CN112007165B - Macrophage polarization regulator and application thereof in promoting thrombopoiesis - Google Patents

Macrophage polarization regulator and application thereof in promoting thrombopoiesis Download PDF

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CN112007165B
CN112007165B CN202010881025.2A CN202010881025A CN112007165B CN 112007165 B CN112007165 B CN 112007165B CN 202010881025 A CN202010881025 A CN 202010881025A CN 112007165 B CN112007165 B CN 112007165B
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黄晓军
孔圆
赵红艳
温琦
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Peking University Peoples Hospital
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Abstract

The present invention relates generally to macrophage polarization modulators and their use in promoting thrombopoiesis. In particular, the invention relates to a pharmaceutical composition for treating a thrombocytopenic disease in a subject comprising a macrophage polarization modulator and a pharmaceutically acceptable carrier. The invention also relates to a kit for diagnosing a thrombocytopenic disease in a subject, comprising a detection reagent for detecting a marker of a thrombocytopenic disease and/or a detection reagent for detecting the level of macrophage polarization. The invention also relates to macrophage polarization regulator, a detection reagent for detecting the marker and application of the detection reagent for detecting the polarization level of the macrophage in thrombocytopenic diseases. The invention has important clinical significance for layered diagnosis of diseases and accurate targeted therapy of diseases.

Description

Macrophage polarization regulator and application thereof in promoting thrombopoiesis
Technical Field
The invention relates to the technical field of biomedicine, in particular to a macrophage polarization regulator and application thereof in promoting thrombopoiesis.
Technical Field
Thrombocytopenic diseases, also called thrombocytopenia, are diseases which are symptomatic by an abnormal reduction in the number of platelets in the peripheral blood. The causes include insufficient platelet production, excessive platelet destruction, abnormal platelet distribution, and the like. Among them, delayed platelet implantation (PT) after transplantation is a common thrombocytopenic disease.
Delayed platelet engraftment after transplantation is one of the serious complications of allogeneic hematopoietic stem cell transplantation, which means that white blood cells and hemoglobin have been stably implanted for +60 days after transplantation on the premise of complete donor chimerism, and platelets continue to be lower than 20X 109/L or still rely on platelet infusion.
At present, the pathogenesis of the transplanted PT is unclear, and no effective clinical treatment means exists. Thrombocytopenic diseases such as post-transplant PT patients are characterized by megakaryocyte maturation disorders and thrombocytopenia, leading to increased associated mortality and increased patient medical costs. Therefore, the discovery of new targets for promoting differentiation of megakaryocytes and thrombopoiesis of patients with thrombocytopenic diseases such as PT after transplantation and the development of new schemes are important clinical scientific problems to be solved urgently.
The maturation of megakaryocytes to thrombopoiesis is a complex biological process. The development of megakaryocytes from hematopoietic stem cells is regulated by various signals of the bone marrow microenvironment during the migration of the bone marrow microenvironment from the endosteum to the vascular microenvironment. Any disorder that results in megakaryocytopoiesis and maturation affects platelet production.
In recent years, animal studies suggest that macrophages play an important regulatory role in maintaining hematopoietic stem cells as an important component of the bone marrow immune microenvironment. However, there are limited and inconsistent reports on the regulation of bone marrow megakaryocytes by bone marrow macrophages. The observation by electron microscope shows that there is direct contact between bone marrow macrophages and megakaryocytes, and it is thought that macrophages exert the function of supporting megakaryocyte maturation and thrombopoiesis through direct contact and substance transfer. On the contrary, the scholars find that the mouse model for idiopathic thrombocytopenic purpura specifically eliminates the mouse bone marrow macrophages to be beneficial to the maturation and differentiation of bone marrow megakaryocytes and promote the platelet recovery in the peripheral circulation of the mouse for idiopathic thrombocytopenic purpura. Therefore, there is a need for more research to define the effect of bone marrow macrophages on megakaryocytes.
Macrophages are differentiated and matured from bone marrow monocytes. The mononuclear macrophage system is an important component of the inherent immunity in the body and has extremely strong heterogeneity. Monocytes are mainly divided into three subtypes according to their phenotype and function: classic, intermediate and non-classic. Macrophages, when stimulated by the environment, also polarize into subpopulations of cells of different phenotypes and functions, mainly classical activated macrophages (M1) and selective activated macrophages (M2). The two cell types have different phenotypes and functions, and M1/M2 polarization imbalance is involved in various autoimmune diseases, metabolic diseases and inflammatory diseases. Functionally, M1 can highly express proinflammatory factors, has strong antimicrobial and antitumor activities, mediates reactive oxygen induced tissue damage, and inhibits tissue regeneration and healing; m2 expresses anti-inflammatory factor, has strong phagocytosis ability, can remove debris and apoptotic cells, promote tissue repair and healing, and has angiogenesis and fibrosis promoting properties. Macrophages undergo polarization changes of different degrees and directions in different tissue microenvironments under different signal transduction pathways. The major signaling pathways currently reported to mediate macrophage polarization to M2 include: [ STAT6/IRF4/PPAR signaling pathway: IL-4 activates intracellular STAT6 to be phosphorylated to form a dimer through being aggregated with IL-4R on a membrane, phosphorylated STAT6 is further combined with KLF-4 and PPAR-gamma, gene transcription is started to regulate and control the expression of specific target genes Arg-1, Mcr-1, Chil3 and the like of M2 type M phi s, and polarization in the direction of M2 is promoted; PI3K/AKT signal path: the knockout of Akt1 gene is proved by gene knockout mice to cause macrophage to be polarized to M1, and tyrosine phosphatidylinositol-3-kinase (PI 3K) can catalyze downstream Akt1 to promote macrophage to be polarized to M2. The activation of the PI3K/Akt signaling pathway in various tumors has been demonstrated to play an important role in the survival, growth, metastasis, angiogenesis and metabolism of tumor cells, so that a large number of PI3K inhibitors have entered phase II and phase III clinical trials and achieved good results.
At present, there is no report on promoting the normalization of the microenvironment of bone marrow megakaryocytes by regulating the polarization of bone marrow macrophages, and finally improving the megakaryocyte function and thrombopoiesis. The research on the signal path of macrophage polarization plays an important role in effectively interfering the conversion between M1 and M2 macrophages, thereby achieving the prevention and treatment of some inflammatory and immune diseases. Therefore, the method for effectively regulating and controlling the bone marrow macrophages to promote the megakaryocyte function or promote the thrombopoiesis has important significance for clinical targeted therapy.
Disclosure of Invention
The inventor finds out through research that the polarization of macrophages in the bone marrow of a PT patient is unbalanced, the polarization ratio of the macrophages to M2 is obviously reduced, and the supporting capacity of the macrophages on bone marrow megakaryocytes is obviously reduced. Specifically, the inventor detects the distribution of each subgroup of PT bone marrow mononuclear macrophage system, and compared with a patient with good hematopoietic reconstruction and no megakaryocyte maturation disorder, the proportion of typical mononuclear cells in the bone marrow of the PT patient is reduced, and the proportion of intermediate and atypical mononuclear cells in the bone marrow of the PT patient is increased; meanwhile, the polarization of the macrophages in the bone marrow of the PT patient to M1 is increased, the polarization of the macrophages to M2 is reduced, and the polarization imbalance state of the macrophages in the direction of the proinflammatory phenotype M1 is presented. In vitro studies confirm that compared with bone marrow macrophages of patients with good hematopoietic reconstitution and no megakaryocyte maturation disorder, the PT bone marrow macrophages have significantly reduced megakaryocyte supporting effect: co-culture experiment found that normal CD34+After the cells are co-cultured with PT bone marrow macrophages, the generation ratio of megakaryocytes is reduced, the maturation and ploidy forming ratio of the megakaryocytes and the colony forming capability are reduced, and the blood is smallThe number of plates produced decreases.
The inventor also finds that compared with M1, M2 has a remarkable supporting effect on megakaryocyte maturation and thrombopoiesis through in vitro induction culture of a human monocyte THP1 line and healthy human bone marrow monocytes. Specifically, the present inventors cultured human monocyte THP1 in vitro and differentiated into primary macrophages, induced the polarization of macrophages to M1 to THP1-M1 using LPS and IFN-gamma, and induced the polarization of macrophages to M2 to THP1-M2 using IL-4 and IL-13. Through the differentiation system of normal hematopoietic stem cells of bone marrow to megakaryocytes, the co-culture of the normal hematopoietic stem cells of bone marrow with THP1-M1 and THP1-M2 respectively shows that the normal CD34 of the bone marrow is compared with the co-culture of the THP1-M1+After the cells are co-cultured with THP1-M2, the megakaryocyte generation rate is increased, the megakaryocyte maturation rate, the ploidy formation rate and the colony forming capability are increased, and the platelet generation quantity is increased, which indicates that the supporting capability of the THP1-M2 on the bone marrow megakaryocyte maturation and the platelet generation is greater than that of the THP 1-M1.
After the in vitro experiment of the cell line, in order to further confirm the supporting effect of M2 on the megakaryocyte maturation, the inventor utilizes healthy human bone marrow mononuclear cells to induce and culture in vitro initial macrophages. Similarly, healthy human primary macrophages are induced by LPS and IFN- γ to BM-M1 and by IL-4 and IL-13 to BM-M2. BM-M1 and BM-M2 were associated with normal bone marrow CD34+Cell co-culture found that bone marrow was normal CD34 compared to BM-M1 co-culture+After the cells are co-cultured with BM-M2, the megakaryocyte generation rate is increased, the megakaryocyte maturation rate, the ploidy formation rate and the colony forming capability are increased, and the platelet generation quantity is increased. In conclusion, M2 has a significant supportive effect on megakaryocyte maturation and thrombopoiesis compared to M1.
The inventor also discovers that PI3K-AKT signal pathway and mTOR signal pathway downstream thereof are activated in macrophages of PT patient bone marrow and macrophages of healthy human bone marrow M2 type through transcriptome level sequencing. Specifically, the inventor carries out transcriptome level sequencing and prospective clinical pairing research to find that the expression levels of components such as macrophage PI3K-AKT signal pathway, vWF, VEGF and collagen of the bone marrow of a PT patient are obviously reduced compared with those of a patient implanted well; meanwhile, the downstream mTOR signaling pathway of PI3K-AKT in BM-M2 with a supporting effect on megakaryocytes is enhanced, which suggests that M2 improves the extracellular matrix of the bone marrow microenvironment through the PI3K-AKT signaling pathway to support megakaryocyte maturation and thrombopoiesis.
The inventor also finds that M2 plays a supporting role in megakaryocyte maturation and thrombopoiesis through a PI3K-AKT signal pathway through in vitro research. Specifically, the inventors of the present invention have determined whether the PI3K-AKT signaling pathway regulates M2 in megakaryocyte maturation and thrombopoiesis supporting effects, by treating THP1-M2 with the classical inhibitors of the PI3K-AKT signaling pathway LY294002 and MK-2206, respectively, and treating with inhibitor treated and untreated THP1-M2 with normal CD34, respectively+And (4) co-culturing the cells. Bone marrow Normal CD34 compared to untreated THP1-M2 group+After the cells are co-cultured with inhibitor-treated THP1-M2, the megakaryocyte production rate is reduced, the megakaryocyte maturation, ploidy formation rate and colony forming ability are reduced, and the platelet production quantity is reduced. The results of the study suggest that the PI3K-AKT signaling pathway plays a key role in the support of M2 for megakaryocyte maturation and thrombopoiesis.
Thus, based on the above findings, the present invention provides in a first aspect a pharmaceutical composition for treating a thrombocytopenic disease in a subject, the pharmaceutical composition comprising a macrophage polarization modulator selected from at least one agent selected from the group consisting of an agent for promoting the polarization of macrophages to M2 and an agent for inhibiting the polarization of macrophages to M1, and a pharmaceutically acceptable carrier.
In some preferred embodiments, the agent for promoting macrophage polarization to M2 and the agent for inhibiting macrophage polarization to M1 are independently selected from the group consisting of an agent that activates the STAT6/IRF4/PPAR signaling pathway and/or an agent that activates the PI3K/AKT signaling pathway; preferably, the agent for promoting macrophage polarization to M2 and the agent for inhibiting macrophage polarization to M1 are independently selected from the group consisting of IL-4, IL-13, and PI3K agonists.
In some preferred embodiments, the PI3K agonist is selected from the group consisting of vitamin D, all-trans retinoic acid, insulin-like growth factor-I, 740Y-P (CAS No.:1236188-16-1), 1-deoxysperamycin (1-Deoxynojirimycin, CAS No.:19130-96-2), 1,3-Dicaffeoylquinic acid (1,3-Dicaffeoylquinic acid, CAS No.:19870-46-3), YS-49(CAS No.:132836-42-1), Erucic acid (CAS No.:305834-79-1), Recilisib (CAS No.:334969-03-8), SC79(CAS No.:305834-79-1), and LM22B-10(CAS No.:342777-54-2), and the like.
The present invention provides in a second aspect the use of a modulator of macrophage polarization selected from at least one agent selected from the group consisting of an agent for promoting polarization of macrophages to M2 and an agent for inhibiting polarization of bone marrow macrophages to M1, in the manufacture of a medicament for treating a thrombocytopenic disease in a subject.
Based on the above-mentioned results of the studies by the present inventors, the drug of the present invention is particularly suitable for regulating bone marrow macrophages to promote megakaryocyte function or to promote thrombopoiesis, particularly megakaryocyte maturation disorder and/or thrombocytopenia occurring in patients with delayed platelet implantation (PT) after transplantation.
In some preferred embodiments, the macrophage is a bone marrow macrophage, i.e., a macrophage derived from bone marrow or referred to as a bone marrow-derived macrophage.
The medicament is particularly suitable for regulating bone marrow macrophages to promote megakaryocyte function or promote thrombocytopoiesis, and especially the megakaryocyte maturation disorder and/or thrombocytopenia occurring in patients with delayed implantation of platelets after transplantation. Thus, in some preferred embodiments, the thrombocytopenic disease is post-transplant delayed platelet implantation.
The invention provides in a third aspect a kit for diagnosing a thrombocytopenic disease in a subject, wherein the test kit comprises a test agent for detecting a marker of a thrombocytopenic disease and/or a test agent for detecting macrophage polarization level.
In some preferred embodiments, the marker is selected from the group consisting of a PI3K-AKT signaling pathway-enhancing marker, an mTOR signaling pathway-enhancing marker, vWF, VEGF, and a collagen component. In some further preferred embodiments, the detection reagent for detecting macrophage polarization level comprises a reagent for detecting M1 macrophage level and a reagent for detecting M2 macrophage level.
In some more preferred embodiments, the PI3K-AKT signaling pathway-enhancing marker is AKT, more preferably a phosphorylated AKT protein and/or AKT1 gene mRNA. It is further preferred that the mTOR signaling pathway enhancement marker is an mTOR gene.
The present invention provides in a fourth aspect the use of a detection reagent for detecting a marker of a thrombocytopenic disease and/or a detection reagent for detecting the level of macrophage polarization in the manufacture of a kit for diagnosing a thrombocytopenic disease in a subject.
In some preferred embodiments, the marker is selected from the group consisting of a PI3K-AKT signaling pathway-enhancing marker, an mTOR signaling pathway-enhancing marker, vWF, VEGF, and a collagen component.
In some further preferred embodiments, the detection reagent for detecting macrophage polarization level comprises a reagent for detecting M1 macrophage level and a reagent for detecting M2 macrophage level.
In some more preferred embodiments, the PI3K-AKT signaling pathway-enhancing marker is AKT, more preferably a phosphorylated AKT protein and/or AKT1 gene mRNA. It is also preferred that the mTOR signaling pathway-enhancing marker is an mTOR gene
In some preferred embodiments, the subject is a thrombocytopenic disease patient, particularly a post-transplant delayed platelet implantation patient. Preferably, the post-transplant delayed implantation of platelets into a patient is such that platelets remain below 20X 10 continuously after 60 days of transplantation9patients/L and/or who still rely on platelet transfusions 60 days after transplantation.
In some other preferred embodiments, the subject is a mammal, more preferably a human.
The present invention provides in a fifth aspect a pharmaceutical composition for treating a thrombocytopenic disease, in particular a post-transplant delayed platelet implantation, in a subject, the pharmaceutical composition comprising a monocyte differentiation modulator and a pharmaceutically acceptable carrier; the monocyte differentiation-regulating agent is at least one agent selected from the group consisting of an agent for promoting canonical monocyte production, an agent for inhibiting intermediate monocyte production, and an agent for inhibiting atypical monocyte production.
The present invention provides in a sixth aspect the use of a monocyte differentiation modulating agent selected from at least one agent selected from the group consisting of an agent for promoting canonical monocytogenes, an agent for inhibiting intermediate monocytogenes and an agent for inhibiting atypical monocytogenes in the manufacture of a medicament for treating a thrombocytopenic disease, in particular a delayed platelet implantation in a subject following transplantation.
The inventor firstly evaluates a mononuclear macrophage system in a PT patient bone marrow microenvironment, finds that abnormal polarization of bone marrow macrophages M1/M2 participates in megakaryocyte maturation disorder of the patient, and can promote maturation and differentiation of bone marrow megakaryocytes and thrombopoiesis by regulating and controlling polarization and functions of the bone marrow macrophages. The biological feature of abnormal polarization and altered function of bone marrow macrophages in PT patients after transplantation is that the excessive polarization of bone marrow macrophages to pro-inflammatory phenotype M1 and the decrease of polarization to M2 result in the significant increase of M1/M2. Through sequencing of a PT patient bone marrow macrophage transcriptome, the PI3K-AKT signal pathway plays an important role in promoting macrophages to polarize and secrete VEGF and vWF and the like to M2 and participating in a bone marrow megakaryocyte maturation supporting effect, and inhibition of the bone marrow macrophage PI3K-AKT signal pathway and remarkable reduction of VEGF and vWF in the macrophages are biomarkers of change of polarization imbalance functions of the bone marrow M1/M2. The polarization of macrophages to M2 is regulated by macrophage polarization regulators such as PI3K-AKT agonists and the like, the polarization imbalance and the dysfunction of M1/M2 in a bone marrow microenvironment are repaired, the support function of the macrophages to megakaryocytes is enhanced, and the maturation of the megakaryocytes and the generation of platelets are promoted.
The inventor finds that the bone marrow macrophages are over-polarized to the proinflammatory phenotype M1, and the decrease of polarization to M2 leads to the remarkable increase of M1/M2, so that the support effect on the bone marrow megakaryocytes is reduced, and the biological characteristics of abnormal polarization and function change of the bone marrow macrophages of the PT patients after transplantation are provided. The marked decrease of the bone marrow macrophage PI3K-AKT signal pathway and VEGF and vWF in macrophages is a biomarker of altered function of the M1/M2 polarization imbalance. Thus, the present method can be used, for example, for: (1) detecting macrophage polarization and functional states in a microenvironment of bone marrow of a PT patient after hematopoietic stem cell transplantation, and evaluating the immune component condition of the microenvironment of bone marrow megakaryocytes, wherein the result has important clinical significance for layered diagnosis and treatment of diseases; (2) on the basis of detecting and evaluating the bone marrow macrophages of patients, regulators for regulating the polarization of the bone marrow macrophages to M2 type through PI3K-AKT signal channel agonists and the like promote the bone marrow megakaryocyte maturation and the thrombocytopoiesis of thrombocytopenic diseases patients such as PT patients and the like, and have important significance for accurate targeted treatment of the diseases.
Drawings
FIG. 1 is a graph showing the ratios of myelomonocytic subpopulations measured in delayed platelet engraftment (PT) patients, in well-functioning implanted (GGF) patients and in Healthy Donors (HD).
FIG. 2 is a graph showing the ratio of bone marrow macrophage polarization subpopulations in patients with delayed platelet engraftment (PT), patients with good engraftment function (GGF) and Healthy Donors (HD).
FIG. 3 is a graph showing the measurement of bone marrow macrophage migration and phagocytosis in patients with delayed platelet engraftment (PT), patients with good engraftment (GGF) and Healthy Donors (HD).
FIG. 4 is a graph showing the effect of bone marrow macrophages on bone marrow megakaryocytes in patients with PT and patients with good engraftment function (GGF).
FIG. 5 shows that human monocyte THP1 system is established to culture THP1-M1 and THP1-M2 in vitro by polarization, and the effect of THP1-M1 and THP1-M2 on megakaryocyte maturation and thrombopoiesis after polarization is detected.
FIG. 6 shows the establishment of healthy human bone marrow mononuclear cell in vitro polarization culture BM-M1 and BM-M2 systems, and the examination of the effects of BM-M1 and BM-M2 on megakaryocyte maturation and thrombopoiesis after polarization.
FIG. 7 is a graph showing the results of analyzing bone marrow macrophage differential gene-enriching pathway of PT patients and GGF patients using GESA.
FIG. 8 is a graph showing the results of analyzing the BM-M1 and BM-M2 macrophage differential gene enrichment pathway using GESA.
FIG. 9 is a graph showing the results of analyzing bone marrow macrophage differential gene enrichment pathway of PT patients and GGF patients using KEGG.
FIG. 10 is a graph showing the difference genes in the PI3K-AKT pathway in bone marrow macrophages of PT patients and GGF patients.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are, however, a subset of the embodiments of the invention and not all embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Examples
The present invention will be further illustrated by way of examples, which are provided for purposes of illustration only and not for purposes of limitation, and the scope of the present invention is not limited to these examples.
Method and device
Detection of monocyte macrophage subpopulation ratio
Taking whole blood 5X 105-1×106One cell to the flow detector tube. Monocyte macrophage cell surface flow antibody (CD 145. mu.l, CD 165. mu.l, CD 1635. mu.l, CCR 25. mu.l, CD 685. mu.l, CX3CR 15. mu.l) was added to the whole blood and incubated at room temperature in the dark for 15 min. A10 Xhemolysin solution (purchased from BD company, erythrocyte lysate 349202) and sterile water for injection are taken to prepare a fresh 1 Xhemolysin solution according to a ratio of 1:9, 2ml of the 1 Xhemolysin solution is added into each tube, and the mixture is fully suspended and shaken on a shaker for 5 seconds. After standing in the dark at room temperature for 8 minutes, the mixture was centrifuged at 2000rpm for 5 minutes. Discarding the supernatant, adding 2ml PBS and mixing, centrifuging at 2000rpm for 5 minutes, discarding the supernatant, adding 200 μ l PBS and shaking and mixing, and detecting on the machine within 4 h. Streaming image splittingAnalysis Using Diva 7.0 software, CD14 was circled in the monocyte subpopulation++CD16-Classical monocyte, CD14++CD16+Intermediate monocytes and CD14+CD16+Non-canonical monocytes; at the same time, CD68 was circled in the monocyte subpopulation+CCR2+M1 type macrophages and CD168+CX3CR1+Macrophage M2 type.
Macrophage phagocytosis assay
The bone marrow mononuclear macrophage is cultured in vitro for 7 days to obtain the bone marrow macrophage. The medium was blotted dry, macrophages were attached to a well plate, 200. mu.l of PBS buffer (pH7.0) and 2. mu.l of diacetylated low density lipoprotein solution (DiI-AcLDL, Life Technologies, Gaithersburg, Md., USA) were slowly added and incubated in an incubator for 4 hours, the PBS buffer was added, washed for 3 minutes and 3 times in total, DAPI (4, 6-diamidine-2-phenylindole) was added and incubated at room temperature for 10 minutes, the PBS buffer was added, washed for 3 minutes and 3 times in total, and then counted by fluorescence microscope excitation with green and blue light.
Macrophage migration function assay
Take 5X 103The cells were cultured in 200. mu.l of liquid culture medium RPMI 1640 containing 10% Fetal Bovine Serum (FBS) in a Transwell chamber, and 500. mu.l of liquid culture medium RPMI 1640 containing 10% FBS in the lower chamber for 24 hours by a conventional method. And (3) sucking the culture medium of the upper chamber and the lower chamber, adding 200 mu l of formaldehyde into the upper chamber, fixing for 10 minutes at room temperature, adding 100 mu l of 0.1% crystal violet dye into the fixing solution of the upper chamber, incubating for 10 minutes at room temperature, sucking the dye solution, washing for 2 times by PBS (phosphate buffer solution), wiping surface cells by a cotton ball, and observing and counting under a microscope.
Co-culture of macrophages and bone marrow megakaryocytes
The supernatant was discarded from the macrophages cultured in vitro, washed once with PBS buffer, and 1X 10 cells were added to each well5A number of CD34+ cells and 1ml of StemBantm SFEM stem cell culture medium (containing 100ng/ml SCF (stem cell factor), 100ng/ml TPO (thrombopoietin) and 10ng/ml IL-3 (Interleukin-3), containing 5% CO2The culture was carried out in a 37 ℃ incubator for 7 days (half every 3 days)Change of solution), on day 7, 5X 10 of the solution was taken out4And (4) carrying out a megakaryocyte colony forming experiment on each cell, and continuously putting the rest cells into an incubator for co-culture. And taking out all the remaining cells on days 12-16 to perform flow detection experiments on the number, ploidy and thrombocytopoiesis of the megakaryocytes. Taking out megakaryocytes from 7 th day of co-culture, and preparing 5X 10 with PBS4Mu.l of single cell suspension, plates containing 5% CO at 37 ℃ according to MegaC. mu.lt-C kit instructions2The incubator is incubated for 10-12 days, and then dehydrated, fixed and subjected to immunocytochemical staining according to the instruction. And (4) observing and photographing under a common microscope, and counting to obtain the GPIIb/IIIa positive red colony, namely the megakaryocyte colony. Secondly, taking the cells with half the volume of the MKs (MK refers to megakaryocyte, and MKs refers to plural number) remained on the day 12-16 of co-culture by using a flow tube, dividing the cells into two tubes, marking as an MK tube and a negative tube, adding 1ml of PBS buffer solution into the two tubes, centrifuging for 300g multiplied by 5min, enabling the sediment to be the MK, enabling the supernatant to be the platelet, sucking the supernatant into a new flow tube, and marking as a platelet tube. MK tube: resuspending with 200. mu.l PBS, adding CD41a-FITC antibody, incubating at room temperature for 15min, adding 1ml PBS, centrifuging at 300g × 5min, and discarding the supernatant; adding 500 μ l 75% ethanol, incubating at 4 deg.C for 1h, washing with 1.5ml PBS for 2 times, centrifuging at 500g × 5min, discarding supernatant, adding 500 μ l PI dye, incubating at room temperature for 10min, and detecting on machine. Platelet tube: resuspend with 200. mu.l PBS, add CD41a-FITC antibody, incubate for 15min at room temperature, add 1ml PBS, centrifuge at 800 g.times.5 min and discard the supernatant, resuspend with 200. mu.l Binding buffer (ensuring the same volume of resuspension per tube), and check on the machine.
Second, result in
Example 1: detecting the ratio of each subgroup of bone marrow mononuclear cells of PT patients, good engraftment (GGF) patients and Healthy Donors (HD)
PT patients, GGF patients and HD bone marrow whole blood with similar clinical characteristics of the external hemogram such as age, sex, basic disease and the like after hematopoietic stem cell transplantation are collected, 200 mu l of marking mononuclear subpopulation surface markers are taken from a flow tube and flow detection is carried out, and a flow type typical graph is shown as a figure 1. A. The analysis of 6 cases of PT, GGF and HD by flow cytometry revealed that the bone marrow of PT patients had a reduced proportion of typical monocytes (FIG. 1.B) and an increased proportion of intermediate and atypical monocytes (FIG. 1.C, D) compared to those of GGF patients and HD.
Example 2: detecting the proportion of bone marrow macrophage polarized subpopulations in PT patients, well-implanted (GGF) patients and Healthy Donors (HD)
PT patients, GGF patients and HD bone marrow whole blood with similar clinical characteristics of the external hemogram such as age, sex, basic disease and the like after hematopoietic stem cell transplantation are collected, 200 mu l of the PT patients, the GGF patients and the HD bone marrow whole blood are marked with macrophage polarization subgroup surface markers in a flow tube and flow detection is carried out, and a flow type typical graph is shown as a figure 2. A. The polarization of macrophages in the bone marrow of PT patients is increased to M1 (figure 2.B), the polarization of macrophages is reduced to M2 (figure 2.C), and the polarization imbalance state of macrophages with the significantly increased M1/M2 is presented (figure 2. D).
Example 3: detecting phagocytosis and migration of bone marrow macrophages of PT patients and patients with good engraftment (GGF)
Collecting whole bone marrow of PT patients and GGF patients with similar clinical characteristics of the external hemogram such as age, sex, basic disease, etc. after hematopoietic stem cell transplantation, extracting bone marrow mononuclear cells and culturing into macrophages in vitro. First, in vitro phagocytosis and migration experiments were performed to assess whether there was a fundamental functional difference between two groups of macrophages. The experimental results showed that there was no significant difference between PT macrophages and GGF macrophages in migration function (fig. 3.a, B) and phagocytic function (fig. 3.C, D).
Example 4: detecting the effect of macrophages of bone marrow of patients with PT and patients with good engraftment (GGF) on bone marrow megakaryocytes
Further, to assess whether there is a difference in the effect of bone marrow macrophages on megakaryocytes in the two groups of patients, bone marrow mononuclear cells were extracted and cultured in vitro to form macrophages. Two groups of macrophages are respectively associated with normal bone marrow CD34+Cells were co-cultured and tested for CD34+The rate of differentiation and maturation of cells into megakaryocytes and the number of thrombocytes produced. The co-culture research shows that compared with GGF bone marrow macrophages, PT bone marrow macrophages have obviously reduced megakaryocyte supporting effect, and the specific expression is normal CD34+After co-culture of cells with PT bone marrow macrophages, the megakaryocyte production rate (FIG. 4.A) is reduced, the megakaryocyte maturation, ploidy formation rate (FIG. 4.B) and colony formation (FIG. 4.C, D) ability are reduced, and the platelet production amount (FIG. 4.E) is reducedLow.
Example 5: establishing human monocyte cell line THP1 in vitro polarization culture THP1-M1 and THP1-M2 systems, and detecting the effect of the polarized THP1-M1 and THP1-M2 on megakaryocyte maturation and thrombopoiesis
Establishing human monocyte THP1 in vitro and inducing to differentiate into initial macrophage, inducing macrophage to be polarized to M1 to be THP1-M1 by using LPS and IFN-gamma, and inducing macrophage to be polarized to M2 to be THP1-M2 by using IL-4 and IL-13. Through the differentiation system of normal hematopoietic stem cells of bone marrow to megakaryocytes, the co-culture of the normal hematopoietic stem cells of bone marrow with THP1-M1 and THP1-M2 respectively shows that the normal CD34 of the bone marrow is compared with the co-culture of the THP1-M1+The ratio of megakaryocytopoiesis (FIG. 5.A), the ratio of megakaryocytopoiesis, ploidy formation (FIG. 5.B) and colony formation ability (FIG. 5.C, D) and the number of thrombocytes (FIG. 5.E) were increased after co-culturing the cells with THP1-M2, indicating that the supporting ability of THP1-M2 for bone marrow megakaryocytopoiesis and thrombopoiesis was higher than that of THP 1-M1.
Example 6: establishing healthy human bone marrow mononuclear cell in-vitro polarization culture BM-M1 and BM-M2 system, and detecting the effect of the polarized BM-M1 and BM-M2 on megakaryocyte maturation and thrombopoiesis
After the in vitro experiment of the cell line, in order to further confirm the supporting effect of M2 on megakaryocyte maturation, an initial macrophage system is established by in vitro induction culture of healthy human bone marrow mononuclear cells. On this basis, healthy human primary macrophages are induced into BM-M1 by LPS and IFN-gamma and BM-M2 by IL-4 and IL-13. BM-M1 and BM-M2 were associated with normal bone marrow CD34+Cell co-culture found that bone marrow was normal CD34 compared to BM-M1 co-culture+After cocultivation of the cells with BM-M2, the megakaryocyte productivity was increased (FIG. 6.A), the megakaryocyte maturation/ploidy formation rate (FIG. 6.B) and colony formation ability (FIGS. 6.C, D) were increased, and the number of platelets produced (FIG. 6.E) was increased.
Example 7: transcriptome level sequencing finds that the activation of bone marrow macrophages and BM-M2 macrophages of GGF patients through PI3K-AKT signal pathway is an important mechanism for promoting the polarization of macrophages to M2 and improving the support effect of the macrophages on megakaryocytes
Transcriptome level sequencing was found comparing bone marrow macrophage differences between PT patients and GGF patients and BM-M1 and BM-M2 macrophages. Analysis of bone marrow macrophages and BM-M1 and BM-M2 macrophages in PT and GGF patients, respectively, using GESA revealed a significant reduction in the PI3K-AKT signaling pathway (fig. 7) of the bone marrow macrophages in PT patients compared to GGF patients; the M-TOR signaling pathway downstream of BM-M1 bone marrow macrophage PI3K-AKT (FIG. 8) was significantly reduced compared to BM-M2. Furthermore, the KEGG pathway enrichment assay found consistent results with a significant reduction in the bone marrow macrophage PI3K-AKT signaling pathway in PT patients compared to GGF patients (figure 9). Bone marrow macrophages of PT patients showed significantly reduced expression levels of vWF, VEGF and collagen components (fig. 10) compared to GGF patients. The bone marrow macrophages of GGF patients and BM-M2 macrophages activate a PI3K-AKT signal channel and a downstream M-TOR channel, which indicates that M2 improves microenvironment extracellular matrix through the PI3K-AKT signal channel to support megakaryocyte maturation and thrombopoiesis.
In conclusion, M2 has a significant supportive effect on megakaryocyte maturation and thrombopoiesis compared to M1.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (3)

1. Use of a macrophage polarization modulator in the preparation of a medicament for treating a myelomegakaryocyte maturation disorder in a subject that occurs in a patient with delayed post-transplant platelets, wherein delayed post-transplant platelets refers to stable engraftment of both leukocytes and hemoglobin +60 days post-transplant with complete donor chimerism and platelets that persist below 20 x 109/L or still dependent on platelet infusion; the macrophageThe polarization modulator is selected from the group consisting of IL-4, IL-13, and PI3K agonists; the PI3K agonist is selected from the group consisting of 1,3-dicaffeoylquinic acid (CAS No.:19870-46-3) and YS-49(CAS No.: 132836-42-1).
2. The use of claim 1, wherein the subject is a mammal.
3. The use of claim 2, wherein the subject is a human.
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