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To study the impact of atypical protein kinase Cι (PKCι) isoform PKC on the pancreatic cancer cells towards the tumoricidal effect of cytokine-induced killer (CIK) cells and explore its mechanisms.CIK cells were prepared by inducing mononuclear cells isolated from the peripheral blood of healthy people with interleukin-2 (IL-2), interferon (IFN) and CD3 mAb and subsequently co-cultured with pancreatic epithelial cell HPDE6-C7, pancreatic cancer cells MiaPaCa and PANC-1 with or without PKC inhibitor named sodium thiomalate (ATM). All cells were divided into control group, ATM group, co-culture group with CIK and co-culture group with CIK+ATM. Cell count was used to detect the growth of each group from 1 to 8 d. Flow cytometry was used to detect the death rate of the cell lines after 48 h cell culture in each group. The small hairpin RNA (shRNA) was used for PKCι knockdown and the recombinant plasmid transfection was for PKCι overexpression in pancreatic cancer cells. Western blot and real-time fluorescent quantitative PCR (qRT-PCR) were utilized to determine the expression of PKCι protein and the impact on gene expression of transforming growth factor-β (TGF-β), a downstream effector modulated by PKC. Different mass concentrations of TGF-β (1, 10, 20 ng/mL) were added into the co-culture of MiaPaCa and PANC-1 with CIK. The cell death rate was detected by flow cytometry 48 h later, so as to explore the possible mechanisms of the impact of PKCι on the tumoricidal effects of CIK cells.ATM and CIK were shown to suppress the growth and induce apoptosis or death of pancreatic cancer cells, meanwhile, ATM can enhance the tumoricidal effect of CIK on pancreatic cancer cells. Moreover, we found that PKCι knockdown in pancreatic cancer cells can down-regulate the gene expression of TGF-β. In return, PKCι overexpression in pancreatic cancer cells can increase the gene expression of TGF-β. The death rate of cancer cells with 10, 20 ng/mL TGF-β was lower compared with the control group (PKCι knockdown in pancreatic cancer cells can not only inhibit the growth of pancreatic cancer cells, but also enhance the tumoricidal effects of CIK on cancer cells. The possible mechanism of PKCι is to affect the immune escape of tumor cells by regulating the expression of TGF-β.

To investigate the influence of a new culture medium added with RGD on cell growth,cell fusion and expression of exogenous gene.A new medium was prepared by adding different concentrations of RGD to ordinary culture medium. The optimum concentration of RGD was determined by observation of the growth of human pancreatic epithelial cell line HPDE6-C7. After determining the optimum concentration of RGD,different concentrations of cells HPDE6-C7 (5×10Firstly,the optimal concentration of RGD was 10 ng/mL. Compared with the normal medium,the cultured cells with RGD had better morphology,adhesion and faster proliferation. In addition,both of the number and positive rate of clones formed in the new medium were significantly higher than that in the ordinary medium (The new culture medium has highlighted advantages in cell growth,cell fusion and expression of exogenous genes. RGD peptide has widely prospect and potential value in the cell culture.

Chilling is a cost-effective and safe method of immobilising flies in Drosophila experiments. However, should condensation form on the plate, it would be fatal to the flies. Here we describe a modified cooling method using reusable commercial ice pack(s) (ca. 400 ml, 2–3 cm tall) rather than crushed ice. The ice pack is covered with a piece of filter paper which absorbs the condensed water formed on the cold surface, and the moistened filter paper does not wet the wings of flies inverted onto it. An ice pack (ca. 400 ml) takes effect in at least 1 h, and its height is shorter than the maximum specimen height of a dissecting microscope, so that flies can be checked while they remain on the cold surface. This method overcomes the disadvantages of the indigenous cooling anaesthetising approach and makes it practically feasible. We also describe an easy-to-make fly-transferring toolkit which will greatly facilitate fly transferral when it is combined with our chilling method. In our design, the flies were dir...