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1.
Zhongguo Gu Shang ; 21(11): 843-5, 2008 Nov.
Article in Chinese | MEDLINE | ID: mdl-19143249

ABSTRACT

OBJECTIVE: To investigate the effects of osteogenic growth peptide (OGP) to the proliferation and differentiation of cultured bone marrow stromal cells (BMSCs) of rats. METHODS: The SD rats (age 6 weeks) were sacrified, and the bone marrow stromal cells as the adherent stromal cell population were separated from the bone marrow culcure. The proliferation curves of bone marrow stromal cells which were conditioned cultured with four kind of different concentrations of osteogenic growth peptide were measured with the MTT method, and the osteogenesis markers including alkaline phosphatase and calcic deposition detected by histochemical staining. RESULTS: Osteogenic growth peptide at the concentration of 10(-10), 10(-11) mol/L promoted the proliferation of bone marrow stromal cells, while at the concentrations of 10(-8), 10(-9) mol/L suppressed the proliferation of bone marrow stromal cells. However,osteogenic growth peptide at the concentration of 10(-9), 10(-8) mol/L advanced the ratio of positive cells in alkaline phosphatase histochemical staining. CONCLUSION: Osteogenic growth peptide shows distinct effects on the proliferation and differentiation of bone marrow stromal cells depending on its concentration. Osteogenic growth peptide at the concentration of 10(-8), 10(-9) mol/L can promote bone marrow stromal cell differentiation to the osteogenic in vitro.


Subject(s)
Bone Marrow/physiology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Histones/pharmacology , Intercellular Signaling Peptides and Proteins/pharmacology , Stromal Cells/physiology , Alkaline Phosphatase/metabolism , Animals , Bone Marrow/drug effects , Bone Marrow/enzymology , Cells, Cultured , Female , Osteogenesis/drug effects , Rats , Rats, Sprague-Dawley , Stromal Cells/drug effects , Stromal Cells/enzymology
2.
Sheng Wu Gong Cheng Xue Bao ; 23(1): 161-5, 2007 Jan.
Article in Chinese | MEDLINE | ID: mdl-17366907

ABSTRACT

Previous methods used for nuclear transplantation were further investigated to develop a method that was both easy to carryout and did not require any special apparatus, such as Piezoimpact or Spindle-View. Following the puncture of zona pellucida with two holes by injection pipette that contained donor nuclei or cells, the injection pipette was pulled back to the perivitelline space while the negative pressure was increased in the holding pipette until the polar body and karyoplasm were wiped off completely. Then a reconstructed embryo was completed by the direct injection of the donor nucleus or cell without pulling out the injection pipette. 200 oocytes were manipulated using this method and it cost about 40 seconds with nucleus injection and about 30 seconds with cell injection to complete a reconstructed embryo. The success rates were 62.6% and 86. 0%, respectively, and enucleation rate was about 73.3% validated by Hoechst 33342. Using this method, the nucleus was completely eliminated and another was injected using the microscope and micromanipulator. Moreover, the efficiency of nuclear transplantation and survival rate of reconstructed embryos were greatly improved. Furthermore, it is very easy to manipulate and popularize in practice.


Subject(s)
Embryo, Mammalian/cytology , Nuclear Transfer Techniques , Oocytes/cytology , Animals , Cell Culture Techniques/methods , Cell Nucleus/metabolism , Cells, Cultured , Cloning, Organism/methods , Embryo, Mammalian/metabolism , Embryonic Development , Female , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Inbred Strains , Oocytes/metabolism , Zona Pellucida/metabolism
3.
Cell Biol Int ; 30(5): 452-8, 2006 May.
Article in English | MEDLINE | ID: mdl-16631391

ABSTRACT

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.


Subject(s)
Cell Proliferation/drug effects , Goats/embryology , Stem Cells/metabolism , Animals , Blastocyst/drug effects , Cell Culture Techniques , Cell Differentiation , Cells, Cultured , Coculture Techniques/methods , Culture Media, Conditioned , Embryo, Mammalian/cytology , Mice , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Stem Cells/cytology
4.
Acta Biochim Biophys Sin (Shanghai) ; 37(7): 480-7, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15999209

ABSTRACT

Embryonic stem (ES) cells can differentiate into neurons in vitro, which provides hope for the treatment of some neurodegenerative diseases through cell transplantation. However, it remains a challenge to efficiently induce ES cells to differentiate into neurons. Here, we show that murine ES cells can efficiently differentiate into neurons when cultured in glial cell-conditioned medium (GCM) under attaching conditions without the formation of embryoid bodies. In comparison with murine embryonic fibroblast-conditioned medium, we found that GCM has a positive effect on limiting the generation of non-neuronal cells, such as astrocytes. In addition, compared with suspension conditions, attaching conditions delay the differentiation process of ES cells.


Subject(s)
Cell Adhesion/physiology , Cell Differentiation/physiology , Culture Media, Conditioned , Neuroglia/cytology , Neurons/cytology , Pluripotent Stem Cells/physiology , Animals , Cell Lineage/physiology , Coculture Techniques , Embryo, Mammalian , Mice , Mice, Inbred C57BL , Neuroglia/physiology , Pluripotent Stem Cells/cytology , Reverse Transcriptase Polymerase Chain Reaction
5.
Chin Med Sci J ; 19(3): 157-63, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15506639

ABSTRACT

OBJECTIVE: To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity. METHODS: The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA. RESULTS: COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity. CONCLUSION: The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.


Subject(s)
DNA-Binding Proteins/pharmacology , Telomerase/biosynthesis , Telomerase/metabolism , Transcription Factors/pharmacology , Transcription, Genetic , COUP Transcription Factor II , COUP Transcription Factors , Cloning, Molecular , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , E-Box Elements/genetics , HeLa Cells , Humans , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Steroid/genetics , Telomerase/genetics , Transcription Factors/genetics , Yeasts/genetics
6.
Cell Biol Int ; 28(6): 457-61, 2004.
Article in English | MEDLINE | ID: mdl-15223022

ABSTRACT

To elucidate the process of fetal liver hematopoiesis, the relationships between stroma and hematopoietic cells involved in maturation were investigated. Cultured mouse fetal liver explants were established for morphological analysis of the interactions between fetal liver stroma and hematopoietic cells ex vivo. Fetal liver stroma comprised epithelial cells and macrophages, which occupied most of the culture surface. Macrophages proliferated extensively in primary culture, but almost disappeared after 3 passages. Close morphological and functional relationships were established between macrophages and hemopoietic cells, whereas epithelial cells did not interact with blood cells. Scanning electron microscopy revealed that macrophages were in close contact with erythroblasts and formed a three-dimensional network. In each erythroblastic island, 2-3 lymphocytes were also in contact with the macrophages; erythroblasts, lymphocytes and macrophages formed close, firm associations through their cytoplasmic membranes. This cell orientation was confirmed by transmission electron microscopy of fetal liver in vivo. In situ hybridization revealed that the macrophages expressed jagged-1, an important ligand of the Notch signaling system in hematopoiesis.


Subject(s)
Erythropoiesis , Hematopoiesis, Extramedullary , Liver/cytology , Liver/embryology , Lymphocytes/ultrastructure , Macrophages/ultrastructure , Animals , Calcium-Binding Proteins , Erythroblasts/ultrastructure , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/ultrastructure , In Situ Hybridization , Intercellular Signaling Peptides and Proteins , Jagged-1 Protein , Liver/metabolism , Macrophages/metabolism , Membrane Proteins , Mice , Mice, Inbred BALB C , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Serrate-Jagged Proteins , Stromal Cells/metabolism , Stromal Cells/ultrastructure
7.
World J Gastroenterol ; 10(3): 389-92, 2004 Feb 01.
Article in English | MEDLINE | ID: mdl-14760764

ABSTRACT

AIM: A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx. METHODS: We synthesized the fully conserved peptide (CP) with nine residues, FVLGGCRHK. HBV-producing 2.2.15 cells were treated with or without 3.5 microM CP for 36 hours. Quantitative detection of viral DNA was performed by real-time PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence. Flow cytometry was performed to detect cell cycle and apoptosis. RESULTS: Both extracellular and intracellular copies of HBV DNA per ml were significantly increased after incubation with 3.5 microM of CP. HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control cells. CP influenced negatively the extracellular viral gene products, and 3.5 microM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5 microM CP could efficiently increase the level of intracellular HBeAg. Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2.15 cells with or without CP. CONCLUSION: Together with the results generated from the synthetic peptide, we address that the conserved region, a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.


Subject(s)
Hepatitis B virus/physiology , Trans-Activators/chemistry , Trans-Activators/pharmacology , Virus Replication/drug effects , Cell Line, Tumor , Conserved Sequence , Hepatitis B virus/genetics , Humans , Peptide Fragments/chemical synthesis , Peptide Fragments/genetics , Trans-Activators/genetics , Viral Regulatory and Accessory Proteins
8.
World J Gastroenterol ; 9(10): 2274-7, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14562392

ABSTRACT

AIM: To evaluate the protective effects of transplanted and mobilized bone marrow stem cells (BMSCs) on mice with severe acute pancreatitis (SAP) and to probe into their possible mechanisms. METHODS: A mouse model of SAP induced by intraperitoneal injections of L-arginine was employed in the present study. Two hundred female Balb/c mice weighing 18-22 g were randomly assigned into 4 groups. Group A was the stem cell mobilized group treated by injection of granulocyte-colony stimulating factor (G-CSF) into mice for 4 days at a dose of 40 microg.kg(-1).d(-1) before induction of SAP. Group B was the group of BMSCs transplantation, in which the mice were given the isolated BMSCs via the tail vein 4 days prior to induction of SAP. Group C served as the model control and only SAP was induced. The mice without induction of SAP in group D acted as the normal control. At the time of animal sacrifice at 24, 48 and 72 h after induction of SAP, blood samples were obtained and prepared to detect serum amylase, while the abdominal viscera were examined both grossly and microscopically for the observation of pathological changes. RESULTS: The mortality of mice in the model control, groups A and B was 34%, 8% and 10% respectively within 72 h after induction of SAP. The serum level of amylase in the model control was significantly increased at all time points after induction of SAP as compared with that of the normal control (P<0.05-0.01). When the mice were pretreated with BMSCs' transplantation or G-CSF injection, their serum level of amylase was significantly reduced at 48 h and 72 h after induction of SAP in comparison with that of the model control (P<0.05-0.01). In accordance with these observations, both gross and microscopic examinations revealed that the pathological changes of SAP in mice pretreated with BMSCs transplantation or G-CSF injection were considerably attenuated as compared with those in the model control at all observed time points. CONCLUSION: Both transplanted allogenic and mobilized autologous BMSCs can protect mouse pancreas from severe damage in the process of SAP.


Subject(s)
Hematopoietic Stem Cell Transplantation , Pancreatitis/therapy , Acute Disease , Amylases/blood , Animals , Female , Granulocyte Colony-Stimulating Factor/pharmacology , Mice , Mice, Inbred BALB C , Pancreas/pathology , Pancreatitis/mortality , Pancreatitis/pathology , Severity of Illness Index , Transplantation, Homologous
9.
World J Gastroenterol ; 9(7): 1521-4, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12854155

ABSTRACT

AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses. METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography. Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 cells. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry. RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time. EGFP fluorescence in HBx expression cells was significantly decreased. CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.


Subject(s)
Apoptosis/physiology , Trans-Activators/metabolism , Antibodies, Monoclonal , Carcinoma, Hepatocellular , Cell Cycle/physiology , Cell Line, Tumor/cytology , Cell Line, Tumor/metabolism , Cell Line, Tumor/virology , Cytoplasm/metabolism , Cytoplasm/virology , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/virology , Flow Cytometry , Genetic Vectors , Green Fluorescent Proteins , Humans , Immunohistochemistry , Indicators and Reagents/metabolism , Liver Neoplasms , Luminescent Proteins/genetics , Trans-Activators/genetics , Trans-Activators/immunology , Transfection , Viral Regulatory and Accessory Proteins
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