阅读说明：本技术 一种肝脏固有和浸润巨噬细胞的分离方法 (Method for separating inherent and infiltrated macrophages of liver ) 是由 季菊玲 季煜华 沈宓 李静 何理 孙玉风 陆鹏 吕秀芳 于 2019-10-22 设计创作，主要内容包括：本发明提供一种肝脏固有和浸润巨噬细胞的分离方法,包括如下步骤：(1)建立并鉴定小鼠异体骨髓移植模型,受体小鼠单核巨噬细胞表面标志物为CD45.2,供体小鼠单核巨噬细胞表面标志物为CD45.1；(2)IHC法和流式细胞术观察照射对肝脏实质细胞和固有巨噬细胞造成的影响；(3)原代分离照射移植组小鼠肝脏单个核细胞,根据供体和受体小鼠单核巨噬细胞表面标志物,流式细胞术分选肝脏固有和浸润巨噬细胞,并描述其数量、分布和表型。本发明通过照射去除CD45.2受体小鼠骨髓来源巨噬细胞,并以CD45.1供体小鼠骨髓补充替代受体小鼠骨髓细胞。从而保证骨髓来源的浸润巨噬细胞细胞和固有巨噬细胞具有不同的分子表型,达到准确高效分离肝脏不同来源巨噬细胞的效果。(The invention provides a method for separating inherent macrophages and infiltrated macrophages of a liver, which comprises the following steps: (1) establishing and identifying a mouse allogeneic bone marrow transplantation model, wherein the surface marker of the recipient mouse mononuclear macrophage is CD45.2, and the surface marker of the donor mouse mononuclear macrophage is CD 45.1; (2) IHC and flow cytometry observe the effects of irradiation on liver parenchymal cells and resident macrophages; (3) primary isolation irradiated transplantation group mouse liver mononuclear cells, flow cytometry sorting liver resident and infiltrating macrophages according to donor and recipient mouse mononuclear macrophage surface markers, and describing the number, distribution and phenotype of the liver resident and infiltrating macrophages. The invention removes CD45.2 acceptor mouse marrow-derived macrophages through irradiation and replaces acceptor mouse marrow cells with CD45.1 donor mouse marrow supplementation. Therefore, infiltrating macrophage cells and inherent macrophages from bone marrow are guaranteed to have different molecular phenotypes, and the effect of accurately and efficiently separating macrophages from different sources of the liver is achieved.)

1. A method for separating intrinsic and infiltrated macrophages from liver is characterized by comprising the following steps:

(1) establishing and identifying a mouse allogeneic bone marrow transplantation model, wherein the surface marker of the recipient mouse mononuclear macrophage is CD45.2, and the surface marker of the donor mouse mononuclear macrophage is CD 45.1;

(2) IHC and flow cytometry observe the effects of irradiation on liver parenchymal cells and resident macrophages;

(3) primary isolation irradiated transplantation group mouse liver mononuclear cells, flow cytometry sorting liver resident and infiltrating liver macrophages according to donor and recipient mouse mononuclear macrophage surface markers, and describing the quantity, distribution and phenotype of the liver resident and infiltrating liver macrophages.

2. The method for separating resident and infiltrated macrophages of the liver according to claim 1, wherein the step (1) comprises the following steps:

preparation of C57BL/6 mouse bone marrow cells of CD45.1:

b. establishing a mouse bone marrow transplantation model:

3. The method for separating intrinsic and infiltrated macrophages of the liver of claim 1, wherein the step (2) comprises the following steps:

a. randomly dividing the C57BL/6 mice into a negative control group which does not receive illumination, a treatment group 24h, 48h and 7d after irradiation, wherein each group comprises 6 mice, the feeding mode is the same as that before, the C57BL/6 mice are subjected to single gamma ray whole body irradiation after one week to destroy bone marrow cells, the mice are continuously fed with antibiotic water for one week and are subjected to standard diet, and after irradiation, the tissues of the mice in the irradiation groups 24h, 48h and 7d and the negative control group are collected to prepare paraffin sections;

b. immunohistochemistry:

4. The method for separating resident and infiltrated macrophages of the liver according to claim 1, wherein the step (3) comprises the following steps:

a. isolation of primary liver mononuclear cells:

cutting liver in a small beaker, and digesting with digestive juice containing collagenase and Dnase for 35min in a water bath at 37 ℃;

uniformly distributing the filtrate into centrifuge tubes, centrifuging at 4 ℃ for 47g, and sucking the supernatant into a new centrifuge tube for 5 min;

centrifuging for 8min at 400g, and discarding the supernatant to obtain precipitate; adding 10ml of 1 XPBS into the cell sediment for resuspension, and repeatedly centrifuging once;

b. flow cytometry sorting:

resuspending the cells with appropriate amount of CSB, adding CD16/32 antibody, and blocking on ice for 5-10 min;

c. cell surface markers detected by flow cytometry indicate: approximately 28.7% of F4/80 and CD11b positive mononuclear macrophages in the liver of bone marrow transplanted mice were CD45.2 positive intrinsic macrophages from the recipient, and 67.5% were CD45.1 positive mononuclear macrophages from the donor.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for separating inherent and infiltrated macrophages of a liver.

Background

The liver is not only an important site of metabolism of substances but also an important immune organ. The liver macrophages are composed of inherent macrophages and infiltrating macrophages, have complex phenotype and function, play an important role in maintaining normal physiological functions of the liver, liver injury caused by various poisons, medicines and pathogenic microorganisms and various liver metabolic diseases, and are always an important field of liver pathological research.

Liver resident macrophages and infiltrating macrophages derived from peripheral blood mononuclear cells are populations of cells of different origin and different biological functions. In recent years it has been investigated that the intrinsic macrophages in the early stages of superficial liver injury decrease in number and then are supplemented by self-proliferation which is independent of peripheral blood mononuclear-derived macrophages chemotactic to local sites. In 2014, zigbee et al in acute liver injury models again demonstrated that infiltrating peripheral blood mononuclear cells do not participate in the recruitment of liver resident macrophages during injury, which can self-renew. The role of each of liver infiltrating macrophages and resident macrophages during liver injury is unknown, but to date, surface markers that effectively distinguish between liver infiltrating and resident macrophages are lacking. The existing method distinguishes different sources of liver macrophages according to the expression levels of mononuclear macrophage surface markers F4/80 and CD11b, but the macrophages are a complex population, and cells with high expression levels or low expression levels of F4/80 and CD11b may exist in both infiltrating and inherent macrophages, so that the two markers cannot accurately distinguish the sources of the macrophages.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for separating intrinsic and infiltrated macrophages of the liver, which is helpful for distinguishing macrophages from different sources, further researches the understanding of the roles of the macrophages in liver local injury repair and immunoregulation and provides a basis for deeply analyzing the roles of the macrophages in various liver pathological processes.

In order to solve the above technical problems, an embodiment of the present invention provides a method for separating intrinsic and infiltrated macrophages from a liver, including the following steps:

(1) establishing and identifying a mouse allogeneic bone marrow transplantation model, wherein the surface marker of the recipient mouse mononuclear macrophage is CD45.2, and the surface marker of the donor mouse mononuclear macrophage is CD 45.1;

(2) IHC and flow cytometry observe the effects of irradiation on liver parenchymal cells and resident macrophages;

(3) primary isolation irradiated transplantation group mouse liver mononuclear cells, flow cytometry sorting liver resident and infiltrating liver macrophages according to donor and recipient mouse mononuclear macrophage surface markers, and describing the quantity, distribution and phenotype of the liver resident and infiltrating liver macrophages.

Wherein, the specific steps of the step (1) comprise:

preparation of C57BL/6 mouse bone marrow cells of CD45.1:

mice were sacrificed and skin was disinfected;

dissociating the long bones of the limbs of the mouse, and soaking in RPMI-1640;

cutting off cartilage at two ends, and flushing out cells in the marrow cavity;

repeatedly blowing and beating the cell suspension uniformly, collecting the bone marrow cell suspension and filtering;

collecting cell suspension, centrifuging for 10min at 250g, removing supernatant, adding 2ml erythrocyte lysate to lyse erythrocyte, standing at room temperature for 3min, centrifuging for 5min at 400g, and removing supernatant;

the cells obtained were resuspended in 200. mu.l of PBS and counted;

b. establishing a mouse bone marrow transplantation model:

irradiating the transplanted mice, destroying bone marrow cells by single gamma-ray total body irradiation, continuously feeding antibiotic water for one week after irradiation, performing standard diet, and receiving intravenous injection of bone marrow cells under isoflurane anesthesia;

non-transplanted mice were irradiated without further treatment as controls;

after the bone marrow transplantation is finished, the observation is continued in the central barrier environment of the animals, and after antibiotic water is drunk for one week, water is freely drunk and fed into a standard diet. Mice that did not receive bone marrow transplantation all died after 2 weeks.

Wherein, the concrete steps of step (2) include:

a. randomly dividing the C57BL/6 mice into a negative control group (not receiving irradiation), treatment groups 24h, 48h and 7d after irradiation, wherein each group comprises 6 mice, the feeding mode is the same as that before, the C57BL/6 mice are subjected to single gamma ray whole body irradiation after one week to destroy bone marrow cells, the mice are continuously fed with antibiotic water for one week and are subjected to standard diet, and after irradiation, the tissues of the irradiation groups 24h, 48h and 7d and the negative control group are collected to prepare paraffin sections;

b. immunohistochemistry:

baking at 60 deg.C for 8 hr;

dewaxing to water: dewaxing with xylene conventionally for 20min × 2 times, dehydrating with gradient ethanol for 5min × 2 times, with 95% ethanol for 5min × 2 times, with 80% ethanol for 5min × 2 times, with 70% ethanol for 5min × 2 times, with 50% ethanol for 5min, and washing with flowing water for 1 min;

antigen retrieval: soaking the slices in 0.01M citric acid buffer solution (pH6.0) in an electric heating pan, heating to boil for 10min, taking out, naturally cooling to room temperature, and washing with 1 × PBS for 5min × 3 times;

blocking endogenous peroxidase: sections at 30% H₂O₂Soaking in methanol solution for 10min, and washing with 1 × PBS for 5min × 3 times;

100ul of primary antibody diluted 1:100 was added to each section, incubated at room temperature for 1h and then incubated overnight at 4 ℃. Dripping 1 × PBS into the negative control section, taking out the negative control section the next day, standing the negative control section at room temperature for 1 hour, and washing the negative control section for 3min × 3 times by using 1 × PBS;

adding corresponding secondary antibody to each section, incubating for 1h at room temperature, washing for 3min with 1 × PBS and multiplying by 3 times;

color development: removing PBS, dripping DAB working solution into the slice, observing and controlling the color development degree under a microscope, when the antigen is obviously expressed,flushing under flowing water to timely stop color development;

counter-staining with hematoxylin for 30s-2min, washing with running water, differentiating with 1% hydrochloric acid-ethanol, and washing with running water;

and (3) dehydrating: 70% ethanol for 3min, 80% ethanol for 3min, 95% ethanol for 3min, anhydrous ethanol for 3min, and xylene for 1 min;

sealing: and (5) sealing the neutral gum.

Wherein, the concrete steps of step (3) include:

a. isolation of primary liver mononuclear cells:

mice were sacrificed and skin was disinfected and fixed to the rat plate;

in a primary culture superclean workbench, opening the abdominal cavity, exposing the heart and the liver, and performing cardiac intubation;

D-Hanks liquid is perfused in situ, 50ml of liquid is perfused until the surface of the liver becomes white, and the liver is isolated. Putting the liver into 1 XPBS solution containing the green-streptomycin, washing for 2 times, and putting the liver into a sterile beaker;

cutting liver in a small beaker, and digesting with digestive juice containing collagenase (0.005%) and Dnase (0.005%) for 35min in a 37 deg.C water bath;

filtering the cell suspension by a 200-mesh filter screen;

uniformly distributing the filtrate into centrifuge tubes, centrifuging at 4 ℃ for 47g, and sucking the supernatant into a new centrifuge tube for 5 min;

centrifuging at 400g for 8min, and discarding the supernatant to obtain precipitate. Adding 10ml of 1 XPBS into the cell sediment for resuspension, and repeatedly centrifuging once;

centrifuging the cell precipitate at gradient density of 25% percoll and 50% percoll at 4 deg.C for 30min at 400 g;

sucking 50% and 25% percoll suspension cells into a new centrifuge tube, adding 8ml of 1 XPBS solution for resuspension, centrifuging for 8min at 4 ℃ at 400g, discarding supernatant, taking precipitate, and repeating the steps once; the resulting pellet was resuspended in 200ul 1 XPBS and counted;

b. flow cytometry sorting:

resuspending the cells with appropriate amount of CSB, adding CD16/32 antibody (0.5 ul for 10^6 cells), and blocking on ice for 5-10 min;

adding 10ul of flow-sorting corresponding antibody, and incubating at 4 deg.C in the dark for 20 min;

adding erythrocyte lysateIncubating 1ml of the hydrolysate for 5min at room temperature, centrifuging 350g for 5min, and removing supernatant;

the pellet was resuspended in 0.5ml Cell Stabilizing Buffer (CSB) and 7-AAD (10) was added⁷Adding 5 ul) into each cell, and incubating for 3-5min in dark; sorting by a flow cytometer;

c. cell surface representation by flow cytometry: two subsets, F4/80 respectively, were found in CD45.2CD11b positive monocyte macrophage cells in normal liver of wild type mice that were not transplanted by irradiation^hiCD11b^low, F4/80^lowCD11b^hi(ii) a About 28.7% of CD11b positive mononuclear macrophages in the liver of bone marrow transplanted mice were CD45.2 positive intrinsic macrophages from the recipient, 67.5% were CD45.1 positive mononuclear macrophages from the donor; the CD45.2 positive macrophage phenotype from the receptor was F4/80^hiCD11b^lowClose to typical resident macrophages; the phenotype of CD45.1 positive macrophage from donor is F4/80^lowCD11b^hiNear infiltrating monocytes.

The technical scheme of the invention has the following beneficial effects:

1. the invention adopts allogeneic bone marrow transplantation mice to establish an acute liver injury model, effectively separates the two groups of cells in the liver injury process, and researches the dynamic changes of the quantity, the phenotype and the biological function of the cells. The separation method of the invention is helpful for distinguishing macrophages from different sources, further researching the understanding of the action of the macrophages in liver local injury repair and immune regulation, and providing a basis for deeply analyzing the action of the macrophages in various liver pathological processes.

2. The invention removes CD45.2 acceptor mouse marrow-derived macrophages through irradiation and replaces acceptor mouse marrow cells with CD45.1 donor mouse marrow supplementation. Thereby ensuring that the macrophage cells derived from the bone marrow and the inherent macrophages have different molecular phenotypes, and achieving the effect of accurately and efficiently separating the macrophages derived from different livers.

3. The bone marrow transplanted mouse liver macrophage is composed of receptor liver inherent macrophage and donor bone marrow derived macrophage, and the separation method can be used for researching the source and the functional characteristics of the liver macrophage under physiological state and liver injury state.

Drawings

FIG. 1 is a schematic diagram of the establishment of a mouse allogeneic bone marrow transplantation model in the present invention;

FIG. 2 is a graph showing that after 24h and 48h irradiation, the liver tissue morphology was substantially normal, the number of CD68 positive cells was reduced compared to that in the non-irradiated group, and the number of CD68 positive cells was significantly increased at 7 d;

FIG. 3 is a schematic of the isolation and identification of C57BL/6 mouse primary liver macrophages;

FIG. 4 is a schematic representation of flow assays of macrophage numbers and phenotypes in bone marrow-only transplanted mice and non-transplanted wild-type mice;

FIG. 5 is a schematic diagram showing the results of flow measurement of peripheral blood mononuclear cells of a CD45.2 recipient mouse after 6 weeks of allogeneic bone marrow transplantation in a mouse according to the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

The invention provides a method for separating inherent and infiltrated macrophages of a liver, which comprises the following steps:

(1) establishing and identifying a mouse allogeneic bone marrow transplantation model (shown in figure 1), wherein the surface marker of the recipient mouse mononuclear macrophage is CD45.2, and the surface marker of the donor mouse mononuclear macrophage is CD 45.1; the method comprises the following specific steps:

preparation of C57BL/6 mouse bone marrow cells of CD45.1:

mice were sacrificed and skin was disinfected;

free out of the four limbs of the mouseSoaking long bone in RPMI-1640;

cutting off cartilage at two ends, and flushing out cells in the marrow cavity;

repeatedly blowing and beating the cell suspension uniformly, collecting the bone marrow cell suspension and filtering;

collecting cell suspension, centrifuging for 10min at 250g, removing supernatant, adding 2ml erythrocyte lysate to lyse erythrocyte, standing at room temperature for 3min, centrifuging for 5min at 400g, and removing supernatant;

the cells thus obtained were resuspended in 200. mu.l of PBS and counted.

b. Establishing a mouse bone marrow transplantation model:

irradiating the transplanted mice, destroying bone marrow cells by single gamma-ray total body irradiation, continuously feeding antibiotic water for one week after irradiation, performing standard diet, and receiving intravenous injection of bone marrow cells under isoflurane anesthesia;

non-transplanted mice were irradiated without further treatment as controls;

after the bone marrow transplantation is finished, the observation is continued in the central barrier environment of the animals, and after antibiotic water is drunk for one week, water is freely drunk and fed into a standard diet. Mice that did not receive bone marrow transplantation all died after 2 weeks.

(2) IHC and flow cytometry observe the effects of irradiation on liver parenchymal cells and resident macrophages; the method comprises the following specific steps:

a. the C57BL/6 mice are randomly divided into a negative control group (not receiving irradiation), treatment groups 24h, 48h and 7d after irradiation, 6 mice in each group are raised in the same way as before, the C57BL/6 mice are subjected to single gamma ray whole body irradiation after one week to destroy bone marrow cells, the mice are continuously fed with antibiotic water for one week and are subjected to standard diet, and after irradiation, the tissues of the irradiation groups 24h, 48h and 7d and the negative control group are collected to prepare paraffin sections.

b. Immunohistochemistry:

baking at 60 deg.C for 8 hr;

dewaxing to water: dewaxing with xylene conventionally for 20min × 2 times, dehydrating with gradient ethanol for 5min × 2 times, with 95% ethanol for 5min × 2 times, with 80% ethanol for 5min × 2 times, with 70% ethanol for 5min × 2 times, with 50% ethanol for 5min, and washing with flowing water for 1 min;

antigen retrieval: soaking the slices in 0.01M citric acid buffer solution (pH 6.0) in an electric heating pan, heating to boil for 10min, taking out, naturally cooling to room temperature, and washing with 1 × PBS for 5min × 3 times;

blocking endogenous peroxidase: sections at 30% H₂O₂Soaking in methanol solution for 10min, and washing with 1 × PBS for 5min × 3 times;

100ul of primary antibody diluted 1:100 was added to each section, incubated at room temperature for 1h and then incubated overnight at 4 ℃. Negative control section is droppedAdding 1 × PBS, taking out the mixture the next day, standing the mixture at room temperature for 1h, and washing the mixture for 3min × 3 times by using 1 × PBS;

adding corresponding secondary antibody to each section, incubating for 1h at room temperature, washing for 3min with 1 × PBS and multiplying by 3 times;

color development: removing PBS, dripping DAB working solution into the slices, observing and controlling the color development degree under a microscope, and rinsing under flowing water to stop color development in time when the antigen is obviously expressed;

counter-staining with hematoxylin for 30s-2min, washing with running water, differentiating with 1% hydrochloric acid-ethanol, and washing with running water;

and (3) dehydrating: 70% ethanol for 3min, 80% ethanol for 3min, 95% ethanol for 3min, anhydrous ethanol for 3min, and xylene for 1 min;

sealing: and (5) sealing the neutral gum.

As shown in FIG. 2, after 24h and 48h irradiation, the morphology of liver tissue was substantially normal, the number of CD68 positive cells was reduced compared to that of the unirradiated group, and at 7d, the number of CD68 positive cells was significantly increased (P < 0.0001).

(3) Primarily separating and irradiating mononuclear cells of mouse liver in a transplantation group by irradiation, sorting inherent and infiltrating liver macrophages by flow cytometry according to surface markers of mononuclear macrophages of donor mice and recipient mice, and describing the quantity, distribution and phenotype of the inherent and infiltrating liver macrophages; the method comprises the following specific steps:

a. isolation of primary liver mononuclear cells:

mice were sacrificed and skin was disinfected and fixed to the rat plate;

in a primary culture superclean workbench, opening the abdominal cavity, exposing the heart and the liver, and performing cardiac intubation;

D-Hanks liquid is perfused in situ, 50ml of liquid is perfused until the surface of the liver becomes white, and the liver is isolated. Putting the liver into 1 XPBS solution containing the green-streptomycin, washing for 2 times, and putting the liver into a sterile beaker;

cutting liver in a small beaker, and digesting with digestive juice containing collagenase (0.005%) and Dnase (0.005%) for 35min in a 37 deg.C water bath;

filtering the cell suspension by a 200-mesh filter screen;

uniformly distributing the filtrate into centrifuge tubes, centrifuging at 4 ℃ for 47g, and sucking the supernatant into a new centrifuge tube for 5 min;

centrifuging at 400g for 8min, and discarding the supernatant to obtain precipitate. Adding 10ml of 1 XPBS into the cell sediment for resuspension, and repeatedly centrifuging once;

centrifuging the cell precipitate at gradient density of 25% percoll and 50% percoll at 4 deg.C for 30min at 400 g;

sucking 50% and 25% percoll suspension cells into a new centrifuge tube, adding 8ml of 1 XPBS solution for resuspension, centrifuging for 8min at 4 ℃ at 400g, discarding supernatant, taking precipitate, and repeating the steps once; the resulting pellet was resuspended in 200ul 1 XPBS and counted.

b. Flow cytometry sorting:

the cells were resuspended in the appropriate amount of CSB and CD16/32 antibody (10) was added⁶Adding 0.5 ul) of cells into ice, and sealing for 5-10min

Then 10ul of flow sorting corresponding antibody is added, and incubation is carried out for 20min at 4 ℃ in the dark;

adding erythrocyte lysate 1ml, incubating at room temperature for 5min, centrifuging at 350g for 5min, and removing supernatant;

the pellet was resuspended in 0.5ml Cell Stabilizing Buffer (CSB) and 7-AAD (10) was added⁷Adding 5 ul) into each cell, and incubating for 3-5min in dark; sorting by a flow sorter.

FIG. 3 shows the isolation and characterization of primary liver macrophages from C57BL/6 mice, where a layer of suspension cells between 50% and 25% percoll are liver macrophage cells, indicated by arrows. FIG. 3B flow assay, successful acquisition of CD45+ F4/80+ liver macrophages; FIG. 3℃ Primary mouse liver macrophages were cultured for 24h, Bright field X100 fold field of view.

FIG. 4 shows the number and phenotype of macrophages in bone marrow-only transplanted mice and non-transplanted wild-type mice were flow-tested. Cell surface representation by flow cytometry: CD45.2CD11b positive monocyte macrophage cell in normal liver of wild type mouse without irradiation transplantationTwo subgroups were seen, F4/80 respectively^hiCD11b^low, F4/80^lowCD11b^hi(ii) a About 28.7% of CD11b positive mononuclear macrophages in the liver of bone marrow transplanted mice were CD45.2 positive intrinsic macrophages from the recipient, 67.5% were CD45.1 positive mononuclear macrophages from the donor; the CD45.2 positive macrophage phenotype from the receptor was F4/80^hiCD11b^lowClose to typical resident macrophages; the phenotype of CD45.1 positive macrophage from donor is F4/80^lowCD11b^hiNear infiltrating monocytes. These results suggest that liver resident macrophages are not completely destroyed by irradiation, and that liver damage caused by irradiation can attract peripheral blood mononuclear cells into the liver and differentiate into macrophages. The model accords with the composition of macrophages in the liver under a natural state, and is an ideal model for carrying out follow-up research.

The invention utilizes mice with different CD45 phenotypes to perform allogeneic bone marrow transplantation, so that tissue-resident macrophages and peripheral blood monocyte-derived infiltrating macrophages in the model have different CD45 surface markers. Intrinsic (CD45.2+) and infiltrating (CD45.1 +) macrophages in the liver can be effectively distinguished by flow sorting. We have now established a C57BL/6 mouse allogeneic bone marrow transplant model with a CD45.1 donor and a CD45.2 recipient. As shown in fig. 5: after 6 weeks of transplantation, CD45.2 recipient mice peripheral blood mononuclear cells were flow-assayed (CD 45.2C57BL/6 for wild type mice, CD45.1/CD45.2C57BL/6 for CD45.2 mice receiving CD45.1 mouse bone marrow transplantation).

The invention removes CD45.2 acceptor mouse marrow-derived macrophages through irradiation and replaces acceptor mouse marrow cells with CD45.1 donor mouse marrow supplementation. Thereby ensuring that the macrophage cells derived from the bone marrow and the inherent macrophages have different molecular phenotypes, and achieving the effect of accurately and efficiently separating the macrophages derived from different livers.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.