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Method for preparing dendritic cells for effectively submitting gastric cancer antigens

A technology for dendritic cells and gastric cancer cells, applied in the field of preparation of dendritic cells, can solve the problems that the preparation methods have not been reported yet

Inactive Publication Date: 2014-11-19
中国人民解放军第一七四医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, dendritic cells that can effectively present antigens through modification of pJAK2(1001–1013) polypeptides and their preparation methods have not been reported yet

Method used

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  • Method for preparing dendritic cells for effectively submitting gastric cancer antigens
  • Method for preparing dendritic cells for effectively submitting gastric cancer antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Extraction and identification of gastric cancer cell MGC-803 total RNA

[0029] 1.1 Extraction of total RNA from gastric cancer

[0030] (1) Place the culture dish covered with about 90% of the gastric cancer cell MGC-803 on the bottom wall on ice, and absorb the culture solution;

[0031] (2) Add 1ml directly Reagent, pipetting repeatedly;

[0032] (3) Transfer the cell lysate to a new 1.5ml Eppendorf tube and let it stand at room temperature for 5 minutes;

[0033] (4) Add 0.2ml chloroform and mix vigorously for 15s;

[0034] (5) Place at room temperature for 2 to 3 minutes;

[0035] (6) Centrifuge at 12,000rpm, 4°C, 15min;

[0036] (7) See the upper water phase, the middle mixed phase, and the lower phenolic phase. Aspirate the aqueous phase and transfer to a new Eppendorf tube, about 0.5ml;

[0037] (8) Add 0.5ml of isopropanol, mix by inverting, and place at room temperature for 10 minutes;

[0038] (9) Centrifuge at 12,000rpm, 4°C, 10min;

[003...

Embodiment 2

[0051] Example 2: Synthesis of SOCS1 antagonist pJAK2(1001-1013) polypeptide

[0052] The amino acid sequence of the pJAK2(1001–1013) polypeptide is 1001 LPQDKE Y YKVKE 1013 , where the underscore Y Phosphorylated tyrosine. The peptide was synthesized by Shanghai Jill Biochemical Company with a purity of over 98.0%. In order to prevent the degradation of the peptide during in vitro manipulation and ensure efficient entry into DC, a lipophilic group (palmitoyl-lysine) was added to the N-terminus of the peptide during synthesis.

Embodiment 3

[0053] Example 3: Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

[0054] Freshly collected 50ml of anticoagulated peripheral blood (20U heparin sodium / ml whole blood) from healthy volunteers, diluted it with 37°C PBS, and slowly poured it into the centrifuge containing the rewarmed Ficoll-Hypaque lymphocyte separation medium along the tube wall. tube (blood:separation fluid=1:1) to make the interface clear. Gradient centrifugation (room temperature, 800g, 20min), take the middle buffy coat cell layer, put it into a 50ml centrifuge tube, suspend the cells with PBS without calcium and magnesium, centrifuge (800g, 10min), wash the cells twice, and centrifuge again (1200rpm , 10 min), and the PBMCs were obtained after the platelets were washed out.

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Abstract

The invention discloses a method for preparing dendritic cells for effectively submitting gastric cancer antigens, and relates to dendritic cells. The method comprises the following steps of: extraction and identification of total RNA (Ribonucleic Acid) of gastric cancer cells MGC-803; synthesis of SOCS1 antagonist pJAK2 (1001-1013) polypeptide; separation of peripheral blood mononuclear cells; acquisition of mononuclear cells; in-vitro induction of immature dendritic cells; RNA transfection of the immature dendritic cells; pJAK2 (1001-1013) polypeptide supported RNA transfection of the dendritic cells; and mature induction of the immature dendritic cells, thus producing mature dendritic cells. By adopting the method, the antigen submission function and the specific immune response level of the dendritic cells can be improved; and the dendritic cells of total RNA transfection of the gastric cancer cells are modified by adopting the SOCS1 antagonist pJAK2 (1001-1013) polypeptide.

Description

technical field [0001] The invention relates to dendritic cells, in particular to a method for preparing dendritic cells that effectively present gastric cancer antigens. Background technique [0002] Dendritic cells (DC) are currently known to have the strongest antigen-presenting ability in vivo and the only antigen-presenting cells that can activate naive T lymphocytes. DCs present antigens in the form of major histocompatibility complex (MHC)-polypeptide complexes, which play an extremely important role in innate immunity and acquired immunity, and have attracted close attention in current tumor immunotherapy. [0003] A large number of studies have shown that DCs are closely related to the occurrence, development and prognosis of tumors, and the occurrence of tumors may be related to the functional defects of tumor-infiltrating DCs or host DCs. In this case, DCs in tumor patients are in an "incapable state". It cannot effectively present tumor antigens, activate T cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10
Inventor 陈玉强王勇军颜江华王生育
Owner 中国人民解放军第一七四医院
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