| Background:Currently, malignant tumor has been a primary cause of death in our country. Some kinds of immunotherapy are increasingly popular on the basis of surgery, radiotherapy and chemotherapy. Adoptive immunotherapy could promote the reconstruction of immune system and eliminate the residual tumor cells. Polysaccharide is a kind of biomacromolecule which has immunomodulatory and anti-tumor activity.Cytokine-induced killer (CIK) cells have rapid proliferation, high efficiency and spectrum in anti-tumor. CD3+CD8+cells and CD3+CD56+ cells are two main kinds of effector cells of CIK. Therefore, how to increase the proliferation and proportion of killing cells is the emphasis of CIK researching.Dendritic cells (DCs) could start the immune response mediated by T lymphocytes and participate in anti-inflammatory as well as antitumor immunity. Some research has showed polysaccharides could stimulate phenotypic and functional maturation of DCs. Meanwhile, co-culture DCs with CIK could promote the maturation of CIK cells.Up to now, no research could prove the polysaccharides had direct effect on CIK cells. While the mechanism of immunomodulatory and anti-tumor effect of polysaccharides on DCs is also undefined.Objectives:’1. To explore the immunomodulatory influence of polysaccharides on the multiplication capacity and antitumor effect of CIK cells.2. To research the effect of four polysaccharides on phenotypic and functional maturation of dendritic cells, as well as to explore the mechanism.3. To research the effect of dendritic cells stimulated by polysaccharides on multiplication capacity and antitumor activity of CIK cells.Methods:1. Umbilical core blood samples from 14 normal parturient donors were obtained after informed consent. Mononuclear cells (MNCs) were isolated from core blood by density-gradient centrifugation using Ficoll-Paque Plus. Achyranthes bidentata polysaccharides, lentinan, ganoderma polysaccharides and astragalus polysacharin were chosen for further research.2. Direct effect of four polysaccharides on multiplication capacity and antitumor effect of CIK cells.2.1.CIK cells cultivations with UCBMNCs:Added O.Olmg/ml PHA and 1000U/ml IFN-y to medium on day 0, and added 500IU/ml IL-2 and 0.1ug/ml mouse anti-humor CD3 on day 1. Medium was changed every 3 days and IL-2 was added until day 16.2.2.Choice of appropriate concentration of polysaccharides:Added Oug/ml、 25ug/ml, 100ug/ml,200ug/ml,300ug/ml,500ug/ml four polysaccharides respectively on day 10 and CCK-8 kit was used to reflect the proliferation of cells after 72 hours.2.3.UCBMNCs were divided into control group and four polysaccharides experimental groups. 100ug/ml polysaccharides were added to the experimental groups respectively with IL-2 supplement. Cell counting was performed on day 0,4,7,10,13,16 and multiplication curve was drawed.2.4.Proportion of CD3+CD8+ cells and CD3+CD56+ cells were detected by flow cytometry on day 16.2.5.CCK-8 kit were used to detect Hela cells after co-cultured with CIK with three kinds of effector-target ratio (10:1,20:1,40:1) on day 16, which reflect the anti-tumor activity of CIK cells.3. Effect of four polysaccharides on phenotypic and functional maturation of DCs3.1.UCBMNCs were cultured 6 hours for selecting anchorage-dependent cells. Add rGM-CSF 50ng/ml, rIL-4 25ng/ml into medium on day 1, and add 50ng/ml TNF-a and IL-1 β on day 5. Cell morphous was observed with light microscope until ended culture on day 7. 100ug/ml polysaccharides were added into the experimental groups respectively and the concentration was maintained.3.2.Scanning Electron Microscope was used on day 7 to detect the ultrastructure of DCs.3.3.The CD14, CD86, CD80, CD83 and CDllc positive cells were detected by flow cytometry on day 7.3.4.HLA-I and HLA-Ⅱ molecule were detected by Western blotting on day 7.3.5.Microarrays analysis of differentially expressed genes:The Whole Human Genome Oligo Microarray was performed to identify differentially expressed genes between DCs stimulated by polysaccharides and control DCs. GO functional categoriesand and KEGG functional pathways were searched.4. Effect of DCs stimulated by polysaccharides on function of CIK cells.4.1.UCBMNCs were cultured 6 hours and then collected the suspension cells for culturing CIK cells use routine methods. Co-cultivation was performed with aforementioned DCs and CIK cells on day 7 and finished until day 16. (DC:CIK=1:5)4.2.Proportion of CD3+CD8+ cells and CD3+CD56+ cells were detected by flow cytometry after co-cultivation on day 16.4.3.CCK-8 kit was used to detect Hela cells after co-cultivation with above-mentioned DCs-CIK cells with three kinds of effector-target ratio (10:1、 20:1、 40:1) on day 16.4.4.RT-PCR was used to detect the mRNA levels of IL-2, Granzyme A, Granzyme B, IFN-γ, TNF-α, TNF-β, Perforin in cells.4.5.Enzyme-Linked Immuno Sorbent Assay (ELISA) was used to detected TNF-P and IFN-y in cell culture supernatants on day 16.Results:1. Direct effect of four polysaccharides on multiplication capacity and antitumor effect of CIK cells.1.1.Choice of appropriate concentration of polysaccharides:No significant difference between four polysaccharides experimental groups and control group as well as proliferation of cells in 0-200ug/ml (P>0.05). Proliferation of CIK cells in 300ug/ml and 500ug/ml polysaccharides were decreased.1.2.Proliferation of cells stimulated by 100ug/ml polysaccharides had no significant difference between four polysaccharides experimental groups and control group (P>0.05)1.3.No significant difference between four polysaccharides experimental groups and control group in aspects of proportion of CD3+CD8+ cells and CD3+CD56+ cells. (P>0.05)1.4.Co-cultivation with CIK and Hela cells:Anti-tumor activity of CIK between control group and four polysaccharides experimental groups had no significant difference (P> 0.05); We observed a significant difference between three kind of effector-target ratio and 40:1 groups had a more obvious killing effect (P< 0.001)2. Effect of four polysaccharides on phenotypic and functional maturation of DCs2.1.Analyzed the morphological change with optical microscopy:Cells turned to suspended and the surface turned more as well as thinner.2.2.Ultrastructure observation of DCs by SEM:Surface of cells had plenty of bifurcate prominence just like branches, which were typical ultrastructural features.2.3.Proliferation of CD14、CD86、CD80、CD83 and CD11c positive cells:CD14, the characteristic surface marker of monocyte, were observed significant decrease on mature DCs, and CD80、CD83% CD86、CD11c, markers of mature DCs, were increased significantly than UCBMNCs (P<0.05).The change of cell marker were more obvious in polysaccharides experimental groups than control group (P<0.05). No significant difference between polysaccharides experimental groups (P>0.05)2.4.Western blotting were performance for expression of HLA-I and HLA-II:The up-regulation were more obvious in polysaccharides experimental groups than control group (P<0.01). No significant difference between polysaccharides experimental groups (P>0.05)2.5.Of all the mRNAs tested in microarray analysis,234 mRNAs were significantly up-regulated in DCs stimulated by lentinan, while 465 mRNAs were down-regulated. GO categories of differentially expressed genes were mainly related to molecular transducer activity, etc. KEGG functional pathways associated with the differentially expressed genes weremainly related to Mineral absorption-Homo sapiens (human), etc.3. Effect of DCs stimulated by polysaccharides on function of CIK cells.3.1.DC-CIK detected by flow cytometry:CD3+CD8+cells and CD3+CD56+cells in experimental groups had a significant higher proportion than control group (P< 0.05) and no significant difference between polysaccharides experimental groups (P>0.05).3.2.Cell viability of Hela after co-cultivation with DC-CIK cells:Polysaccharides experimental groups were observed significant lower viability than control group (P< 0.05), in other words, the antitumor activity of CIK in polysaccharides groups was higher than control group. No significant difference between experimental groups (P>0.05)3.3.The expression of IL-2, GZMA, GZMB, IFN-γ, TNF-α, TNF-β, PFP genes was higher in polysaccharides groups than that in control group (P<0.05). No significant difference between experimental groups (P>0.05)3.4.Concentration of TNF-β、IFN-y in cell culture supernatants was higher in polysaccharides groups than that in control group (P＜0.05). No significant difference between experimental groups (P>0.05)Conclusion:1. Polysaccharides had no direct influence on multiplication capacity and antitumor effect of CIK cells.2. Achyranthes bidentata polysaccharides, lentinan, ganoderma polysaccharides and astragalus polysacharin we chose promoted phenotypic and functional maturation of DCs.3. DCs stimulated by polysaccharides promoted multiplication capacity and antitumor activity of CIK cells. |
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