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Construction method and application of M2-type tumor-associated macrophage model

A technology related to macrophages and tumors, applied in the direction of tumor/cancer cells, animal cells, vertebrate cells, etc., can solve the problems of difficult isolation and culture, lack of experimental models of tumor-related macrophages

Inactive Publication Date: 2016-12-21
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since the maintenance of tumor-associated macrophages depends on the tumor microenvironment in vivo, it is difficult to isolate and culture them. At present, there is still a lack of ideal cell experimental models for tumor-associated macrophages.

Method used

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  • Construction method and application of M2-type tumor-associated macrophage model
  • Construction method and application of M2-type tumor-associated macrophage model
  • Construction method and application of M2-type tumor-associated macrophage model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 U937 cell culture and induction and sorting of U937-derived M2 tumor-associated macrophages

[0037] Human mononuclear lymphoid cell line U937 was cultured in RPMI-1640 medium containing 10% FBS. Dissolve each bag of RPMI-1640 medium (500ml) with deionized water, add 1.7g NaHCO 3 , 1.2g HEPES, 5ml penicillin / streptomycin mixed solution (100×), the volume was adjusted to 450ml, magnetically stirred for 15min, and the pH value was adjusted to 7.3-7.4 to obtain RPMI-1640 basal culture solution. Then add 50ml of fetal bovine serum (FBS), mix thoroughly, and filter with a 0.22 μm microporous membrane to obtain RPMI-1640 medium containing 10% FBS concentration, which is stored at 4°C after aliquoting.

[0038] To induce U937-derived macrophages, we activated U937 cells with PMA (5nM) for 48h. The preparation method of PMA solution is as follows: take PMA (1g) powder, add 32.4ml of DMSO solution, and repeatedly pipette and mix to obtain a concentrated PMA solution ...

Embodiment 2

[0041] Example 2 qRT-PCR identification of the expression of M1 and M2 macrophage markers in U937-derived M2 tumor-associated macrophages

[0042] 2.1 Cell RNA extraction and concentration determination

[0043] (1) Wash the cells 3 times with PBS, take 1×10 6 Each cell was lysed with 1ml RNAiso and placed in a 1.5ml EP tube;

[0044] (2) Add 0.2ml chloroform to each group of samples, shake vigorously for 20sec, and let stand at room temperature for 5min;

[0045] (3) Centrifuge at 12000g for 15min at 4°C;

[0046] (4) Take the upper layer of the layered sample and transfer it to a new 1.5ml EP tube, add 250μl isopropanol, invert it up and down several times to mix well, and let it stand in an ice bath for 20-30min;

[0047] (5) Centrifuge at 12000g for 15min at 4°C;

[0048] (6) Discard the supernatant, add 1ml 75% ethanol, and invert several times to wash the precipitate;

[0049] (7) Centrifuge at 12000g for 5min at 4°C;

[0050] (8) Remove ethanol, open the 1.5ml EP ...

Embodiment 3

[0059] Example 3 Immunofluorescence staining to detect the difference in the expression of M2 macrophage marker CD163 between U937-derived M2 tumor-associated macrophages and control U937 monocytes

[0060] 3.1 Experimental method:

[0061] (1) Collect U937-derived M2 tumor-associated macrophages and control U937 monocytes, place them in a centrifuge tube, centrifuge at 1000 rpm for 3 min, and wash once with PBS;

[0062] (2) Fix with 4% neutral paraformaldehyde (PH=7.4) for 20 min; centrifuge at 1000 rpm for 3 min, wash once with PBS;

[0063] (3) Add 100ul PBS to resuspend the cells, and smear the cells on the poly-lysine-coated detachment-proof sheet by smear method;

[0064] (4) Hydrate slices with PBS, 5min×3 times;

[0065] (5) Block the sections with 10% goat serum, and incubate at room temperature for 30 minutes;

[0066] (6) Primary antibody incubation: add diluted CD163 primary antibody and incubate overnight at 4°C;

[0067] (7) The next day, rinse the slices wi...

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Abstract

The invention relates to a construction method and an application of a M2-type tumor-associated macrophage model. In the invention, an immortalized mononuclear cell (for example, a U937 monocytic series) is induced into a tumor-associated macrophage in vitro environment under stimulation of PMA and IL-4, IL-10 and TGF[beta] growth factor; and on the basis of the mentioned before, biological functions and target therapy value of the tumor-associated macrophage in tumor progression are researched in a manner of in-vivo and in-vitro co-cultivation method of the tumor-associated macrophage and tumor cells. The invention provides important tools and experimental measures for tumor immunology fundamental research and therapy measures based on the tumor-associated macrophage.

Description

technical field [0001] The invention belongs to the field of animal cell lines, and in particular relates to a construction method and application of an M2 tumor-associated macrophage model. Background technique [0002] Tumor-associated macrophages (Tumor-associated Macrophages) are a type of special inflammatory cells derived from mononuclear macrophages in the tumor microenvironment, which widely exist in various solid tumor tissues such as glioma and breast cancer. As the main participants in tumor-associated inflammation, tumor-associated macrophages can regulate malignant biological behaviors such as proliferation, invasion, and angiogenesis, and produce immunosuppression, which has an important impact on the occurrence and development of tumors. [0003] According to the expression of specific membrane surface markers and tumor immunological function, tumor-associated macrophages can be divided into two subtypes, M1 and M2. M1 tumor-associated macrophages highly expr...

Claims

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Application Information

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IPC IPC(8): C12N5/0786C12N5/09
CPCC12N5/0645C12N2501/15C12N2501/2304C12N2501/231C12N2501/727C12N2502/30
Inventor 时雨平轶芳曹棉富张厦卞修武
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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