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Composition and method for stimulating dendritic cell maturation

A technology of dendritic cells and compositions, applied in the fields of tumor immunology, tumor immunotherapy, and cell biology, can solve problems such as reduction, achieve broad clinical application prospects, and effectively resist tumor-specific cytotoxic T lymphocyte responses. , the effect of the simple method

Active Publication Date: 2014-12-24
GUANGZHOU YIYANG BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method only found that DC cells can secrete high levels of IL-12 in the presence of OK-432 stimuli; after the withdrawal of OK-432 stimuli, the ability of DC cells to secrete IL-12 was significantly reduced, Especially in the serum-free culture system, this phenomenon is more prominent

Method used

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  • Composition and method for stimulating dendritic cell maturation
  • Composition and method for stimulating dendritic cell maturation
  • Composition and method for stimulating dendritic cell maturation

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Preparation of mature DC cells (MoDC) derived from peripheral blood mononuclear cells

[0030] Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation. Use Quanta-007 serum-free medium to suspend and isolate PBMC, and use 1×10 8 The number of spread on 75cm 2 culture flask (purchased from BD Company), placed in CO 2 Incubate for 1 hour in the incubator. Gently shake the culture bottle, suck out the non-adherent lymphocytes, place them in a new culture bottle, add 20U IL-2 and double antibody, and continue to culture for later use. Adherent cells were cultured by adding GM-CSF 1000U / ml, IL-4 400U / ml and double antibodies to Cellgro's serum-free DC cell culture medium. The fresh culture medium was replenished once every other day. Routinely cultured in DC culture medium until day 5, the adherent cells differentiated into immature DC cells. Collect the immature DC cells (iDC) growing in suspension, wash by centrifugation ...

Embodiment 2

[0036] Example 2: OK-432 combined with IFN-γ induces DC cells to express higher levels of surface markers and secrete higher levels of cytokines

[0037] First, the effects of different maturation methods on the expression of DC cell surface markers and cytokine secretion were determined. Such as Figure 1A with Figure 1B As shown, OK-432 alone (OK-DC group), OK-432 combined with IFN-γ (OKG-DC group) and αDC1 type maturation factor combination stimulation (αDC1-DC group) can all enhance the expression of DC cell surface markers . There was no significant difference in the expression levels of OK-DC and αDC1-DC. However, the expression levels of surface markers including CD40, CD80, CD86, and HLA-DR in OK-432 combined with IFN-γ-stimulated DC cells (OKG-DC) were significantly higher than those of OK-DC and αDC1-DC.

[0038] Secondly, the levels of cytokines secreted by DC cells stimulated by different maturation agents were further measured. from Figure 1C with Figure ...

Embodiment 3

[0039] Example 3: OK-432 combined with IFN-γ stimulates mature DC cells to have higher migration ability

[0040] Since the migratory ability of DC cells determines the level of T cell activation induced by entering secondary lymph nodes in vivo, the effects of three different maturation methods on the migratory ability of DC cells were determined. from Figure 1E , Figure 1F to Figure 1G It can be seen that all three maturation methods can enhance the expression level of CCR7 in DC cells, and the expression level of CCR7 in DC cells in the OKG-DC group is significantly higher than that in the OK-DC group, but there is no significant difference from the level in the αDC1-DC group. Consistent with this, compared with the DC cells of the OK-DC group, the DC cells of the OKG-DC group showed higher chemotactic migration ability against the CCR7 ligands CCL19 and CCL21, while the DC cells of the αDC1-DC group migrated abilities did not differ significantly.

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Abstract

The invention relates to a composition and method for stimulating dendritic cell maturation. The composition for stimulating dendritic cell maturation comprises OK-432 and IFN-gamma, wherein in every milliliter of dendritic cell culture solution, the amount of OK-432 is 5-10mu g / ml, and the amount of IFN-gamma is 500-1000U / ml. The method for stimulating dendritic cell maturation comprises the following steps: suspending immature dendritic cells by using a fresh dendritic cell culture solution, wherein the cell density is 1*10<6> / ml; adding the OK-432 and IFN-gamma, wherein the addition amount conforms to the condition that the concentration of OK-432 in every milliliter of culture medium is 5-10mu g / ml and the concentration of IFN-gamma is 500-1000U / ml; and performing simulation for 24-48 hours, and harvesting mature dendritic cells. The mature DC cell prepared by the composition and the method has the capacity of continuously secreting IL-12, can be used for continuously keeping the biological activity of the dendritic cells and plays an important role in the application and research fields of tumor immune fundamental research and clinical tumor immunotherapy.

Description

technical field [0001] The invention belongs to the fields of cell biology, tumor immunology and tumor immunotherapy, and relates to a method for culturing human dendritic cells, in particular to a composition for stimulating the maturation of dendritic cells and related and stimulating dendritic cells Sophisticated method. Background technique [0002] Dendritic cells (DCs) are the most powerful antigen-presenting cells (APCs) in the immune system. Through antigen uptake, processing and presentation, DC cells can effectively induce the activation of antigen-specific CD4 and CD8 T cells, thereby activating the adaptive immune response. In addition, DC cells can also activate NK cells, enhance their proliferation ability and killing function, thereby enhancing the innate immune stress response. Therefore, how to improve the biological activity of DC cells, especially how to give full play to the role of DC cells in tumor immunotherapy, has drawn great attention from medical...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0784A61K35/14A61P35/00A61K35/15
Inventor 夏建川潘科
Owner GUANGZHOU YIYANG BIO TECH CO LTD
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