Effects of hypoxia on the expression of autophagy-related molecules in human colon cancer cells / 基础医学与临床

Objective To explore the mechanism of hypoxia on SW480 cell autophagy.Methods We screened the CD133+cell with the characteristics of stem cell by magnetic cell separation. Then SW480 cell and CD133+cell were incubated in hypoxic conditions. Subsequently,we detected HIF-1α expression by immunofluorescence(IF), LC3 expression by Western blot and HIF-2α and Beclin 1 expression in 94 colon cancer tissue samples by immuno-histochemistry. Results Hypoxia induced the activation of HIF-1α in SW480 cell. After hypoxia 24 h, LC3 ex-pression was increased in both SW480 cell and CD133+cell with incubation time. The conversion from LC3Ⅰto LC3Ⅱwas increased in CD133+cell,but decreased in SW480 cell. In olon cancer tissue samples,HIF-2α and Be-clin 1 expression in poorly differentiated group were higher than that of well-differentiated group.HIF-2α expression in non-lymph node metastasis group was higher than high lymphatic metastasis group. However the Beclin expression was in the opposite regulation. Conclusions Hypoxia can induce the SW480 apoptosis and a few of CD133+cell autophagy that can protect the cell from apoptosis by inducing the HIF expression and increasing LC3.

Construction and in vitro study of eukaryotic expression vector carrying GITRL and IL-21 gene / 中国实验血液学杂志

The aim of this study was to construct the eukaryotic expression vector carrying glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) and interleukin-21 (IL-21) gene for transfection into chronic myeloid leukemia (CML) dervied dendritic cell (DC), so as to provide an effective platform for exploring the function of target gene in CML. The recombinant eukaryotic expression vector was transfected to the purified DC by Liposome-mediated method, the interleukin-2 (IL-2) and Interferon-γ (IFN-γ) expression of transfected DC were analyzed by ELISA. Further, the transfected DC with purified NK were mixed and cultured to be DC-CIK, the lactate dehydrogenase release assay was performed to measure the killing activity of DC-CIK. The results indicated that the sequence of cloned target gene was same as that in GenBank. The size of endonuclease products by restriction enzyme were same as the predict one. The concentration of Interleukin-2 (IL-2) and interferon-γ (IFN-γ) in transfected DC all increased. The NK kill activity became stronger while induced by transfected DC. It is concluded that DC transfected by IL-21 and GITRL gene has the ability of self-activation, up-regulate cytokine secretion. Further, the results would be help to provide the theoretical evidence of advanced immunotherapy for treatment of CML patients who showed no reaction to tyrosine kinase inhibitor.

Anti-tumor immune response of dendritic cells derived from lymphoma cells transduced with recombinant adenovirus encoding human P53 / 中国实验血液学杂志

This study was aimed to investigate the immunological effect of modified dendritic cells (DC) which inducing cytotoxic T cells (CTL) against lymphoma cells. The DC were isolated from the lymph node and peripheral blood of patients with diffuse large B cell lymphoma (DLBCL). DC were transfected with recombinant adenovirus vector carrying human p53 gene (rAd-p53-DC). The expression of p53 gene was detected by flow cytometry. Western-blot was used to detect the expression of P53. ELISA was used to detect IL-12 level in supernatant. The mixed lymphocyte reaction (MLR) was used to detect the proliferative ability of auto-lymphocyte stimulated by DC. The lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTL. The results indicates that the expressions of DC surface molecule (except for CD1a) such as CD83, CD80, CD86 and HLA-DR were significantly higher in experiment group than that in control group and blank control group. The secretion of IL-12 in supernatant was higher in experiment group than that in control group. The autologous T lymphocyte proliferation and cytotoxic activity against the same kind of DLBL-cells increased in experiment group as compared with control group and blank control group (P < 0.05). The ability to stimulate T lymphocyte proliferation increased with the rising of the ratio of DC and T lymphocyte. However, there was statistically significant difference between rAd-p53-DC derived from Lymph node and peripheral blood (P < 0.05). It is concluded that rAd-p53-transfected DC can induce CTL response in vitro against lymphoma cells.

Induction of anti-leukemic immunity of dendritic cells derived from multidrug resistant leukemia K562/A02 cells with high expression of P-glycoprotein and sensitive K562 cells / 中国实验血液学杂志

This study was aimed to investigate and compare the anti-leukemic effect mediated by dendritic cells (DC) derived from multidrug resistant (MDR) leukemia K562/A02 cells with high expression of p-glycoprotein (P-gp) and sensitive K562 cells. Multidrug resistant K562/A02 cell line and sensitive K562 cell line from chronic myeloid leukemia (CML) were induced for differentiating to DC in complete RPMI 1640 culture medium supplemented with GM-CSF (1 000 U/ml), IL-4 (500 U/ml) and TNF-alpha (100 ng/ml) for 14 days. The morphologic features of DC were observed by means of optical microscopy and the phenotype of DC was detected by flow cytometry. T-cell stimulating activity was determined by allogeneic lymphocyte reaction (allo-MLR). Cytotoxic activity was measured by MTT assay. The results indicated that DC derived from K562/A02 cells and K562 cells similarly showed the typical morphology of dendritic cell and expressed the surface differentiation antigens and costimulatory molecules CD1a, CD83, HLA-DR, CD80 and CD86 of DC. In allo-MLR, K562/A02-DC had a higher capacity to induce lymphocyte proliferation, compared with K562-DC (P < 0.05). K562/A02-DC and K562-DC could similarly generate specific cytotoxic activity against K562/A02 cells or K562 cells respectively, but low reactivity against HL-60 cells. More importantly, the cytotoxic activity mediated by K562/A02-DC was stronger than that by K562-DC against K562/A02 cells or HL-60/VCR cells (P < 0.01, respectively). It is concluded that functional DC can be differentiated from multidrug resistant leukemia K562/A02 cells as well as sensitive K562 cells in the presence of GM-CSF, IL-4 and TNF-alpha. Especially, DC derived from K562/A02 cells can induced a p-glycoprotein specific anti-leukemic immunity.