Animals ; DNA, Complementary ; genetics ; Genome, Viral ; Humans ; Influenza Vaccines ; genetics ; immunology ; Orthomyxoviridae ; genetics ; immunology ; RNA, Viral ; genetics

AIM:To investigate the stemness of mouse triple-negative breast cancer (TNBC) 4T1 cells induced by doxorubicin (DOX) and the underlying mechanism.METHODS:The 4T1 cells and MDA-MB-468 cells were treated with DOX at different concentrations (0, 0.05, 0.1 and 0.5μmol/L) for 24 h, and the shape and viability of the cells were observed.The concentration of DOX at 0.1μmol/L was chosen as the optimal concentration for the following experiments.The 4T1 cells and MDA-MB-468 cells resistant to DOX were established by continuous stimulation with DOX for 4 weeks, and named as 4T1-DOX and MDA-MB-468-DOX.Sphere formation assay was used to detect the stemness of 4T1cells and MDA-MB-468 cells.The expression of CD133 was observed by immunofluorescence staining.The expression of CD44 was analyzed by flow cytometry.The protein levels of Stat3, phosphorylated Stat3 (p-Stat3) and Oct-4 were determined by Western blot.RESULTS:The sphere formation ability of the 4T1-DOX cells was stronger than that of the 4T1control cells.The 4T1-DOX cells expressed high levels of the stemness markers CD133 and CD44 as compared with the 4 T1 cells (P<0.05).Furthermore, the 4T1-DOX cells exhibited enhanced activation of Stat3 (p-Stat3) and increased expression of Oct-4 (P<0.05) , while the expression of total Stat3 had no obvious variation.In addition, when activation of Stat3 was inhibited by WP1066, the protein levels of p-Stat3, Oct-4 and CD44 were down-regulated (P<0.05).Furthermore, inhibition of Stat3 phosphorylation reduced the sphere formation ability of the 4T1-DOX cells (P<0.05).CONCLUSION:DOX induces the stemness of mouse TNBC 4T1 cells through Stat3-Oct-4 signaling pathway.

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine, acting as a regulator of inflammation and immunity. TNFalpha plays a critical role in the pathogenesis of rheumatoid arthritis. Blocking of TNFa activity suppressed inflammatory tissue damage. In present study, the chimeric gene of soluble TNF receptor and IgG Fc fragment (sTNFR-IgG FC) was cloned into the mammalian cell expression vector pStar. When the plamid pStar/sTNFR-IgGFc-GFP was transfected into endothelial cells, a considerable expression of the sTNFR-IgG Fc fusion protein was detected. Moreover, the product in 100microL expression supernatant could completely antagonize the cytolytic effect of 1ng TNFa on L929 cells, even at 1/64 dilution. Then the plasmid was delivered into CIA-induced rheumatoid arthritis mice by tail vein injection. The expression of sTNFR-IgG Fc was detected in liver by RT-PCR. Animals in treatment group showed reduced symptoms of arthritis and more active. This treatment induced decrease of synovial incrassation and prevented the cartilage destruction of the mice RA model. These results show that tail vein injection is an effective way for gene therapy and sTNFR-IgGFc expression plasmid is potential for the treatment of rheumatoid arthritis.
Animals ; Arthritis, Rheumatoid ; chemically induced ; therapy ; Collagen Type II ; Endothelial Cells ; metabolism ; Escherichia coli ; genetics ; metabolism ; Etanercept ; Genetic Therapy ; Humans ; Immunoglobulin G ; biosynthesis ; genetics ; therapeutic use ; Male ; Mice ; Mice, Inbred DBA ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; therapeutic use ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; therapeutic use ; Transfection ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUNDRecent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma (GBM) cells is also implicated in the failure of current therapies, it is not clear how glioma stem cells (GSCs) are involved in invasiveness. Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement. In this study, we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells, and analyze the migration and invasion potential of these cells.

METHODSA series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011. Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance. Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens. Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression. Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line. Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133 - U87 cells. The migration and invasive ability of CD133+ and CD133 - U87 cells were determined by cell migration and invasion assays in vitro. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.

RESULTSIn the central parts of GBMs, CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining. In the peripheral parts of the tumors, CD133+ cells were lined up along the basement membrane of the vessels and myelinated nerve fibers. Rac1 expression was high and diffused in the central parts of the GBMs, and the Rac1+ cells were distributed basically in accordance with CD133+ cells both in the central and peripheral parts of GBMs. In double-labeling immunofluorescence, Rac1 was expressed in (83.14 ± 4.23)% of CD133+ cells, and CD133 and Rac1 co-expressed cells were located around the vessels in GBMs. Significantly higher amounts of Rac1-GTP were expressed in the CD133+ cells (0.378 ± 0.007), compared to CD133- cells (0.195 ± 0.004) (t = 27.81; P < 0.05). CD133+ cells had stronger ability to migrate (74.34 ± 2.40 vs. 38.72 ± 2.60, t = 42.71, P < 0.005) and invade (52.00 ± 2.28 vs. 31.26 ± 1.82, t = 30.76, P < 0.005), compared to their counterpart CD133- cells in transwell cell migration/invasion assay.

CONCLUSIONSThese data suggest that CD133+ GBM cells highly express Rac1 and have greater potential to migrate and invade through activated Rac1-GTP. The accordance of distribution between Rac1+ cells and CD133+ cells in GBMs implies that Rac1 might be an inhibited target to prevent invasion and migration and to avoid malignant glioma recurrence.

AC133 Antigen ; Antigens, CD ; metabolism ; Cell Line, Tumor ; Glioblastoma ; metabolism ; pathology ; Glioma ; metabolism ; pathology ; Glycoproteins ; metabolism ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Peptides ; metabolism ; rac1 GTP-Binding Protein ; metabolism

BACKGROUNDRecent studies have suggested that cancer stem cells cause tumor recurrence based on their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma cells is also implicated in the failure of current therapies, it is not clear whether cancer stem cells are involved in invasiveness. This study aimed to assess invasive ability of glioma stem cells (GSCs) derived from C6 glioma cell line and the distribution patterns of GSCs in Sprague-Dawley (SD) rat brain tumor.

METHODSSerum-free medium culture and magnetic isolation were used to gain purely CD133(+) GSCs. The invasive ability of CD133(+) and CD133(-) C6 cells were determined using matrigel invasion assay. Immunohistochemical staining for stem cell markers and luxol fast blue staining for white matter tracts were performed to show the distribution patterns of GSCs in brain tumor of rats and the relationship among GSCs, vessels, and white matter tracts. The results of matrigel invasion assay were estimated using the Student's t test and the analysis of Western blotting was performed using the one-way analysis of variance (ANOVA) test.

RESULTSCD133(+) GSCs (number: 85.3 ± 4.0) were significantly more invasive in vitro than matched CD133(-) cells (number: 25.9 ± 3.1) (t = 14.5, P < 0.005). GSCs invaded into the brain diffusely and located in perivascular niche of tumor-brain interface or resided within perivascular niche next to white fiber tracts. The polarity of glioma cells containing GSCs was parallel to the white matter tracts.

CONCLUSIONSOur data suggest that CD133(+) GSCs exhibit more aggressive invasion in vitro and GSCs in vivo probably disseminate along the long axis of blood vessels and transit through the white matter tracts. The therapies targeting GSCs invasion combined with traditional glioblastoma multiforme therapeutic paradigms might be a new approach for avoiding malignant glioma recurrence.

AC133 Antigen ; Analysis of Variance ; Animals ; Antigens, CD ; metabolism ; Blotting, Western ; Brain Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Glycoproteins ; metabolism ; Immunohistochemistry ; Neoplastic Stem Cells ; metabolism ; Peptides ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVESTo dynamically observe how glioma stem cells promote the tumor formation and angiogenesis, and to study the correlation between the distribution of glioma stem cells and microvessels within different growth stages of subcutaneous tumor.

METHODSStem cell medium culture and magnetic activated cell sorting were carried out to obtain CD133+ cells from C6 rat glioma cell line. Sprague Dawley (SD) rat ears model were established to observe glioma stem cells promoting blood vessel formation. Subcutaneous glioma model of C6 and immunohistochemical staining of hypoxia inducible factor-1α (HIF-1α) and CD133 were used to investigate the relationship between distribution of glioma stem cells and microvessels. Expressions of CD133 protein in each stage of the subcutaneous tumor were detected by Western blot.

RESULTSIsolation and identification of glioma stem cells deprived from C6 glioma cell line successfully, the establishment of ears model showed real-time dynamic observation of CD133+ cells involved in angiogenesis and tumor formation. SD rat model of subcutaneous glioma showed the initial of tumor formation, CD133+ cells scattered. With tumor growth, CD133+ cells began to tend to capillaries, in late distributed clusters in perivascular. Meanwhile as tumor growth, CD133 protein expression was gradually increased: the values of Western blot analysis of CD133 expression on 6, 9, 12, 15, 20 d were 0.208±0.004, 0.282±0.003, 0.360±0.004, 0.564±0.135, 0.756±0.007, the differences were significant between different groups (F=2601.681, P<0.01). At a high magnification, the CD133 scores with immunohistochemical staining on 6, 9, 12, 15 d were 0.8±0.4, 2.4±0.5, 4.0 ± 0.7, 6.0±0.7; HIF-1α scores were 0.8±0.4, 2.8±0.8, 5.0±0.7, 6.8±0.4. By Spearman rank correlation analysis found that the relationship between CD133 and HIF-1α expression was positively correlated (r=0.921, P<0.01).

CONCLUSIONSGlioma stem cells promote angiogenesis more than non-stem cells; HIF-1α and its downstream gene product might mediate the distribution of glioma stem cells around the perivascular.

Animals ; Cell Line, Tumor ; Glioma ; blood supply ; metabolism ; pathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Microvessels ; pathology ; Neoplasm Transplantation ; Neoplastic Stem Cells ; pathology ; Neovascularization, Pathologic ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the role of BM2 protein in the life cycle of influenza B virus.

METHODSThe authors screened human kidney MATCHMAKER cDNA library for new binding partners of BM2 of influenza B virus by using the yeast two hybrid system with truncated BM2 (26-109 aa) as the bait.

RESULTSSix positive plasmids encoding N-acetylneuraminate pyruvate lyase, angiopoietin 3, zinc finger protein 251, ribosomal protein S20, protein arginine N-methyltransferase 1 variant 1 (PRMT) and transcription factor-like 1 (TCFL1) were obtained.

CONCLUSIONThe results suggest that BM2 may play an important role in the life cycle of influenza B virus.

Angiopoietin-like Proteins ; Angiopoietins ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Gene Library ; Humans ; Influenza B virus ; genetics ; metabolism ; Kidney ; metabolism ; Oxo-Acid-Lyases ; genetics ; metabolism ; Plasmids ; genetics ; Protein Binding ; Protein-Arginine N-Methyltransferases ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Ribosomal Proteins ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Two-Hybrid System Techniques ; Viral Proteins ; genetics ; metabolism ; Zinc Fingers ; genetics

21-hydroxylase deficiency(21-OHD) is mainly characterized by cortisol deficiency with or without aldosterone deficiency and hyperandrogenemia.The disease requires lifelong exogenous glucocorticoid/salt supplementation.Excessive doses of exogenous glucocorticoids are often needed to control hyperandrogenemia, but the effect is not satisfactory.Corticotropin releasing factor (CRF) type 1 receptor antagonist can directly block the production of adrenocorticotropin, inhibit the generation of adrenogenic androgen, reduce the dose of glucocorticoid therapy, and thus lower the incidence of adverse reactions.In this article, the current research progress on 21-OHD therapy and CRF1 receptor antagonist was reviewed.

Based on the first isolated human H5N1 influenza virus strain A/Anhui/1/2005 in China, HA and HA1 genes were amplified and cloned into the eukaryotic expression vector pStar. The recombinant plasmids pStar-HA and pStar-HA1 were transfected into COS7 cells. Western blot and IFA showed that the two recombinant DNA plasmids were successfully expressed in eukaryotic cells. BALB/c mice were immunized with the plasmids DNA by intramuscular injection. Anti-HA specific antibody in peripheral blood of immunized mice was tested by ELISA. The results showed that the recombinant plasmids successfully induced the anti-HA humoral immune response, and there was no significant difference between HA and HA1 as immunogen. This work provides basis for future development of novel avian flu vaccine.
Animals ; Antibodies, Viral ; blood ; COS Cells ; Cercopithecus aethiops ; Female ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Immunoglobulin G ; blood ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; immunology

OBJECTIVETo generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein.

METHODSM1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1.

RESULTSThe recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals.

CONCLUSIONThese confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.

Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Immunization ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza, Human ; immunology ; Mice ; Recombinant Proteins ; genetics ; immunology ; Viral Matrix Proteins ; genetics ; immunology