[Objective]To investigate the anti-proliferative effects of CEP on HCT116 cells and in mouse xenograft model. [Methods]The in vivo anti-cancer activity of CEP was determined with Xenogen bioluminescence imaging in a xenograft tumor model. The cel-based multiple signaling pathway reporter assays were carried out to determine the effects of CEP on these pathways. [Results] CEP inhibited growth of human cancer cells, the IC 50 was 0.8~11.5 μM. CEP induced cellcycle arrest in S and G2/M phase. CEP also inhibited xenograft tumor growth in athymic nude mice bearing HCT116 cells. The xenograft tumor size was significantly reduced upon the treatment with CEP(10 or 20 mg·kg-1 body weight) for up to 3 weeks. Pathway-spe-cific reporter assays indicated that CEP effectively suppressed the NF-κB and MAPK/ERK signaling pathways. [Conclusions] Our results suggest that the anticancer activity of CEP in colon cancer cells may be mediated through targeting NF-κB and MAPK/ERK signaling pathways.

Objective Uterine epithelial cells were isolated from pregnant mice and cultured in vitro , and exam-ined the effect of CD82 on the expression of integrin αV,β3, E-cadherin and β-catenin in the cells.Methods The uter-ine epithelial cells were primarily isolated from pregnant mouse uterus .The recombinant adenovirus containing mouse CD 82 gene which had been constructed in our lab infected the uterine epithelial cells .Immunocytochemistry was used to detect the protein expressions of integrin αV,β3, E-cadherin and β-catenin in the uterine epithelial cells , which were infected with CD82 adenovirus or not .Results 1.The purity of primary cultured cells was (93.2 ±0.6)%.2.The transfection efficiency of CD82 recombinant adenovirus was ( 92 ±4.5 )%.The adenoviral particles carrying CD 82 gene indeed ex-pressed CD82 gene and protein in the primary uterine epithelial cells after 24 hours or 48 hours.3.The uterine epithelial cells of pregnant mice on d4 expressed integrin αV, β3, E-cadherin and β-catenin.4.In contrast to the control group, when CD82 adenovirus infected cells , the uterine epithelial cells of pregnant mice on d 4 increased the expression of integrinαV,β3 and β-catenin protein, had no significant changes of E-cadherin.Conclusions CD82 may have effect on the ex-pression of integrin αV,β3 and β-catenin in mouse uterine epithelial cells before implantation .

BACKGROUND:Transplantation of exogenous stem cells for functional cardiac celldeath or apoptosis easily induced complications after transplantation. Therefore, cardiac progenitor cells of heart itself have been ideal seed cells. OBJECTIVE:To establish stable celllines of cardiac progenitor cells from mouse heart, and to provide ideal cellmodels for studying proliferation and differentiation factors affecting cardiac progenitor cells during adult myocardial cells were damaged. METHODS:(1) Myocardiocytes were isolated from embryonic 15.5 days mice. (2) The cultured myocardiocytes were immortalized using retrovirus SSR69. Immortalized monoclonal myocardial cells were obtained using antibiotic selection and infinite dilution. (3) Monoclonal cellline with the property of cardiac progenitor cells was screened out. RESULTS AND CONCLUSION:(1) Myocardiocytes were successful y isolated and cultured. Partial cells showed obvious beating. (2) Myocardiocytes infected with retrovirus SSR69 were cloned and got 76 clones, then were named as CP15-#, (3) Screening the first 20 clones, the reasonable clones with the characteristics of cardiac progenitor cells were obtained according to myocardial cellmarker genes. The results suggested that immortalized cardiac progenitor cells were established mediated by reversible SV40 T antigen.

Objective To investigate the effects of human S100A6 on β-catenin in human osteosarcoma cell lines MG63 and U2OS. Methods Cell lines MG63 and U2OS were infected by recombinant adenoviruses carrying human S100A6 and its siRNA gene, AdS100A6 and AdSiS100A6 respectively, to up-regulate and down-regulate the ex-pression of S100A6. Then RT-PCR, Western blot and immunocytochemistry were used to detect mRNA and protein (level and/or distribution) of β-catenin. Results In both cell lines, with up-regulated S100A6, expression of β-catenin mRNA and protein increased(P <0. 05) and β-catenin protein increase was more obvious in nuclear than in cytoplasma; while down-regulating S100A6, both the mRNA and protein level of β-catenin decreased (P<0. 05) ; β-catenin protein decrease was more obvious in nuclear than in cytoplasma, too. Conclusion In-creasing Wnt/β-catenin signaling activity may be a mechanism that S100A6 involves in tumor development.

The purpose of this study is to investigate the effect of superparamagnetic chitosan FGF-2 gelatin microspheres (SPCFGM) on the proliferation and differentiation of mouse mesenchymal stem cells. The superparamagnetic iron oxide chitosan nanoparticles (SPIOCNs) were synthesized by means of chemical co-precipitation, combined with FGF-2. Then The SPCFGM and superparamagnetic chitosan gelatin microspheres (SPCGM) were prepared by means of crosslinking-emulsion. The properties of SPCFGM and SPIONs were measured by laser diffraction particle size analyser and transmisson electron microscopy. The SPCFGM were measured for drug loading capacity, encapsulation efficiency and release pharmaceutical properties in vitro. The C3H10 cells were grouped according to the different ingredients being added to the culture medium: SPCFGM group, SPCGM group and DMEM as control group. Cell apoptosis was analyzed by DAPI staining. The protein expression level of FGF-2 was determined by Western blot. The proliferation activity and cell cycle phase of C3H10 were examined by CCK8 and flow cytometry. The results demonstrated that both of the SPIOCNs and SPCFGM were exhibited structure of spherical crystallization with a diameter of (25 ± 9) nm and (140 ± 12) μm, respectively. There were no apoptosis cells in the three group cells. Both the protein expression level of FGF-2 and cell proliferation activity increased significantly in the SPCFGM group cells (P < 0.05). The SPCFGM is successfully constructed and it can controlled-release FGF-2, remained the biological activity of FGF-2, which can promote proliferation activity of C3H10 cells, and are non-toxic to the cell.
Animals ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Chitosan ; Fibroblast Growth Factor 2 ; pharmacology ; Gelatin ; Magnetite Nanoparticles ; Mesenchymal Stromal Cells ; drug effects ; Mice ; Microspheres ; Plasmids

Objective To investigate the effect of recombinant adenovirus containing CD82/KAI1 on the expressions of integrin ?5,integrin ?3,MMP-9,E-cadherin and ?-catenin in uterine stromal cells.Methods Uterine stromal cells were isolated from pregnant mice during implantation window and cultured primarily.The recombinant adenovirus containing mouse CD82/KAI1 gene was constructed.Protein expression of integrin ?5,integrin ?3,MMP-9,E-cadherin and ?-catenin were detected by immunocytochemical methods in the cultured cells with or without CD82/KAI1 adenovirus transfection.Results When the cells which were cultured for 48 h were transfected with the recombinant adenovirus vector at a titer of 6.5?1012 pfu/ml,CD82/KAI1 were obviously upregulated in the primary uterine stromal cells.In the uterine stromal cells from pregnant mice on the 4 d after gestation,integrin ?5,E-cadherin,?-catenin and MMP-9 were detected,while,integrin ?3 were not.But in the cells of nonpregant rats,no expression of these 5 proteins was found.Compared with the cells transfected with blank adenovirus,the recombinant adenovirus encoding CD82/KAI1 upregulated the expressions of ?-catenin and MMP-9(P0.05)and no integrin ?3 was detected in the stromal cells of pregnant mice on day 4 of gestation.Conclusion The recombinant adenovirus containing mouse CD82/KAI1 gene is successfully constructed.CD82/KAI1 might have certain effect on the expressions of integrin ?5,MMP-9,E-cadherin and ?-catenin in mouse uterine stromal cells before embryo implantation.

Objective To investigate the effects of human S100A6 on ?-catenin in human osteosarcoma cell lines MG63 and U2OS.Methods Cell lines MG63 and U2OS were infected by recombinant adenoviruses carrying human S100A6 and its siRNA gene,AdS100A6 and AdSiS100A6 respectively,to up-regulate and down-regulate the expression of S100A6. Then RT-PCR,Western blot and immunocytochemistry were used to detect mRNA and protein (level and/or distribution) of ?-catenin.Results In both cell lines,with up-regulated S100A6,expression of ?-catenin mRNA and protein increased(P

The HBV X gene was amplified by PCR according to the pecob6 containing the whole fragment of adw subtype of HBV, then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 and the recombinant adenoviral plasmid pAd-X was generated. Then plasmid pAd-X was digested with Pac I and transfected into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein (GFP) expression. With restriction endonuclease analysis and PCR methods, it has been confirmed that HBV X gene was cloned into the adenovirus vector successfully. The expression of X protein in HepG2 cells was detected by Western-blot. The recombined adenovirus Ad-X was constructed successfully, which would contribute to the advanced functional study of HBV X protein.
Adenoviridae ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; metabolism ; Hep G2 Cells ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Trans-Activators ; biosynthesis ; genetics ; Transfection

Objective To characterize the viability and transgene expression of articular chon-drocytes cultured in 3-Dimensional scaffolds provided by four types of carriers.Methods Articular chondrocytes from rabbit knees were cultured and infected with adenovirus that could express green fluo-rescence protein (AdGFP) and GL3 luciferase (AdGL3-Luc).The viability and gene expression were determined with fluorescence microscopy and luciferase assays in four types of scaffolds;type I collagen sponge, fibrin glue, hyaluronan and open-cell polylactic acid (OPLA).Cartilage matrix production was assessed by Alcian blue staining.Results Articular chondrocytes of rabbits were effectively infected by AdGFP and exhibited sustained GFP expression.All the tested scaffolds supported the survival and gene expression of the infected chondrocytes.However, the highest transgene expression was observed in the OPLA carrier (P<0.01).Alcian blue-positive matrix materials were readily detected in OPLA cultures four weeks later.Conclusion OPLA supports the highest transgene expression and is the most conduc-tive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repair.

OBJECTIVETo investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein (BMP)-2 and -3 over expressions on chondrogenesis and osteogenesis of prenatal mouse intervertebral disc cells and provide experimental evidences for the application of BMPs in the therapy of disc diseases.

METHODSThe prenatal mouse intervertebral disc cells were infected with a recombinant adenovirus expressing BMP-2 and BMP-3 for 5-7 days, and the expressions of collagen type I (Col I), collagen type II (Col II), aggrecan, osteocalcin, osteoprotegerin and osteopontin mRNAs were detected with RT-PCR. The expression of cartilage matrix was evaluated with toluidine blue staining, and alkaline phosphatase (ALP) activity was detected with ALP reading and ALP staining.

RESULTSBMP-2 and -3 overexpression did not enhance chondrogenesis and osteogenesis of annulus fibrosus (AF) cells or cause significant increases in the expressions of Col I, Col II or aggrecan mRNA in nucleus pulposus (NP) cells. Adenovirus-mediated overexpression of BMP-2 and BMP-3, however, promoted osteogenesis of NP cells and significantly increased the expression of osteocalcin mRNA; the overexpression of BMP-2, but not BMP-3, enhanced the mRNA expressions of osteoprotegerin and osteopontin. Toluidine blue staining demonstrated that BMP-2 and BMP-3 overexpression did not obviously affect the secretion of cartilage matrix. In NP cells, BMP-2 and -3 overexpression significantly enhanced ALP activity, which was not observed in AF cells.

CONCLUSIONAdenovirus-mediated BMP-2 and -3 overexpression can promote the osteogenic differentiation of NP cells but can not affect osteogenesis of AF cells or chondrogenesis of NP cells.

Animals ; Bone Morphogenetic Protein 2 ; pharmacology ; Bone Morphogenetic Protein 3 ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrogenesis ; Intervertebral Disc ; cytology ; drug effects ; Mice ; Osteogenesis