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A new method to obtain regulatory macrophages

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

Inactive Publication Date: 2017-11-14
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, MattaB, et al. J Immunol, 2005, 175: 342-349.]

Method used

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  • A new method to obtain regulatory macrophages
  • A new method to obtain regulatory macrophages
  • A new method to obtain regulatory macrophages

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 field of biomedicine and discloses a method for obtaining regulatory macrophages in vitro. A method involving the induction of a novel regulatory macrophage using splenic endothelial stromal cells. Specifically, the method materials of the present invention include mouse spleen stromal endothelial cells and bone marrow mononuclear cells. Co-cultivation of the two can differentiate bone marrow mononuclear cells into macrophages, expressing macrophage surface molecular markers, characterized by high expression of CD45RB, moderate expression of CD11c, and the cells have cytokines similar to M2b macrophages expression profile. Relying on contact with ESSCs, 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 have been experimentally confirmed to exhibit regulatory macrophages. Characteristics of phagocytes. This method provides a new method for inducing a large number of macrophages with high secretion of IL-10 in vitro, which can be applied in experiments and clinics.

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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Patent Type & Authority Patents(China)
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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