WO2018196566A1 - Vcam-1+单核细胞及其衍生细胞在促进造血干细胞归巢的应用 - Google Patents

Vcam-1+单核细胞及其衍生细胞在促进造血干细胞归巢的应用 Download PDF

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WO2018196566A1
WO2018196566A1 PCT/CN2018/081682 CN2018081682W WO2018196566A1 WO 2018196566 A1 WO2018196566 A1 WO 2018196566A1 CN 2018081682 W CN2018081682 W CN 2018081682W WO 2018196566 A1 WO2018196566 A1 WO 2018196566A1
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hematopoietic stem
cells
vcam
stem cells
monocytes
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PCT/CN2018/081682
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French (fr)
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潘巍峻
李丹彤
薛文志
李美
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中国科学院上海生命科学研究院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types

Definitions

  • the present invention relates to the field of biomedicine, and in particular to the use of VCAM-1 + monocytes and their derived cells for promoting hematopoietic stem cell homing.
  • the normal blood system plays an important role in the body, mainly involved in the transport of oxygen, nutrients and waste, and is also an important part of the immune system.
  • the establishment of the adult blood system relies on hematopoietic stem cells.
  • Long-range hematopoietic stem cells gradually differentiate into common myeloid precursor cells (CMP) and common lymphoid precursor cells (CLP), and finally differentiate into mature blood cells, including red blood cells, platelets, neutrophils, monocytes, and T lymphocytes.
  • CMP common myeloid precursor cells
  • CLP common lymphoid precursor cells
  • Hematopoietic stem cells have self-renewal, proliferation, differentiation, aging and other behaviors, and rely on the regulation of the microenvironment.
  • the bone marrow microenvironment of hematopoietic stem cells is very complex, including numerous cell types, such as vascular endothelial cells, perivascular cells, CAR cells, stromal cells, osteoblasts, osteoclasts, and some monocytes.
  • the bone marrow microenvironment participates in the regulation of the fate of hematopoietic stem cells by secreting cytokines or interacting with hematopoietic stem cells.
  • the diseases that require hematopoietic stem cell transplantation mainly include three types: malignant blood diseases (commonly such as leukemia, anemia, etc.), non-hematological diseases such as autoimmune diseases (such as systemic lupus erythematosus), and solid tumors (such as Hodgkin's lymph). tumor).
  • cord blood has a greater advantage: on the one hand, the cord blood source is more extensive; on the other hand, the cord blood acquisition process has almost no effect on the donor compared to the side effects of bone marrow and peripheral blood acquisition. .
  • the next critical step after entering the donor is the process by which hematopoietic stem cells homing to the bone marrow microenvironment.
  • the homing of hematopoietic stem cells is a prerequisite for hematopoietic stem cells to rebuild the blood system.
  • Homing is a complex multi-step process. It is affected by many factors. By regulating the homing process, accelerating the hematopoietic stem cell re-building blood system speed and shortening the anemia period, it can effectively improve the graft survival rate. Therefore, if effective cells or molecules can be found in the process of homing, the utilization rate of hematopoietic stem cells will be greatly increased, and the problem of insufficient sources of hematopoietic stem cells will be solved to some extent.
  • a first aspect of the invention provides the use of a VCAM-1 + monocyte and a cell derived therefrom, or a promoter thereof, for the preparation of a composition or formulation for promoting hematopoietic stem cell homing.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and (a) VCAM-1 + monocytes and derived cells thereof.
  • the composition is a liquid composition.
  • the concentration of the VCAM-1 + monocytes and derived cells thereof is 1.0 ⁇ 10 3 - 1.0 ⁇ 10 9 /ml, preferably 1.0 ⁇ 10 4 . - 1.0 ⁇ 10 8 / ml, more preferably 1.0 ⁇ 10 5 - 1.0 ⁇ 10 7 / ml.
  • composition further comprises (b) hematopoietic stem cells.
  • the concentration of the hematopoietic stem cells in the composition is 1.0 ⁇ 10 3 -1.0 ⁇ 10 10 /ml, preferably 1.0 ⁇ 10 4 -1.0 ⁇ 10 9 /ml, More preferably, it is 1.0 ⁇ 10 5 - 1.0 ⁇ 10 8 /ml.
  • the composition further comprises an additional component selected from the group consisting of prostaglandin (PGE2), hyaluronic acid (HA), antimicrobial peptide LL-37, or a combination thereof.
  • PGE2 prostaglandin
  • HA hyaluronic acid
  • antimicrobial peptide LL-37 or a combination thereof.
  • the carrier is selected from the group consisting of an infusion medium carrier and/or an injection vehicle carrier.
  • the carrier is one or more carriers selected from the group consisting of physiological saline, glucose. Saline, or a combination thereof.
  • VCAM-1 + cells and monocyte-derived cells expressing surface markers selected from the group of VCAM-1 + cells and monocyte-derived cells: mfap4, mpeg1, or combinations thereof.
  • the VCAM-1 + monocytes and their derived cells are not endothelial cells.
  • the VCAM-1 + monocytes and their derived cells are not stromal cells.
  • the promoter is a substance that promotes expression of VCAM-1 protein by VCAM-1 + monocytes and their derived cells.
  • the component (a) is from 0.01 to 90% by weight, preferably from 0.1 to 60% by weight, more preferably from 1 to 40% by weight, based on the total weight of the composition.
  • the composition is a liquid or semi-solid.
  • the composition is in a unit dosage form, and the unit dosage form has a volume of from 1 to 500 ml, preferably from 2 to 200 ml, more preferably from 5 to 100 ml.
  • the composition is an injectable preparation.
  • the promoting hematopoietic stem cell homing comprises: promoting the retention of hematopoietic stem cells in the hematopoietic microenvironment, increasing the residence time of hematopoietic stem cells in the hematopoietic microenvironment, and/or increasing hematopoietic stem cells that remain in the microenvironment for a long time. proportion.
  • the promoting the retention of hematopoietic stem cells in the hematopoietic microenvironment means: the ratio of the number S1 of hematopoietic stem cells that stay in the hematopoietic microenvironment to the number S1 of hematopoietic stem cells that stay in the hematopoietic microenvironment in the control group. (S1/S0) ⁇ 150%, preferably ⁇ 200%, more preferably ⁇ 250% (e.g., 150-500%).
  • the hematopoietic stem cell growth is promoted such that about 50% or more of the hematopoietic stem cells stay in the hematopoietic microenvironment.
  • the increase in the residence time of the hematopoietic stem cells in the hematopoietic microenvironment refers to the ratio of the time T1 at which the hematopoietic stem cells stay in the hematopoietic microenvironment to the time T0 at which the hematopoietic stem cells stay in the hematopoietic microenvironment in the control group (T1/ T0) ⁇ 150%, preferably ⁇ 200%, more preferably ⁇ 350% (e.g., 150% - 400%).
  • the increase of the residence time of the hematopoietic stem cells in the hematopoietic microenvironment means that the residence time of the hematopoietic stem cells in the hematopoietic microenvironment is ⁇ 6 minutes, preferably ⁇ 30 minutes, optimally ⁇ 120 minutes (such as 5 -240 minutes, 10-240 minutes, 30-200 minutes)
  • the hematopoietic stem cells can remain in the hematopoietic microenvironment for 6-30 min, preferably 30-120 min, and more preferably >120 min.
  • the increase in the proportion of hematopoietic stem cells that remain in the microenvironment for a long time means that the number W1 of hematopoietic stem cells having a residence time of ⁇ 120 min in the hematopoietic microenvironment stays in the hematopoietic microenvironment under the same conditions as in the control group.
  • the proportion of hematopoietic stem cells that remain in the microenvironment for a prolonged period (more than 30 minutes) is increased from 2% to 20% compared to the control group.
  • composition or formulation may be used alone or in combination in the application to promote hematopoietic stem cell homing.
  • the combined use comprises: in combination with other drugs that promote hematopoietic stem cell homing.
  • the other drug that promotes hematopoietic stem cell homing is selected from the group consisting of prostaglandin (PGE2), hyaluronic acid (HA), antimicrobial peptide LL-37, or a combination thereof.
  • PGE2 prostaglandin
  • HA hyaluronic acid
  • antimicrobial peptide LL-37 or a combination thereof.
  • VCAM-1 + monocytes and their derived cells have one or more characteristics selected from the group consisting of:
  • VCAM-1 + monocytes and their derived cells have a surface antigen mfap4, and/or mpeg1;
  • VCAM-1 + monocytes and their derived cells are nearly circular in shape and have few tentacles.
  • a second aspect of the invention provides a cellular reagent comprising:
  • a first pharmaceutical composition comprising (a) a first active ingredient, said first active ingredient being VCAM-1 + monocytes and derived cells thereof, and pharmaceutically acceptable Carrier
  • a second pharmaceutical composition comprising (b) a second active ingredient, said second active ingredient being a hematopoietic stem cell; and a pharmaceutically acceptable carrier;
  • first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.
  • the cellular reagent is a liquid reagent.
  • the cells in the cellular reagent are substantially ( ⁇ 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) or all of (a) VCAM -1 + monocytes and their derived cells and (b) hematopoietic stem cells.
  • the ratio of the number of cells of the component (a) to the component (b) is from 10,000:1 to 1:10000, preferably from 1000:1 to 1:1000, more preferably, 100:1-1:100, or 10:1-1:10 or 1:10-1:10000.
  • the concentration of the VCAM-1 + monocytes and derived cells thereof is 1.0 ⁇ 10 3 - 1.0 ⁇ 10 9 /ml, preferably 1.0 ⁇ 10 4 . - 1.0 ⁇ 10 8 / ml, more preferably 1.0 ⁇ 10 5 - 1.0 ⁇ 10 7 / ml.
  • the concentration of the hematopoietic stem cells in the composition is 1.0 ⁇ 10 3 -1.0 ⁇ 10 10 /ml, preferably 1.0 ⁇ 10 4 -1.0 ⁇ 10 9 /ml, More preferably, it is 1.0 ⁇ 10 5 - 1.0 ⁇ 10 8 /ml.
  • the carrier is selected from the group consisting of an infusion medium carrier and/or an injection vehicle carrier.
  • the carrier is one or more carriers selected from the group consisting of physiological saline, glucose. Saline, or a combination thereof.
  • VCAM-1 + cells and monocyte-derived cells expressing surface markers selected from the group of VCAM-1 + cells and monocyte-derived cells: mfap4, mpeg1, or combinations thereof.
  • the VCAM-1 + monocytes and their derived cells are not endothelial cells.
  • the VCAM-1 + monocytes and their derived cells are not stromal cells.
  • the cellular agent further includes other drugs or promoters that promote homing of hematopoietic stem cells.
  • the promoter promotes expression of the VCAM-1 protein by VCAM-1 + monocytes and their derived cells.
  • the other drug or promoter that promotes homing of hematopoietic stem cells is selected from the group consisting of prostaglandin (PGE2), hyaluronic acid (HA), antimicrobial peptide LL-37, or a combination thereof.
  • PGE2 prostaglandin
  • HA hyaluronic acid
  • antimicrobial peptide LL-37 antimicrobial peptide LL-37
  • a third aspect of the invention provides a kit comprising:
  • first container and the second container may be the same and may be different.
  • a fourth aspect of the invention provides a method of promoting hematopoietic stem cell homing, comprising:
  • VCAM-1 + monocytes and their derived cells are contacted with hematopoietic stem cells to promote homing of the hematopoietic stem cells.
  • the method is non-therapeutic and non-diagnostic.
  • the method is therapeutic.
  • a fifth aspect of the invention provides a method for promoting hematopoietic stem cell migration in vitro, comprising:
  • VCAM-1 + monocytes and their derived cells are contacted with hematopoietic stem cells under suitable culture conditions in vitro, and the VCAM-1 + monocytes and their derived cells are combined with the hematopoietic stem cells, and the said Hematopoietic stem cell migration.
  • a sixth aspect of the invention provides a method of determining a potential therapeutic agent that promotes homing of hematopoietic stem cells, comprising:
  • VCAM-1 + monocytes and their derived cells in the test group clearly bind and move with the hematopoietic stem cells compared with the control group, it indicates whether the test compound is a potential therapeutic agent for promoting hematopoietic stem cell homing.
  • the method is non-therapeutic and non-diagnostic.
  • the method further comprises the step (iii) of administering the potential therapeutic agent identified in step (ii) to the mammal to observe VCAM-1 + monocytes in the mammal and The combination of derived cells and hematopoietic stem cells and the movement after binding.
  • the mammal comprises a human or a non-human mammal.
  • the non-human mammal comprises a rodent, a primate, preferably a mouse, a rat, a rabbit, a monkey.
  • a seventh aspect of the invention provides a method for promoting hematopoietic stem cell homing, comprising the steps of:
  • VCAM-1 + monocytes and their derived cells are administered to the subject in need, before, during, or after administration of the hematopoietic stem cells, thereby promoting homing of the hematopoietic stem cells.
  • the subject comprises a human or a non-human mammal.
  • the preceding and following time intervals are ⁇ 2 days, preferably ⁇ 1 day, more preferably ⁇ 6 hours.
  • VCAM-1 + monocytes and their derived cells and the hematopoietic stem cells are administered simultaneously.
  • Figure 1 shows a marked reduction in the homing of hematopoietic stem cells in the VCAM-1 mutant, in which, in the VCAM-1 mutant, A, the number of hematopoietic stem cell homing (arrows) is reduced; B, hematopoietic stem cells are long in the hematopoietic microenvironment The proportion of cells that stayed at the time was significantly reduced.
  • FIG. 2 shows that VCAM-1 + cells help hematopoietic stem cell homing, where A, in the immunofluorescence results, VCAM-1 + cells (triangles) are adjacent to the homing hematopoietic stem cells (arrows); B, we established VCAM-1 direct-labeled antibody-specific labeling of VCAM-1 + cells in vivo observation system; triangles indicate specifically labeled VCAM-1 + cells, asterisks indicate autofluorescence pigments; C, in long-term observation, VCAM-1 + Cells (triangles) and hematopoietic stem cells (arrows) undergo long-term intercellular interactions in the hematopoietic microenvironment.
  • VCAM-1 + cells promote hematopoietic stem cells to be coated by vascular endothelial cells, and hematopoietic stem cells stay in the hematopoietic microenvironment for a long time. Homing.
  • the right side shows the process by which hematopoietic stem cells are gradually coated by endothelial cells.
  • FIG. 3 shows that VCAM-1 + cells are a subset of monocytes and their derived cells.
  • A micro-cell sequencing results show that VCAM-1 + cells overexpress the molecular markers mfap4 of monocytes and their derived cells.
  • B immunofluorescence results show that VCAM-1 + cells co-localize with some monocytes and their derived cells, triangles indicate co-localization, and arrows indicate non-VCAM-1 + monocytes and their derived cells ;
  • C VCAM-1 direct-labeled antibody in vivo labeled monocytes and their derived cells.
  • Figure 4 shows that removal of monocyte-macrophages resulted in a significant reduction in hematopoietic stem cell homing, in which A, hematopoietic stem cells (arrows) were returned after removal of mononuclear-macrophages using the clomarosome coated with clodronate.
  • A hematopoietic stem cells (arrows) were returned after removal of mononuclear-macrophages using the clomarosome coated with clodronate.
  • the nest to the hematopoietic microenvironment is significantly reduced;
  • B the proportion of hematopoietic stem cells that stay in the microenvironment for a long time is significantly reduced.
  • VCAM-1 + monocytes and their derived cells have an extremely excellent effect of promoting hematopoietic stem cell homing.
  • VCAM-1 + monocytes and their derived cells can significantly promote hematopoietic stem cell retention, increase the residence time of hematopoietic stem cells in the hematopoietic microenvironment, and increase the proportion of hematopoietic stem cells that remain in the microenvironment for a long time.
  • the inventors completed the present invention.
  • VCAM-1 + monocytes and their derived cells are mononuclear cells expressing VCAM-1 protein and their derived cells in hematopoietic tissues, which can detect the specificity of VCAM-1 + monocytes and their derived cells.
  • the marker is identified and is selected from the group consisting of mfap4, mpeg1 or a combination thereof.
  • VCAM-1 + cells mpeg1 + cells. 94% of VCAM-1 + cells are round and the remaining small part is dendritic. In the circular monocytes and their subpopulations, the VCAM-1 + group only accounted for 61.8%. Therefore, VCAM-1 + cells can be considered to be a subset of monocytes and their derived cells.
  • Hematopoietic stem cells have the ability to self-renew, proliferate, and differentiate.
  • Long-range hematopoietic stem cells gradually differentiate into common myeloid precursor cells (CMP) and common lymphoid precursor cells (CLP), and finally differentiate into mature blood cells, including red blood cells, platelets, neutrophils, monocytes, and T lymphocytes.
  • CMP common myeloid precursor cells
  • CLP common lymphoid precursor cells
  • Homing of hematopoietic stem cells refers to the process by which hematopoietic stem cells in peripheral blood return to the bone marrow microenvironment, which is the prerequisite for hematopoietic stem cells to rebuild the blood system.
  • the patient During the process of hematopoietic stem cell transplantation, the patient has to undergo an anemia period accompanied by leukopenia and thrombocytopenia, which means that high incidence of infection and bleeding at this stage is a serious threat to the patient's life and is a critical period for the success of hematopoietic stem cell transplantation.
  • Homing is a complex multi-step process. It is affected by many factors.
  • homing is crucial for the reconstruction of the hematopoietic system of donor hematopoietic stem cells after transplantation. The higher the homing efficiency, the higher the survival rate after transplantation.
  • composition (cell reagent)
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of VCAM-1 + monocytes and derived cells thereof; hematopoietic stem cells, and a pharmaceutically acceptable carrier.
  • VCAM-1 + monocytes and their derived cells and hematopoietic stem cells can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, wherein the pH is typically about 5-8. Preferably, the pH is about 7-8.
  • the term "effective amount” or “effective amount” refers to an amount that can produce a function or activity on a human and/or animal and that can be accepted by a human and/or animal.
  • the effective amount is: 1.0 x 10 3 - 1.0 x 10 9 / ml, preferably 1.0 x 10 4 - 1.0 x 10 8 / ml, more preferably 1.0 x 10 5 - 1.0 ⁇ 10 7 / ml.
  • the effective amount of cells is injected in one shot.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including.
  • the pharmaceutically acceptable carrier which can be used is not particularly limited and may be one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must have sufficient Purity and low enough toxicity.
  • compatibil it is meant herein that the components of the composition are capable of intermingling with the adipose mesenchymal progenitor cells of the invention without significantly reducing the therapeutic effect thereof.
  • Examples of pharmaceutically acceptable carrier parts of the invention are physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
  • optimized vectors can also be designed based on the nature of adipose mesenchymal progenitor cells.
  • the carrier is preferably an infusion carrier and/or an injection carrier.
  • the pharmaceutical compositions of the present invention comprise a safe and effective amount of VCAM-1 + monocytes and their derived cells and hematopoietic stem cells, and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.
  • the effective amount of the VCAM-1 + monocytes and their derived cells and hematopoietic stem cells of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like.
  • the selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the condition to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.
  • the pharmaceutical composition of the present invention is preferably an intravenous injection.
  • the concentration of the VCAM-1 + monocytes and the cells derived therefrom is 1.0 ⁇ 10 3 - 1.0 ⁇ 10 9 /ml, preferably 1.0 ⁇ 10 4 - in the intravenous injection reagent.
  • 1.0 ⁇ 10 8 /ml more preferably 1.0 ⁇ 10 5 - 1.0 ⁇ 10 7 /ml
  • the concentration of the hematopoietic stem cells is 1.0 ⁇ 10 3 - 1.0 ⁇ 10 10 /ml, preferably 1.0 ⁇ 10 4 - 1.0 ⁇ 10 9 / ml, more preferably 1.0 ⁇ 10 5 - 1.0 ⁇ 10 8 / ml.
  • the injection method of the pharmaceutical composition is not particularly limited, and may be a single injection preparation or a combination of preparations for multiple injections. In a preferred embodiment of the invention, the pharmaceutical composition is a single injection.
  • the pharmaceutical composition is preferably an intravenous preparation.
  • VCAM-1 + monocytes and their derived cells are contacted with hematopoietic stem cells to promote homing of the hematopoietic stem cells.
  • the method for promoting hematopoietic stem cell homing comprises the steps of:
  • VCAM-1 + monocytes and their derived cells are administered to the subject in need, before, during, or after administration of the hematopoietic stem cells, thereby promoting homing of the hematopoietic stem cells.
  • a method of determining a potential therapeutic agent that promotes homing of hematopoietic stem cells comprising:
  • VCAM-1 + monocytes and their derived cells in the test group clearly bind and move with the hematopoietic stem cells compared with the control group, it indicates whether the test compound is a potential therapeutic agent for promoting hematopoietic stem cell homing.
  • VCAM-1 + monocytes and their derived cells can significantly promote hematopoietic stem cell homing in a hematopoietic microenvironment.
  • the present invention provides, for the first time, a cellular reagent or pharmaceutical composition for significantly promoting hematopoietic stem cell homing.
  • the present invention provides for the first time a cellular reagent or pharmaceutical composition having the potential to promote hematopoietic stem cell mobilization.
  • Hematopoietic stem cells and VCAM-1 + cells were obtained from zebrafish and labeled by different means. Hematopoietic stem cells are labeled by the zebrafish transgenic line Tg (kdrl:dendra2) in vivo labeling system from directed hematopoiesis; VCAM-1 + cells are labeled by anti-VCAM-1 direct labeling antibody.
  • the clodrosome coated with clodronate was purchased from Encapsula NanoSciences (SKU: 8909).
  • Example 1 Vcam-1 mutants were observed in vivo to observe abnormal homing of hematopoietic stem cells (HSC)
  • the transgenic line Tg (kdrl:dendra2) (obtained from the Luo Lingfei Laboratory of Southwest Jiaotong University) drives the expression of the light converting protein Dendra2 by a vascular endothelial promoter.
  • 32hpf is the peak period of hematopoietic stem cell sprouting in the zebrafish aorta-gonad-medullary region.
  • the zebrafish induced hematopoiesis by the confocal microscope (Olympus FV1000) with ultraviolet light (wavelength 405nm) to produce regional aorta-gonad-medium In the region (AGM), the blood vessels are irradiated for 30 s, and the blood vessels can be changed from green to red, and endogenously labeled with hematopoietic stem cells.
  • Example 2 In vivo labeling of VCAM-1 + cells and observing how they promote hematopoietic stem cell homing
  • 1nl Anti-VCAM-1 direct-labeled antibody (wavelength 647 fluorophore-labeled protein kit, Thermo fisher A30009) or 1nl IgG fluorescent secondary antibody was injected from the main cardiac main vein into the wild-type zebrafish embryo (Fig. 2B).
  • Anti-VCAM-1 direct-labeled antibodies can label cells, while IgG fluorescent secondary antibodies are not specifically labeled. If the same method was used to inject 1 nl of Anti-VCAM-1 direct-labeled antibody into the vcam-1 mutant, there were no labeled cells (Fig. 2B). Therefore, the Anti-VCAM-1 direct-labeled antibody can specifically label VCAM-1 + cells in vivo (Fig. 2B).
  • VCAM-1 + cells promote hematopoietic stem cell homing. It has been observed that VCAM-1+ cells interact with hematopoietic stem cells, or push hematopoietic stem cells into small blood vessels, or make the interaction between hematopoietic stem cells and vascular endothelial cells closer (Fig. 2C), prolonging the hematopoietic stem cell residence time. Can stay for more than 30 minutes or even more than 2 hours.
  • VCAM-1 + cells are a subset of monocytes and their derived cells
  • VCAM-1 + cells were labeled in vivo by direct labeling antibodies (wavelength 647) as described in the methods of the above examples.
  • the zebrafish were digested at 50-52 hpf, and VCAM-1 + cells (wavelength 647) were enriched by flow sorting (Beckman Moflo Astrios), and microRNA sequencing was performed to detect RNA expression levels. Comparing the whole tail sample and the mpeg1 + labeled monocytes and their derived cell samples of the tail hematopoietic tissue, it was found that the marker mfap4 of monocytes and their derived cells was higher in VCAM-1 + cells than in the whole tail sample. Expression was similar to that of mpeg1 + labeled monocytes and their derived cell samples of tail hematopoietic tissue (Fig. 3A).
  • Tg monocytes and their derived cells are labeled as green.
  • VCAM-1 + cells were identified using anti-VCAM-1 rabbit antibody (obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) (Fig. 3B).
  • VCAM-1 + cells can be considered to be a subset of monocytes and their derived cells.

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Abstract

本发明提供了VCAM-1 +单核细胞及其衍生细胞在促进造血干细胞归巢的应用，具体地，本发明提供了促进造血干细胞归巢的组合物或制剂，本发明还提供了确定促进造血干细胞归巢的潜在治疗剂的方法。

Description

VCAM-1+单核细胞及其衍生细胞在促进造血干细胞归巢的应用 技术领域
本发明涉及生物医药领域，具体地，涉及VCAM-1 +单核细胞及其衍生细胞在促进造血干细胞归巢的应用。
背景技术
正常的血液系统在机体中承担重要功能，主要参与氧气运输、养分和废物的转运，同时也是免疫系统的重要组成部分。成体血液系统的建立依赖于造血干细胞。长程造血干细胞逐步分化为共同髓系前体细胞(CMP)和共同淋系前体细胞(CLP)，最后分化形成成熟的血液细胞，包括红细胞、血小板、中性粒细胞、单核细胞、T淋巴细胞和B淋巴细胞，进而构成完整有效的血液系统。
造血干细胞具有自我更新、增殖、分化、衰老等行为，并且依赖于微环境的调控。造血干细胞的骨髓微环境十分复杂，包括众多的细胞种类，如血管内皮细胞、血管周细胞、CAR细胞、基质细胞、成骨细胞、破骨细胞，其中还有一部分单核细胞。骨髓微环境通过分泌细胞因子或与造血干细胞之间产生互作，参与造血干细胞的命运调控。
当造血干细胞自身或所处的微环境出现异常时，往往会导致严重的血液疾病，影响病人身体健康。在这种情况下，病人需要进行清髓处理，外源输入健康的造血干细胞，帮助自身重建正常的血液系统，即为造血干细胞移植。目前，需要造血干细胞移植的疾病主要包括三类：恶性血液疾病(常见如白血病、贫血等)，非血液系统疾病如自身免疫性疾病(如系统性红斑狼疮)、实体瘤(如霍奇金淋巴瘤)。
近几十年来，造血干细胞移植的数目逐年增加。然而，造血干细胞的来源仍然十分有限，目前主要有三个来源：骨髓来源、外周血来源、脐带血来源。骨髓移植的供体有限，在找不到合适配型的情况下难以开展。而外周血来源需要的动员剂目前应用较多的主要有两种，G-CSF和AMD3100，并且动员得到的造血干细胞数目有限。相对于以上两种来源，脐带血具有更大的优势：一方面，脐带血来源更加广泛；另一方面，相比骨髓和外周血获取产生的副作用，脐带血获取过程几乎对供体没有任何影响。然而，目前脐带血移植仍存在亟待解决的问题，即一份脐带血所含的造血干细胞数量不足以满足一个成人的移植要 求，而同时使用两份脐带血时则面临不相容的问题。此外，体外扩增虽然具有比较大的潜力，但在体外扩增的情况下，造血干细胞容易出现干性消失的情况，降低移植效率。
对于造血干细胞移植，输入供体之后的下一个关键步骤就是造血干细胞归巢到骨髓微环境的过程。造血干细胞归巢是造血干细胞重建造血系统的前提。归巢是一个复杂的多步骤过程，受到很多因素的影响，通过调控归巢过程，加快造血干细胞重建造血系统速度，缩短贫血期，可以有效提高移植存活率。因此，如果在归巢的过程中能够找到有效的细胞或分子，将会大大增加造血干细胞的利用率，在一定程度上解决造血干细胞来源不足的问题。
因此，本领域迫切需要开发一种促进造血干细胞归巢的有效的细胞或分子。
发明内容
本发明的目的在于提供一种促进造血干细胞归巢的有效的细胞或分子。
本发明第一方面提供了一种VCAM-1 +单核细胞及其衍生细胞或其促进剂的用途，用于制备一组合物或制剂，所述组合物或制剂用于促进造血干细胞归巢。
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述的药物组合物含有药学上可接受的载体以及(a)VCAM-1 +单核细胞及其衍生细胞。
在另一优选例中，所述的组合物为液态组合物。
在另一优选例中，所述的组合物中，所述VCAM-1 +单核细胞及其衍生细胞的浓度为1.0×10 3-1.0×10 9个/ml，较佳地1.0×10 4-1.0×10 8个/ml，更佳地1.0×10 5-1.0×10 7个/ml。
在另一优选例中，所述的组合物还含有(b)造血干细胞。
在另一优选例中，所述的组合物中，所述造血干细胞的浓度为1.0×10 3-1.0×10 10个/ml，较佳地1.0×10 4-1.0×10 9个/ml，更佳地1.0×10 5-1.0×10 8个/ml。
在另一优选例中，所述的组合物还包括选自下组的额外组分：前列腺素(PGE2)、透明质酸(HA)、抗菌肽LL-37、或其组合。
在另一优选例中，所述的载体选自下组：输液剂载体和/或注射剂载体，较佳地，所述的载体是选自下组的一种或多种载体：生理盐水、葡萄糖盐水、或其组合。
在另一优选例中，所述VCAM-1 +单核细胞及其衍生细胞为表达选自下组的表 面标记的VCAM-1 +单核细胞及其衍生细胞：mfap4、mpeg1、或其组合。
在另一优选例中，所述VCAM-1 +单核细胞及其衍生细胞不是内皮细胞。
在另一优选例中，所述VCAM-1 +单核细胞及其衍生细胞不是基质细胞。
在另一优选例中，所述促进剂为促进VCAM-1 +单核细胞及其衍生细胞表达VCAM-1蛋白的物质。
在另一优选例中，所述组分(a)占所述组合物总重量的0.01-90wt％,较佳地0.1-60wt％，更佳地1-40wt％。
在另一优选例中，所述组合物为液态或半固体。
在另一优选例中，所述组合物为单元剂型，且所述单元剂型的体积为1-500ml，较佳地，2-200ml，更佳地5-100ml。
在另一优选例中，所述组合物为注射制剂。
在另一优选例中，所述促进造血干细胞归巢包括：促进造血干细胞在造血微环境的停留、增加造血干细胞在造血微环境的停留时间、和/或增加长时间停留在微环境的造血干细胞的比例。
在另一优选例中，所述促进造血干细胞在造血微环境的停留指：在造血微环境发生停留的造血干细胞的数量S1与对照组中在造血微环境发生停留的造血干细胞的数量S0的比值(S1/S0)≥150％，较佳地≥200％，更佳地≥250％(如150-500％)。
在一个实施例中，当在对照组仅有20％造血干细胞在造血微环境发生停留时，促进造血干细胞停留使得约50％或更多的造血干细胞在造血微环境发生停留。
在另一优选例中，所述增加造血干细胞在造血微环境的停留时间指：造血干细胞在造血微环境停留的时间T1与对照组中造血干细胞在造血微环境停留的时间T0的比值(T1/T0)≥150％，较佳地，≥200％，更佳地，≥350％(如150％-400％)。
在另一优选例中，所述的增加造血干细胞在造血微环境的停留时间指造血干细胞在造血微环境的停留时间≥6分钟，较佳地≥30分钟，最佳地≥120分钟(如5-240分钟，10-240分钟，30-200分钟)
在一个实施例中，造血干细胞在造血微环境可停留6-30min，较佳地，30-120min，更佳地，＞120min。
在另一优选例中，所述增加长时间停留在微环境的造血干细胞的比例指：在造血微环境停留时间≥120min的造血干细胞的数量W1与对照组中同等条件下在造血微环境发生停留的造血干细胞的数量W0的比值(W1/W0)≥200％，较佳地≥500％，更佳地≥1000％(如200-3000％)。
在一个实施例中，与对照组相比，长时间(超过30min)停留在微环境的造血干细胞的比例由2％增加到20％。
在另一优选例中，所述组合物或制剂在促进造血干细胞归巢的应用中，可单独使用，或联合使用。
在另一优选例中，所述的联合使用包括：与其它促进造血干细胞归巢的药物联合使用。
在另一优选例中，所述其它促进造血干细胞归巢的药物选自下组：前列腺素(PGE2)、透明质酸(HA)、抗菌肽LL-37、或其组合。
在另一优选例中，所述VCAM-1 +单核细胞及其衍生细胞具有选自下组的一种或多种特征：
(i)VCAM-1 +单核细胞及其衍生细胞具有表面抗原mfap4、和/或mpeg1；
(ii)VCAM-1 +单核细胞及其衍生细胞在形态上接近圆形，触手少。
本发明第二方面提供了一种细胞试剂，包括：
(i)第一药物组合物，所述第一药物组合物含有(a)第一活性成分，所述第一活性成分为VCAM-1 +单核细胞及其衍生细胞，以及药学上可接受的载体；
(ii)第二药物组合物，所述第二药物组合物含有(b)第二活性成分，所述第二活性成分为造血干细胞；以及药学上可接受的载体；
其中，所述的第一药物组合物和第二药物组合物为不同的药物组合物，或同一药物组合物。
在另一优选例中，所述细胞试剂为液体试剂。
在另一优选例中，所述的细胞试剂中的细胞基本上(≥90％、95％、96％、97％、98％、99％、99.5％、99.9％)或全部由(a)VCAM-1 +单核细胞及其衍生细胞和(b)造血干细胞构成。
在另一优选例中，所述组分(a)与组分(b)的细胞数量之比比为10000：1-1:10000，较佳地，1000：1-1:1000，更佳地，100：1-1:100，或10：1-1:10或1:10－1：10000。
在另一优选例中，所述的组合物中，所述VCAM-1 +单核细胞及其衍生细胞的浓度为1.0×10 3-1.0×10 9个/ml，较佳地1.0×10 4-1.0×10 8个/ml，更佳地1.0×10 5-1.0×10 7个/ml。
在另一优选例中，所述的组合物中，所述造血干细胞的浓度为1.0×10 3-1.0×10 10个/ml，较佳地1.0×10 4-1.0×10 9个/ml，更佳地1.0×10 5-1.0×10 8个/ml。
在另一优选例中，所述的载体选自下组：输液剂载体和/或注射剂载体，较佳地，所述的载体是选自下组的一种或多种载体：生理盐水、葡萄糖盐水、或其组合。
在另一优选例中，所述VCAM-1 +单核细胞及其衍生细胞为表达选自下组的表面标记的VCAM-1 +单核细胞及其衍生细胞：mfap4、mpeg1、或其组合。
在另一优选例中，所述VCAM-1 +单核细胞及其衍生细胞不是内皮细胞。
在另一优选例中，所述VCAM-1 +单核细胞及其衍生细胞不是基质细胞。
在另有一优选例中，所述细胞试剂还包括其他可促进造血干细胞归巢的药物或促进剂。
在另一优选例中，所述促进剂可促进VCAM-1 +单核细胞及其衍生细胞表达VCAM-1蛋白。
在另一优选例中，所述其他可促进造血干细胞归巢的药物或促进剂选自下组：前列腺素(PGE2)、透明质酸(HA)、抗菌肽LL-37、或其组合。
本发明第三方面提供了一种药盒，包括：
(i)第一容器，以及装于该第一容器中的活性成分(a)VCAM-1 +单核细胞及其衍生细胞，或含有活性成分(a)的药物；
(ii)第二容器，以及装于该第二容器中的活性成分(b)造血干细胞，或含有活性成分(b)的药物；和
(iii)说明书，所述说明书中记载了联合给予活性成分(a)和活性成分(b)从而促进造血干细胞归巢的说明。
在另一优选例中，所述第一容器与第二容器可以相同，可以不同。
本发明第四方面提供了一种促进造血干细胞归巢的方法，包括：
在造血微环境下，将VCAM-1 +单核细胞及其衍生细胞与造血干细胞接触，从而促进所述造血干细胞的归巢。
在另一优选例中，所述方法为非治疗和非诊断性的。
在另一优选例中，所述方法为治疗性的。
本发明第五方面提供了一种体外促进造血干细胞迁移的方法，包括：
在体外适合的培养条件下，将VCAM-1 +单核细胞及其衍生细胞与造血干细胞接触，使所述VCAM-1 +单核细胞及其衍生细胞与所述造血干细胞结合，并促进所述造血干细胞迁移。
本发明第六方面提供了一种确定促进造血干细胞归巢的潜在治疗剂的方法，包括：
(i)在测试组中，在造血微环境下，提供一测试化合物和VCAM-1 +单核细胞及其衍生细胞与造血干细胞，观察VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况；
并且在不存在所述测试化合物且其他条件相同的对照组中，观察对照组所述造血微环境下的VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况；
(ii)将上一步骤观察的VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况进行比较，从而确定所述测试化合物是否是促进造血干细胞归巢的潜在治疗剂；
其中，如果与对照组相比，测试组中的VCAM-1 +单核细胞及其衍生细胞与造血干细胞明显结合并移动，则表明所述测试化合物是否是促进造血干细胞归巢的潜在治疗剂。
在另一优选例中，所述方法为非治疗性和非诊断性的。
在另一优选例中，所述方法还包括步骤(iii)，将步骤(ii)所确定的潜在治疗剂施用于哺乳动物，从而观察所述哺乳动物中的VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况。
在另一优选例中，所述哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物、灵长目动物，较佳地，包括小鼠、大鼠、兔、猴。
本发明第七方面提供了一种促进造血干细胞归巢的方法，包括步骤：
给需要的对象，在施用造血干细胞之前、之中、或之后，施用VCAM-1 +单核细胞及其衍生细胞，从而促进所述造血干细胞的归巢。
在另一优选例中，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述的之前和之后的时间间隔≤2天，较佳地≤1天，更佳地≤6小时。
在另一优选例中，将VCAM-1 +单核细胞及其衍生细胞和所述造血干细胞同时施用。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了VCAM-1突变体中造血干细胞归巢明显减少，其中，在VCAM-1突变体中，A、造血干细胞归巢(箭头)的数目减少；B、造血干细胞在造血微环境中长时间停留的细胞所占比例明显降低。
图2显示了VCAM-1 +细胞帮助造血干细胞归巢，其中，A、在免疫荧光结果中，VCAM-1 +细胞(三角形)与归巢的造血干细胞(箭头)位置邻近；B、我们建立了VCAM-1直标抗体特异性标记VCAM-1 +细胞的活体观察系统；三角形指示被特异标记的VCAM-1 +细胞，星号指示自发荧光色素；C、在长时程观察中，VCAM-1 +细胞(三角形)与造血干细胞(箭头)在造血微环境发生长时间细胞间相互作用，VCAM-1 +细胞推动造血干细胞被血管内皮细胞包被，造血干细胞在造血微环境中长时间停留，完成归巢。右侧显示造血干细胞被内皮细胞逐渐包被的过程。
图3显示了VCAM-1 +细胞是单核细胞及其衍生细胞的一个亚群，其中，A、微量细胞测序结果显示，VCAM-1 +细胞高表达单核细胞及其衍生细胞的分子标志mfap4；B、免疫荧光结果显示VCAM-1 +细胞与部分单核细胞及其衍生细胞共定位，三角形所指示的是共定位细胞，箭头所指示的是非VCAM-1 +的单核细胞及其衍生细胞；C、VCAM-1直标抗体活体标记单核细胞及其衍生细胞。
图4显示了去除单核-巨噬细胞导致造血干细胞归巢明显减少，其中，使用包裹有氯膦酸盐的脂质体Clodrosome去除单核-巨噬细胞之后，A、造血干细胞(箭头)归巢到造血微环境明显减少；B、长时间停留在微环境的造血干细胞比例明显减少。
具体实施方式
本发明人经过长期而深入的研究，意外地发现，VCAM-1 +单核细胞及其衍生细胞具有极其优异的促进造血干细胞归巢的作用。具体地，在造血微环境下，VCAM-1 +单核细胞及其衍生细胞可显著促进造血干细胞停留、增加造血干细胞在造血微环境的停留时间、增加长时间停留在微环境的造血干细胞的比例。在此基础上，发明人完成了本发明。
VCAM-1 +单核细胞及其衍生细胞
VCAM-1 +单核细胞及其衍生细胞是一类造血组织中存在的表达VCAM-1蛋白的单核细胞及其衍生细胞，可通过检测VCAM-1 +单核细胞及其衍生细胞的特异性标记物对其进行鉴定，所述标记物选自下组：mfap4、mpeg1或其组合。
几乎所有的VCAM-1 +细胞是mpeg1 +细胞。94％的VCAM-1 +细胞是圆形的，剩余的一小部分是树突状的。而在圆形的单核细胞及其亚群中，VCAM-1 +的类群仅占61.8％。因此，可以认为VCAM-1 +细胞是单核细胞及其衍生细胞中的一个亚群。
造血干细胞
成体血液系统的建立依赖于造血干细胞。造血干细胞具有自我更新、增殖、分化的能力。长程造血干细胞逐步分化为共同髓系前体细胞(CMP)和共同淋系前体细胞(CLP)，最后分化形成成熟的血液细胞，包括红细胞、血小板、中性粒细胞、单核细胞、T淋巴细胞和B淋巴细胞，进而构成完整有效的血液系统。
归巢
造血干细胞归巢指的是外周血中的造血干细胞回到骨髓微环境的过程，是造血干细胞重建造血系统的前提。在造血干细胞移植过程中，病人须经历一段贫血时期，伴随白细胞减少症和血小板减少症，意味着此阶段高发感染及出血，严重威胁病人的生命，是造血干细胞移植能否成功的关键期。归巢是一个复杂的多步骤过程，受到很多因素的影响，通过调控归巢过程，加快造血干细胞重建造血系统速度，缩短贫血期，可以有效提高移植存活率。由此可见，归巢对于移植后供体造血干细胞重建病人造血系统至关重要。归巢效率越高，移植后的存活率相对更高。
药物组合物(细胞试剂)
本发明还提供了一种药物组合物，它含有有效量的VCAM-1 +单核细胞及其衍生细胞；造血干细胞，以及药学上可接受的载体。
通常，可将VCAM-1 +单核细胞及其衍生细胞和造血干细胞配制于无毒的、惰性的和药学上可接受的水性载体介质中，如生理盐水中，其中pH通常约为5-8，较佳地，pH约为7-8。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生 功能或活性的且可被人和/或动物所接受的量。在本发明的优选实施例中，所述的有效量为：1.0×10 3-1.0×10 9个/ml，较佳地1.0×10 4-1.0×10 8个/ml，更佳地1.0×10 5-1.0×10 7个/ml。优选地，所述的有效量细胞一次性注射完毕。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括。在本发明中，可以使用的药学上可接受的载体并没有特别的限制，可以是一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的脂肪间充质祖细胞相互掺和，而不明显降低其治疗效果。本发明药学上可以接受的载体部分例子有生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末，适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。除了上述的常规载体外，也可以根据脂肪间充质祖细胞的性质设计优化的载体。所述的载体优选为输液剂载体和/或注射剂载体。
本发明的药物组合物含有安全有效量的VCAM-1 +单核细胞及其衍生细胞和造血干细胞，以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
本发明所述VCAM-1 +单核细胞及其衍生细胞和造血干细胞的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。
本发明的药物组合物优选为静脉注射试剂。在另一优选例中，所述的静脉注射试剂中，VCAM-1 +单核细胞及其衍生细胞的浓度为1.0×10 3-1.0×10 9个/ml，较佳地1.0×10 4-1.0×10 8个/ml，更佳地1.0×10 5-1.0×10 7个/ml，所述造血干细胞的浓度为1.0×10 3-1.0×10 10个/ml，较佳地1.0×10 4-1.0×10 9个/ml，更佳地 1.0×10 5-1.0×10 8个/ml。所述的药物组合物的注射方式没有特别限制，可以是单次注射制剂，也可以是多次注射的制剂组合。在本发明的一种优选实施例中，所述的药物组合物为单次注射剂。
在本发明中，所述的药物组合物优选为静脉注射制剂。
促进造血干细胞归巢的方法
在本发明中，还提供了一种促进造血干细胞归巢的方法，包括步骤：
在造血微环境下，将VCAM-1 +单核细胞及其衍生细胞与造血干细胞接触，从而促进所述造血干细胞的归巢。
在一优选实施方式中，所述促进造血干细胞归巢的方法，包括步骤：
给需要的对象，在施用造血干细胞之前、之中、或之后，施用VCAM-1 +单核细胞及其衍生细胞，从而促进所述造血干细胞的归巢。
确定促进造血干细胞归巢的潜在治疗剂的方法
在本发明中，还提供了一种确定促进造血干细胞归巢的潜在治疗剂的方法，包括：
(i)在测试组中，在造血微环境下，提供一测试化合物和VCAM-1 +单核细胞及其衍生细胞与造血干细胞，观察VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况；
并且在不存在所述测试化合物且其他条件相同的对照组中，观察对照组所述造血微环境下的VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况；
(ii)将上一步骤观察的VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况进行比较，从而确定所述测试化合物是否是促进造血干细胞归巢的潜在治疗剂；
其中，如果与对照组相比，测试组中的VCAM-1 +单核细胞及其衍生细胞与造血干细胞明显结合并移动，则表明所述测试化合物是否是促进造血干细胞归巢的潜在治疗剂。
本发明的主要优点包括：
(1)本发明首次发现，在造血微环境下，VCAM-1 +单核细胞及其衍生细胞 可显著促进造血干细胞归巢。
(2)本发明首次提供了一种用于显著促进造血干细胞归巢的细胞试剂或药物组合物。
(3)本发明首次提供了一种具有促进造血干细胞动员潜能的细胞试剂或药物组合物。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例中用到的材料和试剂如无特殊说明，均为市售产品。
造血干细胞以及VCAM-1 +细胞均来自斑马鱼体内，通过不同方式标记后进行观察。造血干细胞通过斑马鱼转基因系Tg(kdrl:dendra2)活体标记系统标记，来自于定向造血；VCAM-1 +细胞则通过anti-VCAM-1直标抗体进行标记观察。
包裹有氯膦酸盐的脂质体(clodrosome)购自EncapsulaNanoSciences(SKU:8909)。
实施例1 Vcam-1突变体活体观察发现造血干细胞(HSC)归巢异常
1)造血干细胞内源标记系统
转基因系Tg(kdrl:dendra2)(获自西南交通大学罗凌飞实验室)中血管内皮启动子(promoter)驱动光转换蛋白Dendra2的表达。32hpf是斑马鱼主动脉-性腺-中肾区域造血干细胞出芽的高峰期，在这个时期通过共聚焦显微镜(OlympusFV1000)用紫外光(波长405nm)对斑马鱼定向造血产生区域主动脉-性腺-中肾区域(AGM)的血管照射30s，可以将血管由绿色转变为红色，进而内源特异标记出芽的造血干细胞。
2)Vcam-1突变体造血干细胞归巢异常
在Tg(kdrl:dendra2)造血干细胞标记系统中，从发育时期的52h开始，利用 共聚焦显微镜(Zeiss 880)对斑马鱼尾部造血组织区域进行长时程活体成像。3um的z轴间隔和小于3min的时间间隔使得我们可以实时观察细胞的微小行为和细胞之间的相互作用。利用Imaris软件，可以对每个造血干细胞的停留时间进行统计，从而分析归巢事件。
通过成像，我们发现如图1A所示，相对于野生型中移动速度较慢且成像为圆形的造血干细胞，vcam-1突变体中大部分造血干细胞快速流过尾部造血组织，由于共聚焦显微镜线扫成像方式，成像结果显示为红线。通过分析发现，相对于野生型，vcam-1突变体中造血干细胞在尾部造血组织停留时间明显减少,其中停留时间较长(>30min)的造血干细胞数目明显减少，即造血干细胞的归巢行为发生明显异常(图1B)。
实施例2活体标记VCAM-1 +细胞并观察其如何促进造血干细胞归巢
1)免疫荧光
在Tg(kdrl:dendra2)中，32hpf将AGM进行光转换，标记造血干细胞。在54hpf，利用4％多聚甲醛固定胚胎，进行免疫荧光。利用anti-VCAM1-1兔抗(获自中国科学院上海生命科学研究院)识别VCAM-1 +细胞(二抗Thermo fisher anti-rabbit-647，A27040)，结果显示，VCAM-1 +细胞与造血干细胞位置临近(图2A)，显示其可能与造血干细胞之间存在相互作用。
2)直标抗体活体标记
在52hpf，从心脏总主静脉注射1nl Anti-VCAM-1直标抗体(波长647荧光基团标记蛋白试剂盒，Thermo fisher A30009)或1nl IgG荧光二抗到野生型斑马鱼胚胎(图2B)，Anti-VCAM-1直标抗体可以标记细胞，而IgG荧光二抗无特异性标记。如果同样方法注射1nl Anti-VCAM-1直标抗体到vcam-1突变体中，没有被标记的细胞(图2B)。因此Anti-VCAM-1直标抗体可以特异性活体标记VCAM-1 +细胞(图2B)。从54 hpf开始，利用共聚焦显微镜(Zeiss 880)对尾部造血组织区域进行长时程活体成像，观察VCAM-1 +细胞如何促进造血干细胞归巢。通过观察发现，VCAM-1+细胞与造血干细胞相互作用，或将造血干细胞推入小血管，或使得造血干细胞与血管内皮细胞之间的相互作用更紧密(图2C)，使造血干细胞停留时间延长，可以停留超过30分钟甚至超过2小 时。
实施例3 VCAM-1 +细胞是单核细胞及其衍生细胞中的亚群
1)微量细胞测序
如上述实施例的方法所述，通过直标抗体(波长647)活体标记VCAM-1 +细胞。在50-52hpf，将斑马鱼进行切尾消化，利用流式分选(Beckman Moflo Astrios)的方法富集VCAM-1 +细胞(波长647)，进行微量细胞测序检测RNA表达水平。对比全尾的样本和尾部造血组织的mpeg1 +标记的单核细胞及其衍生细胞样本，结果发现，单核细胞及其衍生细胞的标记物mfap4在VCAM-1 +细胞中相对于全尾样本高表达，并且与尾部造血组织的mpeg1 +标记的单核细胞及其衍生细胞样本表达水平相近(图3A)。
2)免疫荧光及活体成像
在Tg(mpeg1:egfp)中，单核细胞及其衍生细胞被标记成绿色。
54 hpf利用4％多聚甲醛固定胚胎，进行免疫荧光。利用anti-VCAM-1兔抗(获自中国科学院上海生命科学研究院)识别VCAM-1 +细胞(图3B)。
在52hpf，从心脏总主静脉注射1nl Anti-VCAM-1直标抗体到Tg(mpeg1:egfp)中，特异性活体标记VCAM-1 +细胞(图3C)。
免疫荧光及活体成像结果均显示，mpeg1 +的细胞有树突状和圆形两类，二者数目接近。几乎所有的VCAM-1 +细胞是mpeg1 +细胞。94％的VCAM-1 +细胞是圆形的(图3B、3C)，剩余的一小部分是树突状的。而在圆形的单核细胞及其亚群中，VCAM-1 +的类群仅占61.8％。因此，可以认为VCAM-1 +细胞是单核细胞及其衍生细胞中的一个亚群。
实施例4利用包裹有氯膦酸盐(clodronate)的脂质体去除单核细胞及其衍生细胞会影响造血干细胞归巢
在45hpf Tg(mpeg1:egfp)中，从斑马鱼的心脏总主静脉注射3nl空脂质体 或3nl包裹氯膦酸盐(clodronate)的脂质体，单核细胞及其衍生细胞吞噬脂质体，脂质体释放氯膦酸盐导致单核细胞及其衍生细胞凋亡。在60-72hpf期间进行长时程活体成像，观察是否去除单核细胞及其衍生细胞对造血干细胞归巢行为的影响。
结果显示，与VCAM-1突变体类似，相对于对照组(注射空脂质体)中移动速度较慢且成像为圆形的造血干细胞，去除单核细胞及其衍生细胞后，大部分造血干细胞快速流过尾部造血组织，由于共聚焦显微镜线扫成像方式，成像结果显示为红线(如图4A所示)。通过分析发现，相对于对照组，去除单核细胞及其衍生细胞后，在尾部造血组织CHT区域停留时间较长(>30min)的造血干细胞数目明显减少，即造血的归巢行为发生明显异常(图4B)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种VCAM-1 +单核细胞及其衍生细胞或其促进剂的用途，其特征在于，用于制备一组合物或制剂，所述组合物或制剂用于促进造血干细胞归巢。
  2. 如权利要求1所述的用途，其特征在于，所述的组合物中，所述VCAM-1 +单核细胞及其衍生细胞的浓度为1.0×10 3-1.0×10 9个/ml，较佳地1.0×10 4-1.0×10 8个/ml，更佳地1.0×10 5-1.0×10 7个/ml。
  3. 如权利要求1所述的用途，其特征在于，所述的组合物还含有(b)造血干细胞。
  4. 如权利要求3所述的用途，其特征在于，所述的组合物中，所述造血干细胞的浓度为1.0×10 3-1.0×10 10个/ml，较佳地1.0×10 4-1.0×10 9个/ml，更佳地1.0×10 5-1.0×10 8个/ml。
  5. 如权利要求1所述的用途，其特征在于，所述VCAM-1 +单核细胞及其衍生细胞为表达选自下组的表面标记的VCAM-1 +单核细胞及其衍生细胞：mfap4、mpeg1、或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述促进造血干细胞归巢包括：促进造血干细胞在造血微环境的停留、增加造血干细胞在造血微环境的停留时间、和/或增加长时间停留在微环境的造血干细胞的比例。
  7. 一种细胞试剂，其特征在于，包括：
    (i)第一药物组合物，所述第一药物组合物含有(a)第一活性成分，所述第一活性成分为VCAM-1 +单核细胞及其衍生细胞，以及药学上可接受的载体；
    (ii)第二药物组合物，所述第二药物组合物含有(b)第二活性成分，所述第二活性成分为造血干细胞；以及药学上可接受的载体；
    其中，所述的第一药物组合物和第二药物组合物为不同的药物组合物，或同一药物组合物。
  8. 一种药盒，其特征在于，包括：
    (i)第一容器，以及装于该第一容器中的活性成分(a)VCAM-1 +单核细胞及其衍生细胞，或含有活性成分(a)的药物；
    (ii)第二容器，以及装于该第二容器中的活性成分(b)造血干细胞，或含有活性成分(b)的药物；和
    (iii)说明书，所述说明书中记载了联合给予活性成分(a)和活性成分(b)从 而促进造血干细胞归巢的说明。
  9. 一种体外促进造血干细胞迁移的方法，其特征在于，包括：
    在体外适合的培养条件下，将VCAM-1 +单核细胞及其衍生细胞与造血干细胞接触，使所述VCAM-1 +单核细胞及其衍生细胞与所述造血干细胞结合，并促进所述造血干细胞迁移。
  10. 一种确定促进造血干细胞归巢的潜在治疗剂的方法，其特征在于，包括：
    (i)在测试组中，在造血微环境下，提供一测试化合物和VCAM-1 +单核细胞及其衍生细胞与造血干细胞，观察VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况；
    并且在不存在所述测试化合物且其他条件相同的对照组中，观察对照组所述造血微环境下的VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况；
    (ii)将上一步骤观察的VCAM-1 +单核细胞及其衍生细胞与造血干细胞的结合情况以及结合后的移动情况进行比较，从而确定所述测试化合物是否是促进造血干细胞归巢的潜在治疗剂；
    其中，如果与对照组相比，测试组中的VCAM-1 +单核细胞及其衍生细胞与造血干细胞明显结合并移动，则表明所述测试化合物是否是促进造血干细胞归巢的潜在治疗剂。
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CN103784940A (zh) * 2014-03-03 2014-05-14 湖北华赛生物医药技术有限公司 提高造血干细胞归巢及植入率的方法和试剂

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