CN115671136A - Application of M0 or M1 type Ly6C + CX3CR1+ monocyte-derived macrophage in treating hepatic fibrosis - Google Patents

Abstract

The invention provides M0 or M1 type Ly6C ⁺ CX3CR1 ⁺ Application of monocyte-derived macrophage in preparing hepatic fibrosis medicine is provided. Experiments prove that the M0 and M1 CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR2 ⁺ BMDM and CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ The CCR2-BMDM four groups of cells can effectively improve the weight and the organ index (reducing the liver body ratio and the spleen body ratio) of a carbon tetrachloride hepatic fibrosis mouse, reduce inflammatory response and reduce the hepatic fibrosis degree, prove that the four groups of cells can obviously reduce the hepatic inflammation and the hepatic fibrosis degree, play a positive auxiliary role in regeneration after hepatic injury and provide a new way for treating the hepatic fibrosis.

Description

Application of M0 or M1 type Ly6C + CX3CR1+ monocyte-derived macrophage in treating hepatic fibrosis

Technical Field

The invention belongs to the field of biomedicine, and relates to M0 or M1 type Ly6C ⁺ CX3CR1 ⁺ Application of monocyte-derived macrophages in preparation of hepatic fibrosis drugs, a preparation method of the macrophages and a pharmaceutical composition containing the macrophages.

Background

The incidence rate of the liver cirrhosis in the population of China is up to 0.51 percent, about 700 million patients with liver cirrhosis exist in the country at present, wherein 1-8 percent of the patients progress to the hepatocellular carcinoma every year, and the liver cirrhosis disease seriously threatens the life health of people. Cirrhosis can be caused by a variety of etiologies, including: HBV and HCV infection, alcoholic liver disease, non-alcoholic fatty liver disease, autoimmune liver disease, genetic and metabolic diseases, drugs or chemical poisons and the like, parasitic infection, circulatory disturbance and the like, which cause gradual loss of healthy hepatocyte tissue and liver structure, diffuse fibrosis of the liver, pseudolobular formation and intrahepatic and extrahepatic vascular proliferation. If the factors (such as virus and alcohol) causing liver damage are removed, hepatic fibrosis can be partially reversed, thereby allowing liver regeneration to occur.

The experimental animal model of liver regeneration after liver injury shows that the macrophage plays a key role in controlling and repairing the fibrotic liver disease. Liver repair can be treated by macrophages differentiated from myelomonocytes, but undifferentiated myelomonocytes have no therapeutic effect. Macrophage therapy can mitigate carbon tetrachloride (CCL 4) -mediated liver damage by modulating hepatic stellate cell apoptosis and increasing the production of anti-inflammatory cytokines. In addition, macrophage-mediated Matrix Metalloproteinase (MMP) release and phagocytosis are critical for the resolution of fibrotic scarring. Macrophages are also capable of stimulating hepatic progenitor proliferation, differentiation and recruitment of lost hepatocytes by Wnt and TWEAK signaling. Adoptive macrophage therapy therefore provides an important potential therapeutic strategy for patients with cirrhosis to promote regression of fibrosis.

Disclosure of Invention

The invention is carried out based on the research, and aims to provide M0 or M1 type Ly6C ⁺ CX3CR1 ⁺ Monocyte-derived macrophage, preparation method thereof and application thereof in preparing hepatic fibrosis drugs, and also provides a pharmaceutical composition containing the macrophage.

In a first aspect of the invention, an M0 or M1 type Ly6C is provided ⁺ CX3CR1 ⁺ Monocyte-derived macrophages. Preferably, the macrophage cell comprises M0 or M1 type CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR2 ⁺ Monocyte-derived macrophage, M0 or M1 type CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR 2-monocytes are derived from four cells, macrophages.

The preparation method of the macrophage is summarized as follows: monocyte is separated from bone marrow cells, cultured for 7 days in vitro by macrophage induction culture medium, cultured for 24 hours by complete culture medium without adding stimulant to obtain M0 type monocyte-derived macrophage (BMDM), and cultured for 24 hours by complete culture medium with Lipopolysaccharide (LPS) + interferon gamma (IFN gamma) stimulant to obtain M1 type BMDM.

Specifically, the macrophage of the invention is respectively extracted from a wild type C57 mouse and CCR2 ^-/- The bone marrow cells of the C57 mouse are obtained after being cultured, induced and differentiated, and the preparation method comprises the following steps:

wild type C57B/L mice and CCR2 ^-/- C57 mice, 6-8 weeks old, euthanized (cervical dislocation); wiping the two lower limbs of the mouse by using a 75% alcohol cotton ball; cutting off fur of lower limbs of the mouse by using scissors, then stripping off muscles of tibia and femur of the mouse, taking off the tibia and the femur, and putting the tibia and the femur into a DMEM-F/12 culture solution precooled at 4 ℃; clamping the bone with forceps, extracting 10ml DMEM-F/12 culture solution with 10ml syringe, replacing 1ml syringe needle, and slowly flushing medullary cavity until the bone becomes thoroughlyWhite; uniformly blowing and beating the flushed bone marrow cells, and filtering by using a 700 mu m nylon membrane; centrifuging at 4 deg.C for 10min at 500g, mixing cell precipitate, counting, and inoculating about 4 × 10 cells in 10cm culture dish ⁶ And (4) cells.

Macrophage induction medium (80% DMEM-F/12+10% FBS +10% L929 cell supernatant) containing L929 cell line cell culture supernatant was used at 37 ℃ and 5% CO ₂ Culturing in an incubator for 4 days; after 4 days, replacing new macrophage induction culture medium for continuous culture; and after 7 days, discarding the old culture solution, adding 2ml PBS into a culture dish for washing, discarding the PBS, adding 3ml culture solution, scraping cells by using a cell scraper, transferring the cells into a 15ml centrifuge tube, culturing the cells for 24 hours at 400 Xg, 10min and 4 ℃ by remaining cell sediment, obtaining M0 type BMDM by taking a part of the cells respectively and adding LPS + IFN gamma stimulant into the complete culture medium for culturing for 24 hours to obtain M1 type BMDM.

The culture and cryopreservation method of the L929 cell strain comprises the following steps: using MEM culture medium containing 10% horse serum as medium, 175cm ² Culturing in an adherent culture bottle, carrying out passage for 1 time every 2-3 days, and carrying out digestion passage by trypsin; cryopreserved as system at 40% horse serum +10% DMSO +50% MEM.

The results of the assay showed CD45 in both cell types ⁺ The cell proportion reaches 97.5% (M0) and 92% (M1); in CD45 ⁺ Under the cell population, it is F4/80 ⁺ CD11b ⁺ The cell ratio was 98.6% (M0), 97.4% (M1), indicating that this method allows the isolation of BMDM of high purity (fig. 1); in CD45 ⁺ F4/80 ⁺ CD11b ⁺ In cells, ly6C ⁺ The cell ratio was 95.8% (M0), 93.0% (M1); in CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ In cells, CX3CR1 ⁺ CCR2 ⁺ The proportion of macrophages was 93.7% (M0), 95.3% (M1). CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR2 ⁺ Macrophages are the major cell type of wild type mouse BMDM.

In a second aspect of the present invention, there is provided a use of the above-mentioned macrophage in the preparation of a medicament for treating liver fibrosis.

Preferably, the medicament for treating hepatic fibrosis is a medicament for reducing the liver body ratio and the spleen body ratio, reducing inflammatory reaction and relieving the hepatic fibrosis degree.

More preferably, the agent for reducing inflammatory response is an agent for reducing ALT, AST, IL6, TNF α, CCL2, TGF β, PDGF levels, reducing infiltration of inflammatory cells in the zone of the sink;

the medicine for relieving the degree of hepatic fibrosis is a medicine for reducing hepatocyte necrosis, reducing the damage degree of hepatic lobule structure, relieving pathological change of choriohyaloid degeneration, reducing tissue collagen deposition and promoting the thinning and narrowing of collagen fiber.

In a third aspect of the invention, a pharmaceutical composition for treating hepatic fibrosis is provided, wherein the pharmaceutical composition is M0 or M1 type Ly6C ⁺ CX3CR1 ⁺ The monocyte-derived macrophage is the only active component, or comprises the macrophage and also comprises pharmaceutically acceptable auxiliary materials. Adjuvants help cells to exert their therapeutic effects more stably, and these preparations can ensure the conformational integrity of the cells disclosed in the present invention while preventing their inactivation.

In the form of medicine, the medicine composition is an injection, and the treatment is carried out in an intravenous injection mode.

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

the invention provides M0 or M1 type Ly6C ⁺ CX3CR1 ⁺ Application of monocyte-derived macrophage in preparing hepatic fibrosis medicine is provided. Experiments prove that the M0 and M1 CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR2 ⁺ BMDM and CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ The CCR2-BMDM can effectively improve the weight and the organ index (reducing the liver body ratio and the spleen body ratio) of a carbon tetrachloride hepatic fibrosis mouse, reduce inflammatory response and reduce the hepatic fibrosis degree, and proves that the four groups of cells can obviously reduce the hepatic inflammation and the hepatic fibrosis degree, play a positive auxiliary role in regeneration after liver injury, and provide a new way for treating the hepatic fibrosis.

Drawings

FIG. 1 shows the results of isolated culture and flow assay of M0-type (A) and M1-type (B) BMDM;

FIG. 2 shows the effect of BMDM cell infusion on liver fibrosis mouse serum AST, ALT, P < 0.001;

figure 3 is the effect of BMDM cell infusion on serum inflammation in liver fibrosis mice, P <0.05, P <0.01, P < 0.001;

FIG. 4 shows HE staining results (200X) of mouse liver tissues;

FIG. 5 shows the results of sirius red staining of mouse liver tissues;

FIG. 6 is a graph showing the effect of BMDM cell infusion on liver tissue alpha-SMA expression in liver fibrosis mice;

FIG. 7 is a graph of the effect of BMDM cell infusion on TGF β and PDGF expression in liver fibrosis mouse sera.

Detailed Description

The following further description of the present invention, taken in conjunction with the accompanying examples and drawings, is not to be construed as limiting the invention, as the examples do not include a detailed description of the conventional methods.

Example 1 isolation and Induction of monocyte-derived macrophages (BMDM)

In vitro separate culture of wild mouse and CX3CR1 ^-/- Mouse, CCR2 ^-/- The mouse bone marrow mononuclear cells are cultured for 7 days in a macrophage inducing culture medium containing a cell culture supernatant of an L929 cell line, then cultured for 24 hours in a complete culture medium without adding a stimulant to obtain M0 type bone marrow mononuclear cell-derived macrophages (BMDM), and cultured for 24 hours in a complete culture medium with LPS + IFN gamma stimulants to obtain M1 type BMDM.

Example 2 therapeutic Effect of monocyte-derived macrophages (BMDM) on mouse hepatic fibrosis induced by carbon tetrachloride

1. Experimental methods

(1) The carbon tetrachloride mouse hepatic fibrosis model and experiment are divided into groups: preparation of carbon tetrachloride (CCl) ₄ ) The induced hepatic fibrosis model of wild mouse is divided into normal group and CCl according to weight stratification method ₄ Model group, WTMBMDM M0+ CC1 ₄ Group, WTBBMDM M1+ CC1 ₄ Group, CX3CR1 ^-/- BMDM M0+CC1 ₄ Group, CX3CR1 ^-/- BMDM M1+CC1 ₄ Group, CCR2 ^-/- BMDM M0+CC1 ₄ Group, CCR2 ^-/- BMDM M1+CC1 ₄ Groups, 6 per group. Wild type mice are treated with CCl ₄ After 2 weeks of molding, macrophage infusion therapy was performed by tail vein infusion of various M0 and M1 BMDM, and then CCl was continued ₄ The model was made for 4 weeks, and finally the mice were sacrificed and the material was collected for testing.

(2) Biochemical liver function index detection: serum ALT, AST levels.

(3) Histopathological histology of liver was performed to observe the condition of inflammatory injury of liver (HE staining) and the degree of hepatic fibrosis (sirius red staining).

(4) Immunohistochemical staining of hepatic stellate cell activation marker protein alpha-SMA in liver tissue.

(5) Liver fibrosis-promoting cytokines TGF β and PDGF assays in serum.

(6) Statistical analysis: normal distribution metrology data is expressed as mean + -standard deviation (x + -s), and two-way T-test is used for pairwise comparisons between groups. P <0.05 is statistically significant.

2. Results

(1)CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR2 ⁺ Macrophages are the major cell type of BMDM in wild-type mice

The mouse bone marrow derived monocytes were isolated and cultured in vitro for 7 days without adding a stimulator for 24 hours to obtain M0 type monocyte derived macrophages (BMDM) (FIG. 1A), and after culturing with LPS + IFN γ stimulator for 24 hours to obtain M1 type BMDM cells (FIG. 1B). Flow assay results suggest that two types of cells are CD45 ⁺ The cell proportion reaches 97.5% (M0) and 92% (M1); under the CD45+ cell population, F4/80 thereof ⁺ CD11b ⁺ The cell ratio was 98.6% (M0), 97.4% (M1), and the results showed that this method could isolate BMDM with high purity (fig. 1); in CD45 ⁺ F4/80 ⁺ CD11b ⁺ In cells, ly6C ⁺ The cell ratio was 95.8% (M0), 93.0% (M1); in CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ In cells, CX3CR1 ⁺ CCR2 ⁺ The macrophage proportion was relatively high, 93.7% (M0), 95.3% (M1). From this, the separation methodHigh purity and CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR2 ⁺ Macrophages are the major cell type of wild-type mouse BMDM.

(2) Effect of BMDM macrophage treatment on body weight and organ index of liver fibrosis mice

After the organ indexes are calculated, the following findings are obtained: CCl compared to wild-type control mice ₄ The liver body ratio of the model group mice is increased, and the spleen body ratio is obviously increased; and CCl ₄ Model group comparison, infusion BMDM treatment experimental group (wild type BMDM M0 type, wild type BMDM M1 type, CX3CR 1) ^-/- BMDM M0 type, CX3CR1 ^-/- BMDM M1 type, CCR2 ^-/- BMDM M0 type, CCR2 ^-/- BMDM type M1) liver and spleen body ratios were decreased. And CCl ₄ Comparison of model groups, where CCR2 ^-/- The liver body ratio of BMDM M0 type and M1 type is obviously reduced, and wild BMDM M0 type, M1 type and CCR2 ^-/- BMDM M0 and M1, CX3CR1 ^-/- The BMDM M1 type splenomeial ratio decreased significantly (table 1).

TABLE 1 influence of BMDM cell infusion on the body weight and organ index of liver fibrosis mice

Note: * P <0.01N vs M; # P <0.05MF vs M

(3)CX3CR1 ⁺ BMDM macrophage treatment can reduce wild-type CCl ₄ Inflammatory response in liver fibrosis mice

The serum liver function results show that: in comparison with the normal group, CCl ₄ The serum ALT and AST of the model group are obviously increased; and CCl ₄ Compared with the model group, the serum ALT and AST of the wild BMDM M0 type and the M1 type are obviously reduced (P is less than 0.001), and the CCR2 is obviously reduced ^-/- The BMDM M0 type and M1 type serum ALT and AST of the mouse are reduced, CX3CR1 ^-/- The BMDM M0 type and M1 type of the mice have no influence on the biochemistry of the serum of the CCl4 hepatic fibrosis mice (figure 2).

ELISA detection suggested: and CCl ₄ Model group comparison, M0-type and M1-type wild-type mice BMDM, M0-type and M1-type CCR2 infusion ^-/- The mouse inflammation related indexes IL6, TNF alpha and CCL2 of the mouse BMDM are all reduced; infusion of M0-and M1-form CX3CR1 ^-/- Mouse BMDM mouse inflammation associated indicators IL6, TNF alpha, CCL2 were not statistically different (FIG. 3).

Liver tissue HE results show: the normal group of mice has complete liver cells, the liver cells are arranged in a plate shape in a single row in a radial manner by taking a central vein as the center, the hepatic lobule structure is clear, and hepatic cell necrosis and inflammatory cell infiltration are not seen; CCl ₄ The mouse in the model group has disorder arrangement of liver cells, liver cell lysis and necrosis, liver lobule structure damage, a large amount of inflammatory cell infiltration in a sink region and gross vitreous degeneration pathological change; and CCl ₄ Model group comparison, wild type mouse BMDM infusion group and CCR2 ^-/- Hepatic cell necrosis of a mouse BMDM infusion group is obviously reduced, hepatic lobular structure damage is reduced, a large amount of inflammatory cell infiltration is reduced in a sink region, and pathological changes of the ground glass-like lesion are obviously reduced (figure 4); CX3CR1 compared with CCl4 model group ^-/- There was no significant improvement in hepatocytes, lobular architecture, and regions of the sink in the BMDM-infused mice (fig. 4).

(4)CX3CR1 ⁺ The BMDM cell treatment can obviously reduce the hepatic fibrosis degree of CCl4 hepatic fibrosis wild mice

Results of Sirius red staining of liver tissues showed: the liver tissue structure of the normal group of mice is normal, and the deposition of collagen fibers is rare; CCl ₄ The hepatic lobule structure of the mouse in the model group is damaged, a great amount of collagen fibers are deposited in a sink area and extend to the periphery to form fiber intervals, and the normal hepatic lobules are partially wrapped and segmented; and CCl ₄ Model group comparison, wild type mouse BMDM infusion group and CCR2 ^-/- The tissue collagen deposition of the mouse BMDM infusion group is obviously reduced, the collagen fiber is thinned and narrowed, and the part disappears (figure 5); and CCl ₄ Comparison of model groups, CX3CR1 ^-/- Liver tissue collagen deposition, collagen fiber thickness did not significantly change in the mice BMDM-infused groups (fig. 5).

Immunohistochemistry of liver tissues showed: the alpha-SMA of the liver tissue of the wild type normal group mouse is not expressed; and normal groupIn contrast, CCl ₄ The model group is mainly distributed around the fibrous septum of the hepatic tissue manifold area, and particularly, the inflammatory cell infiltration area is more obvious; and CCl ₄ Comparison of model groups, wild type BMDM infusion group and CCR2 ^-/- The expression of the mouse infusion group tissue alpha-SMA around the hepatic tissue sink area interval is obviously reduced; and CCl ₄ Comparison of model groups, CX3CR1 ^-/- There was no significant change in α -SMA expression in the mice infusion group (fig. 6).

ELISA detection suggested: compared with the model group CCl4, the wild BMDM M0 type and BMDM M1 type mice infusion inflammation related indexes of the pro-fibrosis factors TGF beta and PDGF are obviously reduced. CX3CR1 ^-/- BMDM M0 type and M1 type groups are increased, and PDGF is not obviously increased; CCR2 ^-/- There was a decrease in BMDM type M0 and M1 (FIG. 7).

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. Type M0 or M1 Ly6C ⁺ CX3CR1 ⁺ Application of monocyte-derived macrophage in preparing medicine for treating hepatic fibrosis is provided.
2. 2. Use according to claim 1, characterized in that:

   wherein the macrophage is M0 or M1 type CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR2 ⁺ Monocyte-derived macrophage and M0 or M1 type CD45 ⁺ F4/80 ⁺ CD11b ⁺ Ly6C ⁺ CX3CR1 ⁺ CCR2 ^- Monocytes are derived from any of the macrophages.
3. 3. Use according to claim 1, characterized in that:

   wherein the mononuclear cells are derived from bone marrow cells.
4. 4. Use according to claim 1, characterized in that:

   wherein, the medicament for treating the hepatic fibrosis is a medicament for reducing the liver body ratio and the spleen body ratio, relieving inflammatory reaction and relieving the hepatic fibrosis degree.
5. 5. Use according to claim 4, characterized in that:

   wherein the agent that reduces inflammatory response is an agent that reduces ALT, AST, IL6, TNF α, CCL2, TGF β, PDGF levels or reduces infiltration of inflammatory cells in the zone of the sink;

   the medicine for relieving the degree of hepatic fibrosis is a medicine for reducing hepatocyte necrosis, reducing the degree of hepatic lobule structure damage, relieving pathological change of choriohyaloid degeneration, reducing tissue collagen deposition and promoting thinning and narrowing of collagen fibers.
6. 6. Use according to any one of claims 1 to 5, wherein the macrophage is prepared by the following process:

   monocyte is separated from bone marrow cells, after in vitro macrophage induction culture medium is used for culturing for 7 days, M0 type monocyte source macrophage is obtained by culturing in complete culture medium without adding stimulant for 24 hours, and M1 type BMDM is obtained by culturing in complete culture medium with lipopolysaccharide and interferon gamma stimulant for 24 hours.
7. 7. Use according to claim 6, characterized in that:

   wherein said macrophage-inducing medium consists of 80% DMEM-F/12, 10% FBS, 10% L929 cell supernatant, and the complete medium consists of 90% DMEM-F/12, 10% FBS.
8. 8. Use according to claim 1 or 2, characterized in that:

   whereinThe medicine is Ly6C in M0 or M1 type ⁺ CX3CR1 ⁺ Monocyte-derived macrophages are the only active component or comprise the macrophages.
9. 9. Use according to claim 1, characterized in that:

   wherein, the medicine is injection.
10. 10. A pharmaceutical composition for treating hepatic fibrosis, which comprises M0 or M1 type Ly6C ⁺ CX3CR1 ⁺ Macrophage derived from monocyte and pharmaceutically acceptable adjuvants.

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