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New method for obtaining regulatory macrophage

A macrophage, regulatory technology, applied in the field of biomedicine, can solve problems such as changing biological functions

Inactive Publication Date: 2015-02-11
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific surface markers expressed by these different subtypes of macrophages have not yet been determined, because the biological characteristics of macrophages are very plastic, and different microenvironments can significantly change their biological functions and differentiate into different types. Functional subgroups [Svensson M, Kaye PM. Trends Immunol, 2006, 27: 580-587. Stout RD, Jiang C, Matta B, et al. J Immunol, 2005, 175: 342-349.]

Method used

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  • New method for obtaining regulatory macrophage
  • New method for obtaining regulatory macrophage
  • New method for obtaining regulatory macrophage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Culture, passage and identification of endothelial-like splenic stromal cells (ESSC)

[0024] 1. Materials and methods

[0025] 1. Materials

[0026] C57BL / 6J mice, 1 week old, female, were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences and raised at SPF level. 75% ethanol, normal saline, RPMI-1640 (purchased from Hyclone Company), fetal bovine serum (FBS, Beijing Yuanheng Jinma Biotechnology Development Co., Ltd.), 0.25% trypsin, FACS washing solution, mAnti-PE CD106 (purchased from eBioscience). Vials, test tubes, scissors, tweezers, droppers, pipettes, centrifuges, flow cytometer FACS Calibur (BD, USA), CO 2 Cell incubator (Forma3111 type, Thermo, USA), optical inverted microscope.

[0027] 2. Experimental method:

[0028] (1) Culture and passage of ESSC:

[0029] Aseptically isolate the spleen of 1-week-old C57BL / 6J mice, cut it to a size of 1-2 mm with sterile scissors, and stick it evenly and firmly to the...

Embodiment 2

[0035] Example 2 Co-cultivation of ESSC and bone marrow mononuclear cells

[0036] 1. Materials and methods

[0037] 1. Materials

[0038] C57BL / 6J mice, 6-8 weeks old, female, were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences and raised at SPF level. 75% ethanol, normal saline, RPMI-1640 (purchased from Hyclone Company), fetal bovine serum (FBS, Beijing Yuanheng Jinma Biotechnology Development Co., Ltd.), 0.25% trypsin, FACS washing solution, mAnti-PE CD106 (purchased from eBioscience). Vial, test tube, scissors, tweezers, dropper, pipette, centrifuge, 2ml sterile syringe, flow cytometer FACS Calibur (BD, USA), CO 2 Cell incubator (Forma3111 type, Thermo, USA), optical inverted microscope.

[0039] 2. Experimental method:

[0040] (1) Obtaining bone marrow mononuclear cells

[0041] C57BL / 6J mice were sacrificed by cervical dislocation, soaked in 75% ethanol for 2-3 minutes, wiped off the alcohol with paper, separated the fem...

Embodiment 3

[0044] Characterization of differentiated cells after co-cultivation in Example 3

[0045] 1. Materials and methods

[0046] 1. Materials

[0047] Anti-mouse F4 / 80, CD14, CD40, CD80, CD86, Ia, CD11b, CD11c, CD45RB, CD4, CD8, CD19 and isotype control antibodies were purchased from eBioscience. IL-6, IL-12, TNF-α ELISA kit (purchased from eBioscience), IL-10 ELISA kit (purchased from Daktronics), LPS (purchased from Sigma). Giemsa reagent.

[0048] 2. Experimental method:

[0049] (1) Giemsa dyeing

[0050] Take differentiated cells 2×10 5 / 50μl 10% RPMI-1640 and throw the cells onto the glass slide with a slide machine; when they are about to dry, fix them with methanol for 10min; When drying, add 100 μl Giemsa stain to the cells and cover it, and stain at room temperature for 30 minutes; after half an hour, pour off the Giemsa stain, wait for it to dry quickly and rinse gently with water, during which time the staining can be observed with a microscope.

[0051] (2)ELIS...

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Abstract

The invention belongs to the biomedical field and discloses a method for obtaining regulatory macrophage in vitro and relates to a method for obtaining new regulatory macrophage by virtue of induction of endothelial matrix cells of spleen. Specifically, the material used in the method comprises endothelial matrix cells of spleen and bone marrow mononuclear cells of a mouse. The matrix cells and the bone marrow mononuclear cells are co-cultured, so that the bone marrow mononuclear cells are differentiated into macrophage a molecular marker for expressing the macrophage is characterized by high expressive CD45RB and medium expressive CD11c. Moreover, the macrophage has a cytokine expression spectrum which is similar to that of an M2b macrophage. Dependent on ESSC contact culture, the macrophage can secrete high-level IL-10 and inhibit T cell proliferation and induce T apoptosis in vitro; verified through experiments, the macrophage sorted by the method has the characteristics of the regulatory macrophage. The method provides a new method for obtaining high-secretion IL-10 macrophage by in-vitro massive induction and can be applied to experiments and clinic.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a method for inducing regulatory macrophages by using spleen endothelial stromal cells. Specifically, the method materials of the present invention include mouse spleen stromal endothelial cells and bone marrow mononuclear cells. The co-culture of the two can make the bone marrow mononuclear cells differentiate into macrophages, and express macrophage surface molecular markers, characterized by high expression of CD45RB and moderate expression of CD11c. The macrophages can secrete high levels of IL-10, and inhibit T cell proliferation and induce T cell apoptosis in vitro, indicating that the macrophages sorted by this method are experimentally confirmed to exhibit the characteristics of regulatory macrophages. Background technique [0002] Macrophages are a class of multifunctional innate immune cells distributed in various organs, connective tissues, and serous cavities in the body [van...

Claims

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Application Information

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IPC IPC(8): C12N5/0786
Inventor 王庆阳张纪岩王小茜肖鹤张雪莹黎燕沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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