ABSTRACT
Objective:To explore the effects of miR-27a on the phenotype and cytokine secretion in LPS-stimulated dendritic cells.Methods:Murine bone marrow-derived dendritic cells were transfected with miR-27a mimics and negative control mimics,and then stimulated by LPS for 24 hours.Dendritic cells exposed to LPS were collected for analysis of the DC immunophenotype by flow cy-tometry and supernatants were collected to determine cytokine lever.Moreover,the capability of stimulating allogeneic CD4 T+cell prolif -eration was measured by MLR ( mixed lymphocyte reaction) .Results: The levels of MHCⅡ, CD80, and CD86 were significantly increased in LPS-stimulated dendritic cells when compared with imDC ( P<0.001).Transfection with miR-27a mimics resulted in sig-nificantly lower expression levels in levels of MHCⅡ,CD80,and CD86 (P<0.001).For cytokine secretion,transfection with miR-27a mimics enhanced IL-10 production (P<0.01) and reduced the production of IL-12 (P<0.05).For MLR,transfection with miR-27a mimics suppressed allogeneic CD4+T cell proliferation.Conclusion: MiR-27a may play critical roles in regulating the maturation process and cytokine secretion in LPS-stimulated dendritic cells.
ABSTRACT
Objective:To construct a mutant D314A of Escherichia coli cytosine deaminase (EC-CD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. Methods: Eukaryotic expression plasmid containing EC-CD gene (pcDNA3.1-CD~(wt)) was constructed, and the mutant pcDNA3.1-CD~(D314A) plasmid, with aspartic acid (D) at position 314 of EC-CD gene substituted by alanine (A) (EC-CD~(D314A)), was established by site-directed mutation. EC-CD~(wt) and EC-CD~(D314A) were transfected into human colon cancer cell line LoVo via Lipofectamine~(tm) 2000, and positive LoVo-CD~(wt) and LoVo-CD~(D314A) cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of EC-CD and EC-CD~(D314A) genes on LoVo cells were e-valuated by MTT assay. Results: The mutant D314A was confirmed by sequence analysis. EC-CD and EC-CD~(D314A) mRNA were expressed after transfected into LoVo cells. The IC_(50) of Lovo-CD~(D314A) cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVo-CD~(wt) cells ([689.76±0.45] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVo-CD~(wt) cells and Lovo-CD~(D314A) cells were (48.5±0.49)% and (17.3±0.40) % (P = 0.000), respectively. Conclusion: Mutatant EC-CD gene (EC-CD~(D314A)) has a significantly in-creased antitumor effect on LoVo cells compared with wild type EG-CD gene, and it may become a new candidate gene for tumor gene therapy.
ABSTRACT
ObjectiveTo evaluate the effect of combined cytosine deaminase(CD) gene therapy and chemical reagents on colon cancer cell line.MethodsLovo cells transducted with CD gene in a recombinant adenovirus AdCMVCD vector were treated with 5 fluorocytosine(5 FC) and ciplatin(CDDP) or mitomycine C(MMC). Cytotoxic effect was assayed with MTT method. ResultsLow dose of CDDP or MMC reduces the IC 50 of 5 FC significantly on Lovo cells transducted with CD gene and enhances the bystander effect of AdCMVCD/5 FC system.ConclusionCombined therapy with CD/5 FC system and chemical reagents may have a potential application in the treatment of colon cancer.
ABSTRACT
Objective This study was to investigate the expression of human interferon ?(huIFN ?) gene in colon cancer cells. MethodsThe whole length cDNA of huIFN ? was subcloned into the expression vector pcDNA 3 after sequence measurement. Lipofectamine was used to transfer huIFN ? gene into three colon cancer cell lines(LOVO?SW620?HCT116BG) .The integration and expression of huIFN ? were identified by PCR and RT PCR respectively, the activity of IFN ? expressed in different cell lines was measured by ELISA. Results PCR and RT PCR showed successful integration and sustained expression of huIFN ? in 3 colon cancer cell lines. Conclusion The huIFN ? can be effectively transduced into human colon cancer cells, and sustained expression was obtained.
ABSTRACT
Objective To investigate the anti-tumor effect of interferon-?(IFN-?) gene therapy on colon cancer. Methods BALB/c mice were inoculated subcutaneously with CT26 (murine colon carcinoma cell line) cells to prepare an in vivo tumor model. Eight days after tumor inoculation,the tumor-bearing mice were divided into 3 groups and injected intra-tumorally with one of the following preparations: pcDNA3-IFN-?/Lipofectamine,pcDNA3 /Lipofectamine,and PBS respectively. The expression of IFN-?,the immunity function of mice,the histological changes of the tumor,the tumor volume in tumor-bearing mice were tested after gene therapy. ResultsThe level of IFN-? in the serum and the CTL activity increased significantly( P