阅读说明：本技术 一种巨噬细胞向m2型巨噬细胞分化的检测方法 (Method for detecting differentiation of macrophages into M2 type macrophages ) 是由 褚一凡 江嘉豪 何美第 陈炽锋 刘锐 王进辉 于 2019-10-18 设计创作，主要内容包括：本发明提供一种巨噬细胞向M2型巨噬细胞分化的检测方法,涉及干细胞及再生医学领域。该检测方法,包括以下步骤：获取单核巨噬细胞：将稀释血液加入淋巴细胞分离液中,离心,去掉上层液体,保留沉淀,加入清洗液,离心,去掉上层液体,保留沉淀,分选出单核巨噬细胞；分组培养：将获取的单核巨噬细胞分为共培养组和对照组,向共培养组中加入脐带间充质干细胞,将共培养组和对照组放置于相同条件下培养；检测：检测分组培养后巨噬细胞CD163、CD204、CD206、HLA-DR的表达情况。本发明能够全面地评价脐带间充质干细胞对巨噬细胞向M2型巨噬细胞分化的影响。(The invention provides a method for detecting the differentiation of macrophages to M2 type macrophages, and relates to the field of stem cells and regenerative medicine. The detection method comprises the following steps: obtaining mononuclear macrophages: adding diluted blood into lymphocyte separation liquid, centrifuging, removing upper layer liquid, retaining precipitate, adding cleaning solution, centrifuging, removing upper layer liquid, retaining precipitate, and separating out mononuclear macrophage; grouping culture: dividing the obtained mononuclear macrophages into a co-culture group and a control group, adding umbilical cord mesenchymal stem cells into the co-culture group, and culturing the co-culture group and the control group under the same condition; and (3) detection: detecting the expression of macrophages CD163, CD204, CD206 and HLA-DR after the grouping culture. The method can comprehensively evaluate the influence of the umbilical cord mesenchymal stem cells on the differentiation of the macrophages to M2 type macrophages.)

1. A method for detecting macrophage differentiation to M2 type, comprising the steps of:

obtaining mononuclear macrophages: diluting blood to obtain diluted blood, adding the diluted blood into lymphocyte separation liquid, centrifuging, removing upper layer liquid, retaining precipitate, adding cleaning liquid, centrifuging, removing upper layer liquid, retaining precipitate, and separating out mononuclear macrophage;

grouping culture: dividing the obtained mononuclear macrophages into a co-culture group and a control group, adding umbilical cord mesenchymal stem cells into the co-culture group, and culturing the co-culture group and the control group under the same condition;

and (3) detection: detecting the expression conditions of macrophages CD163, CD204, CD206 and HLA-DR after the grouping culture, defining CD206 positive, CD204 positive, CD163 positive and HLA-DR negative as M2 type macrophages, and judging the influence of the umbilical cord mesenchymal stem cells on the differentiation of the macrophages to the M2 type macrophages according to the expression conditions of a co-culture group and a control group.

2. The assay of claim 1, wherein in the step of obtaining mononuclear macrophages, the step of diluting the blood comprises: mixing and diluting the obtained blood and physiological saline to obtain diluted blood, wherein the volume ratio of the blood to the physiological saline is 1: (1-2).

3. The assay of claim 1, wherein in the step of obtaining mononuclear macrophages, the conditions of the diluted blood centrifugation are: the centrifugal temperature is 20 +/-1 ℃, the centrifugal force is 400-500 g, the centrifugal time is 18-22 min, the speed is increased to 7, and the speed is decreased to 0.

4. The detection method according to claim 1, wherein in the step of obtaining the mononuclear macrophage, the washing solution is a phosphate buffer solution, and the centrifugation conditions after adding the phosphate buffer solution are as follows: the centrifugal temperature is 4 +/-1 ℃, the centrifugal force is 200-300 g, the centrifugal time is 7-9 min, the speed is increased to 9, and the speed is decreased to 9.

5. The method of claim 1, wherein the step of obtaining mononuclear macrophages comprises sorting mononuclear macrophages with magnetic beads.

6. The detection method according to claim 1, wherein the concentration of mononuclear macrophages in the step of the group culture is 5 to 10X 10⁵one/mL.

7. The detection method according to claim 1, wherein in the step of grouping culture, the ratio of the number of the umbilical cord mesenchymal stem cells added into the co-culture group to the number of the mononuclear macrophages is 1 (0.8-1.2).

8. The detection method according to claim 6 or 7, wherein in the step of performing the group culture, the culture time is 6 to 8 days.

Technical Field

The invention relates to the field of stem cells and regenerative medicine, in particular to a method for detecting the differentiation of macrophages to M2 type macrophages.

Background

Macrophages are important constitutional cells of body immunity, are widely distributed in all parts of the body, and have strong capabilities of deforming movement, recognizing phagocytosis, killing and eliminating pathogens and the like. Under the influence of local microenvironment, macrophages can differentiate into different subgroups, such as M1 type macrophages and M2 type macrophages, etc., wherein M2 type macrophages are mainly involved in the repair of damaged tissues. The mesenchymal stem cells are pluripotent stem cells, have multidirectional differentiation capacity, can be differentiated into osteoblasts, chondrocytes, adipocytes and the like, and can promote the repair and regeneration of damaged tissues.

At present, most studies on the influence of stem cells on tissue regeneration focus on the differentiation of stem cells into other cells, and the study on the regulation of immune cells by stem cells to promote tissue repair is lacking. Therefore, the invention aims to provide a method for detecting that mesenchymal stem cells influence the differentiation of macrophages to M2 type macrophages.

Disclosure of Invention

In view of the above, it is necessary to provide a method for detecting the differentiation of macrophages into M2-type macrophages, which can comprehensively detect and evaluate the effect of umbilical cord mesenchymal stem cells on the differentiation of macrophages into M2-type macrophages.

A method for detecting macrophage differentiation to M2 type macrophage, comprising the following steps:

obtaining mononuclear macrophages: diluting blood to obtain diluted blood, adding the diluted blood into lymphocyte separation liquid, centrifuging, removing upper layer liquid, retaining precipitate, adding cleaning liquid, centrifuging, removing upper layer liquid, retaining precipitate, and separating out mononuclear macrophage;

grouping culture: dividing the obtained mononuclear macrophages into a co-culture group and a control group, adding umbilical cord mesenchymal stem cells into the co-culture group, and culturing the co-culture group and the control group under the same condition;

and (3) detection: detecting the expression conditions of macrophages CD163, CD204, CD206 and HLA-DR after the grouping culture, defining CD206 positive, CD204 positive, CD163 positive and HLA-DR negative as M2 type macrophages, and judging the influence of the mesenchymal stem cells on the differentiation of the macrophages to the M2 type macrophages according to the expression conditions of a co-culture group and a control group.

The detection method provides four detection indexes, namely CD163, CD204, CD206 and HLA-DR, and when the expressions of the macrophages CD163, CD204 and CD206 are increased and the expression of the HLA-DR is reduced after co-culture, the mesenchymal stem cells have strong capacity and good quality of influencing the differentiation of the macrophages to M2 type macrophages, and have strong capacity of regulating tissue regeneration. The invention selects the four detection indexes, wherein CD163 is a scavenger receptor and mediates macrophage phagocytosis of a hemoglobin complex; CD204 is also a scavenger family member, a cell involved in the clearance of apoptosis; CD206 is a mannose receptor, recognizes proteoglycan expressed by pathogenic microorganisms, and eliminates external harmful substances; the HLA-DR is histocompatibility protein, mediates antigen presentation of macrophages, is mainly expressed on the surface of M1 type macrophages, and can comprehensively and efficiently detect the influence of mesenchymal stem cells on the differentiation of the macrophages to the M2 type macrophages by combining the four surface markers.

In one embodiment, in the step of obtaining mononuclear macrophages, the step of diluting blood is: mixing and diluting the obtained blood and physiological saline to obtain diluted blood, wherein the volume ratio of the blood to the physiological saline is 10: (12-18). Preferably, the volume ratio of blood to saline is 10: 15.

In one embodiment, in the step of obtaining mononuclear macrophages, the conditions of the diluted blood centrifugation are as follows: the centrifugal temperature is 20 +/-1 ℃, the centrifugal force is 400-500 g, the centrifugal time is 18-22 min, the speed is increased to 7, and the speed is decreased to 0.

In one embodiment, in the step of obtaining mononuclear macrophages, the washing solution is phosphate buffer solution, and the centrifugation conditions after adding the phosphate buffer solution are as follows: the centrifugal temperature is 4 +/-1 ℃, the centrifugal force is 200-300 g, the centrifugal time is 7-9 min, the speed is increased to 9, and the speed is decreased to 9.

In one embodiment, in the step of obtaining mononuclear macrophages, magnetic beads are used for sorting the mononuclear macrophages.

In one embodiment, the concentration of the mononuclear macrophage in the step of grouping culture is 5-10 × 10⁵one/mL.

In one embodiment, in the step of grouping culture, the ratio of the number of the mesenchymal stem cells added into the co-culture group to the number of the mononuclear macrophages is 1 (0.8-1.2). Preferably, the ratio of the number of mesenchymal stem cells to the number of mononuclear macrophages added to the co-cultured group is 1: 1.

In one embodiment, in the step of grouping culture, the culture time is 6-8 days.

Compared with the prior art, the invention has the following beneficial effects:

the detection method of the invention provides four detection indexes, namely CD163, CD204, CD206 and HLA-DR, when the expressions of macrophages CD163, CD204 and CD206 are increased and the expression of HLA-DR is reduced after co-culture, the mesenchymal stem cells have strong capacity and good quality for influencing the differentiation of the macrophages to M2 type macrophages, and the umbilical cord mesenchymal stem cells have strong capacity for regulating tissue regeneration. The invention selects the four detection indexes, wherein CD163 is a scavenger receptor and mediates macrophage phagocytosis of a hemoglobin complex; CD204 is also a scavenger family member, a cell involved in the clearance of apoptosis; CD206 is a mannose receptor, recognizes proteoglycan expressed by pathogenic microorganisms, and eliminates external harmful substances; the HLA-DR is histocompatibility protein, mediates antigen presentation of macrophages, is mainly expressed on the surface of M1 type macrophages, and can comprehensively and efficiently detect the influence of mesenchymal stem cells on the differentiation of the macrophages to the M2 type macrophages by combining the four surface markers.

Drawings

FIG. 1 is a graph of the change in macrophage expression of CD 163;

FIG. 2 is a graph showing the change in CD204 expression by macrophages;

FIG. 3 is a graph showing the change in CD206 expression by macrophages;

FIG. 4 shows the change in HLA-DR expression by macrophages.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The following is a description of preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.