) were collected from 15 donors before mobilization and on fifth day of mobilization with rhG-CSF. After the PBMNC were activated with 500 ng/ml of CD3 monoclonal antibody and 500 microg/ml of rhIL-2 for 96 hours, the activated T cells were collected for testing proliferation, cytotoxicity, Fas expression, perforin and Fas ligand (FasL) mRNA expression, the IFN-gamma concentration in the culture medium of the activated T cells was determined by radioimmunoassay. The results showed that the proliferation activity of T lymphocytes and the cytotoxicity of T cells activated with CD3 monoclonal antibody and rhIL-2 were reduced markedly after mobilization with rhG-CSF (P < 0.05). The Fas molecule expression in the activated T cells was very high both before and after mobilization with rhG-CSF (P > 0.10). The activated T cells expressed perforin mRNA and didn't express FasL mRNA both before and after mobilization with rhG-CSF. The concentration of IFN-gamma in the culture medium of the activated T cells decreased significantly after mobilization with rhG-CSF (P < 0.01). It is concluded that activity of proliferation and cytotoxicity of donor's T cells is impaired after mobilization with rhG-CSF.

Influence of hepatocyte growth factor on biological characteristics of bone marrow-derived mesenchymal stem cells / 中国实验血液学杂志

Hepatocyte growth factor (HGF) is one of major growth factors in the bone marrow microenvironments with which the proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells were closely contacted. However, its roles in the regulation of proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells remain unclear. This study was aimed to investigate the effect of HGF on biological characteristics of bone marrow-derived mesenchymal stem cells. Expression of c-Met, the receptor for HGF was detected by immunohistochemistry assay, cell proliferation was determined by MTT, activity of ALP was quantitatively assayed, cell migration and anoikis-induced MSC apoptosis were analyzed. The results showed that HGF not influenced the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Treatment of bone marrow-derived mesenchymal stem cells with recombinant human hepatocyte growth factor resulted in inhibition of anoikis-induced apoptosis. HGF significantly stimulated the migration of bone marrow-derived mesenchymal stem cells. Both PI-3 kinase and MAPK kinase were proved to be involved in HGF-induced migration. It is concluded that HGF/c-Met signal regulates the apoptosis and migration of bone marrow-derived mesenchymal stem cells.

Hepatocyte growth factor recruits endothelial progenitor cells from bone marrow into blood circulation / 中国应用生理学杂志

<p><b>AIM</b>To assess whether hepatocyte growth factor recruits bone marrow-derived endothelial progenitor cells into blood circulation to participate in postnatal angiogenesis and endothelium repair.</p><p><b>METHODS</b>The adenovirus vector encoding HGF gene (Ad-HGF) were intravenous administrated into BALB/c mice, and then serum HGF was determined by enzyme-linked immunosorbent assay, the number of CD34+ cells in peripheral blood was assayed by flow cytometry, and the nucleated cells in peripheral blood were isolated, cultured and the endothelial cell colonies were characterized by staining with antibodies against tie-2, vWF. The carbon tetrachloride-induced liver damage model of female mice was established. The peripheral blood nucleated cells of Ad-HGF treated male mice were intravenous administrated into these mice, and 4 weeks later, in situ hybridization for the sry gene was used to identify the implanted cells in the damaged tissues.</p><p><b>RESULTS</b>Intravenous administration of Ad-HGF resulted in significant elevation of serum hepatocyte growth factor level and induced profoundly increase of endothelial progenitor cells in the peripheral blood, which were characterized by their ability to form endothelial cell colonies in culture and expression of CD34, tie-2, and vW factor. HGF-mobilized endothelial progenitors could incorporate into sites of neovascularization in a liver regeneration model.</p><p><b>CONCLUSION</b>Hepatocyte growth factor could markedly recruit bone marrow-derived endothelial progenitor cells into blood circulation.</p>

Suppressive effect of Notch signal activation on apoptosis of multiple myeloma cells / 中国实验血液学杂志

The proliferation and apoptosis of multiple myeloma (MM) cells were regulated by bone marrow microenvironments in which Notch signal plays important role in mediating cell-cell communication. However, the regulatory effect of Notch signal on the proliferation and apoptosis of multiple myeloma cells remains unclear. In this study, regulatory effect of Notch signal on the apoptosis of MM cells induced by DMS (N, N-dimethylsphingosine) was investigated. RT-PCR was used to identify the expression of Notch receptor and related molecules such as Dll-1, Jagged-1, Deltex-1 in MM cell lines. The intracellular domain of Notch (ICN), active form of Notch, was transferred into MM cells by retrovirus. The apoptosis of MM cells was determined by trypan blue exclusion tests and TdT-mediated dUTP nick end labeling (TUNEL) assay. The results showed that multiple myeloma cells expressed the Notch-1 and its related molecules. Notch activated multiple myeloma cell lines were obtained. Activation of Notch protected the multiple myeloma cells from the apoptosis induced by DMS,which was determined by cell viability and TUNEL assay. In conclusion, Notch signal suppressed the apoptosis of multiple myeloma cells and would possibly be a novel therapeutic target.

The extracellular domain of human delta-like-1 expressed and purified from CHO cells promotes expansion of hematopoietic progenitor cells / 中国实验血液学杂志

Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.