WO2019075782A1 - 一种重塑骨髓微环境的方法 - Google Patents

一种重塑骨髓微环境的方法 Download PDF

Info

Publication number
WO2019075782A1
WO2019075782A1 PCT/CN2017/108943 CN2017108943W WO2019075782A1 WO 2019075782 A1 WO2019075782 A1 WO 2019075782A1 CN 2017108943 W CN2017108943 W CN 2017108943W WO 2019075782 A1 WO2019075782 A1 WO 2019075782A1
Authority
WO
WIPO (PCT)
Prior art keywords
bone marrow
msc
leukemia
cells
subject
Prior art date
Application number
PCT/CN2017/108943
Other languages
English (en)
French (fr)
Inventor
王金勇
夏成祥
董勇
王童洁
刘晓飞
杜鹃
耿阳
刘丽娟
吴红玲
Original Assignee
中国科学院广州生物医药与健康研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院广州生物医药与健康研究院 filed Critical 中国科学院广州生物医药与健康研究院
Priority to CN201780027447.XA priority Critical patent/CN109069543B/zh
Publication of WO2019075782A1 publication Critical patent/WO2019075782A1/zh

Links

Images

Classifications

    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • 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
    • A61P7/06—Antianaemics
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0669—Bone marrow stromal cells; Whole bone marrow

Definitions

  • the present invention relates to the field of bone marrow microenvironment remodeling and, in particular, to a method of remodeling the bone marrow microenvironment and its use in the treatment of cancer, particularly leukemia.
  • CMML Chronic granulocyte-monocytic leukemia
  • JMML juvenile granulocyte-monocytic leukemia
  • MDS myelodysplastic disorders
  • MPN myeloproliferative disorders
  • CMML and JMML are involved in oncogenic point mutations in genes involved in RAS/MAPK signaling, and 25% to 40% of CMML/JMML patients carry somatic mutations in NRAS. It has been reported that mice bearing an endogenous mutation of the Nras allele develop a CMML-like disease with a long incubation period.
  • Aplastic anemia is abbreviated as aplastic anemia, which is caused by bone marrow hematopoietic stem cell defects, hematopoietic microenvironment damage and immune mechanism changes, leading to bone marrow hematopoietic failure and the occurrence of whole blood cell reduction. disease.
  • the bone marrow microenvironment is a complex network structure with multiple functions consisting of several cell types, including mesenchymal stromal cells (MSC), endosteal osteoblasts (OB), endothelial cells, and other rare cell types.
  • MSCs can be isolated and propagated in vitro.
  • MSC is a precursor cell of bone marrow stromal cells, accounting for 0.01% to 0.001% of bone marrow mononuclear cells, equivalent to 1% of the number of HSCs, and can be directed to osteoblasts, chondrocytes, adipocytes, and myocytes under different induction conditions. Differentiation of pericytes, reticular fibroblasts, and nerve cells.
  • MSC is commonly used to treat systemic graft-versus-host disease (GVHD), improve wound healing and promote recovery of lung injury, kidney damage and myocardial damage.
  • GVHD systemic graft-versus-host disease
  • MSCs are pluripotent cells that are injected into the recipient. The host can be differentiated into adipocytes, osteoblasts, chondrocytes, myoblasts, and/or neuron-like cells. Therefore, it is difficult to predict the accurate results after injection of MSC into the BM microenvironment, such as the role of MSC injection in the normalization of HSC pool, recovery of normal hematopoietic function, and inhibition of disease progression.
  • the treatment of malignant hematological tumors mainly includes chemotherapy, radiotherapy and combined hematopoietic stem cell transplantation.
  • the above treatment methods are accompanied by various degrees of side effects, including severe damage to non-tumor healthy cells, particularly germ cells, and graft-versus-host disease (GVHD), which seriously degrades the quality of life of patients.
  • GVHD graft-versus-host disease
  • the bone marrow microenvironment of patients with malignant hematologic tumors is usually destroyed by tumors, which in turn leads to the weakening or even depletion of normal hematopoiesis.
  • the present invention is directed to a method of reshaping the bone marrow microenvironment and its use.
  • the method of the invention realizes the remodeling of the pathological bone marrow microenvironment for the first time, and effectively reverses the bone marrow hematopoietic weakening and failure caused by the destruction of the bone marrow stromal cells and the disappearance of the endosteal osteoblasts caused by the destruction of the bone marrow microenvironment.
  • Phenomenon successful recovery of normal hematopoiesis in the bone marrow microenvironment significantly prolonged the survival of the subject.
  • the method of the present invention can be used for the treatment of hematological tumors and Aplastic anemia (AA); preferably, the hematological tumor is leukemia; further preferably, the hematological tumor is juvenile granulocyte monocytic leukemia ( Juvenile Myelomonocytic Leukemia) and Chronic myelomonocytic leukemia; safe, effective, and no side effects.
  • AA Aplastic anemia
  • the invention provides a method of remodeling a bone marrow microenvironment in a subject with impaired bone marrow microenvironment, It comprises implanting a composition comprising isolated mesenchymal stromal cells (MSC) into the medullary cavity of the subject.
  • MSC mesenchymal stromal cells
  • the mesenchymal stromal cells are obtained from the endosteal and/or bone marrow nucleated cells of a healthy subject.
  • the subject is a mammal.
  • the subject is a mouse; when the subject is a mouse, the mesenchymal stromal cells having the following phenotype: TER119 - CD45 - CD31 - Sca1 + CD51 + CD146 +.
  • the subject is a human; when the subject is a human, the mesenchymal stromal cells have the following phenotype: CD235ab - CD45 - CD34 - CD31 - CD271 + CD146 + .
  • the composition comprising isolated mesenchymal stromal cells further comprises a pharmaceutically acceptable carrier, diluent or vehicle; preferably, the pharmaceutically acceptable carrier, diluent Or the vehicle is phosphate buffer or physiological saline.
  • the implantation is a multiple, local injection.
  • the implantation is carried out at a dose of 1.0 x 10 5 to 3.0 x 10 7 MSC/kg body weight, preferably 2.5 x 10 7 MSC/kg body weight.
  • the implantation is performed once a week, once every two weeks, once every three weeks or once a month; preferably, the implantation is performed every two weeks; preferably, The implantation is performed within a time window of 12 to 24 weeks, preferably 16 weeks.
  • the subject has a disease selected from the group consisting of a hematological tumor and an Aplastic Anemia (AA); preferably, the hematological tumor is leukemia; further preferably, the blood The tumor is juvenile granulocyte monocytic leukemia or chronic myelomonocytic leukemia.
  • AA Aplastic Anemia
  • the bone marrow microenvironment exhibits one or more selected from the group consisting of Conditions: recovery of osteoblasts, partial recovery of functional MSCs.
  • the subject after implanting a composition comprising isolated mesenchymal stromal cells (MSC) into the medullary cavity of the subject, the subject exhibits one or more selected from the group consisting of Multiple conditions: normal hematopoietic recovery, tumor growth inhibition, and prolonged survival.
  • MSC mesenchymal stromal cells
  • the invention provides the use of the method of the first aspect in the treatment of hematological tumors and aplastic anemia.
  • the blood tumor is leukemia; further preferably, the blood tumor is juvenile myelomonocytic leukemia or chronic myelomonocytic leukemia.
  • the invention firstly develops a method for locally and multiple injections of donor bone marrow stromal cells in the bone marrow cavity from the bone marrow microenvironment under the tumor burden, and realizes the remodeling of the pathological bone marrow microenvironment for the first time, effectively reversing the suffering of the bone marrow microenvironment.
  • the stem cell treatment method of the invention has the characteristics of safe, effective and no side effects for treating leukemia.
  • the invention partially implants donor bone marrow stromal cells through the bone marrow cavity, optimizes the injection dose and the injection interval time, and successfully reverses the gradual depletion of the bone marrow microenvironment in the mouse model of leukemia, thereby realizing the remodeling and recovery of the bone marrow microenvironment.
  • Normal bone marrow hematopoiesis inhibits/delays the pathological process of leukemia and significantly prolongs survival.
  • Figure 1 shows hematoxylin-eosin staining (Fig. 1A) and osteocalcin histochemical staining of the tibia of the mouse in the pre-leukemic phase of the representative control mice and NrasG12D receptor mice (Fig. 1B). ).
  • FIG. 2 shows the fibroblast colony forming unit experiment (CFU-F) showing the depletion of functional MSCs in NrasG12D receptor mice;
  • Figure 2A Wright-Gemsa staining of CFU-F experiments;
  • Figure 2B NrasG12D-induced Dynamic analysis of CFU-F colonies during the development of leukemia;
  • WT BM wild-type bone marrow
  • Figure 3 shows the number of MSCs and TER119-CD45-CD31-CD51+Sca1-phenotype osteoblasts (OB) in the TER119-CD45-CD31-CD51+Sca1+CD146+ phenotype of bone marrow in NrasG12D mice by flow cytometry.
  • Figure 3A shows the circled strategy of MSC and OB in flow cytometry; the graph shows the flow pattern from a representative WT mouse (above, as a control) and NrasG12D receptor mice (below)
  • Figure 3B shows the statistical results of the absolute number of MSC cells in all bone marrow nucleated cells;
  • Figure 3C shows the statistical results of the absolute number of OB cells in all bone marrow nucleated cells.
  • Figure 4 shows the increase in proinflammatory cytokine levels in the serum of NrasG12D receptor mice at the latent stage of leukemia;
  • Figure 4A TGF-[beta]1
  • Figure 4B TNF-[alpha]
  • Figure 4C GM-CSF.
  • Figure 5 shows the identification of surface markers for isolated cultured MSCs.
  • Figure 6 shows a schematic diagram of the MSC transplantation experiment of the examples.
  • Figure 7 shows hematoxylin-eosin staining (A and B) and osteocalcin histochemical staining (C and D) showing partial recovery of osteoblasts near the endosteal of NrasG12D receptor mice after MSC injection.
  • Figure 8 shows hematoxylin-eosin staining (A) and osteocalcin histochemical staining (B) showing that at 3 weeks after MSC injection, osteoblasts near the endosteal of NrasG12D receptor mice began to recover.
  • Figure 10 is a statistical analysis showing that the total number of white blood cells in the peripheral blood of NrasG12D receptor mice after MSC treatment was significantly decreased (A), platelets were significantly elevated (B), and the total number of red blood cells was not significantly changed (C).
  • FIG. 12 shows the ELISA detection of mouse serum (Fig. 12A) and intraluminal TGF- ⁇ 1 levels (Fig. 12B).
  • the level of TGF- ⁇ 1 in the serum and bone marrow cavity of MSC-treated mice was significantly lower than that of untreated mice.
  • Figure 13 shows the effect of TGF- ⁇ 1 on osteogenesis in an in vitro osteogenic differentiation experiment
  • Figure 13A shows the results of alizarin red staining after 21 days of MSC-induced osteogenic differentiation
  • Figure 14 shows flow cytometry analysis of the cell cycle of HSC and MPP in the bone marrow of untreated and MSC-treated NrasG12D receptor mice (Fig. 14A), cell cycle statistics of HSC (Fig. 14B) and the absolute number of HSC and MPP Statistical analysis (Fig. 14C).
  • bone marrow microenvironment refers to a cellular component composed of supporting cells adjacent to hematopoietic stem cells in the bone marrow that participate in the maintenance, self-renewal, and directed differentiation of hematopoietic stem cells.
  • the bone marrow microenvironment herein mainly refers to bone marrow mesenchyme. Stromal cells and osteoblasts close to the endosteal.
  • meenchymal stromal cells refers to a stromal cell in the bone marrow that supports hematopoietic stem cells and is capable of secreting a variety of factors to support hematopoiesis.
  • endosteal refers to a membrane tissue that grows adherently on the inside of a bone.
  • NrasG12D receptor mouse and “NrasG12D leukemia mouse” as used herein are used interchangeably and refer to a mouse model of chronic phylogenetic leukemia (CMML/JMML-like) constructed by introducing a NrasG12D mutation, which The construction method is detailed in Example 1.
  • CMML/JMML-like chronic phylogenetic leukemia
  • hematoxylin and eosin staining For hematoxylin and eosin staining, the tibia and femur bones of control mice and NrasG12D receptors were fixed in 4% paraformaldehyde and then processed for histological hematoxylin and eosin staining (pathology laboratory, GIBH) ).
  • the tibia was decalcified in 14% EDTA and embedded in paraffin for sectioning (pathology laboratory, GIBH). Endogenous peroxidase activity was quenched and blocked by blocking buffer (5% normal goat serum and 0.3% Triton X-100 in PBS). Slides were incubated with the relevant primary rabbit anti-mouse polyclonal antibody overnight at 4 °C followed by secondary biotinylated anti-rabbit antibody (goat anti-rabbit IgG-HRP, sc-2004, Santa Cruz, 1:1000) Dilute) Incubate for 1 hour at room temperature.
  • anti-osteocalcin polyclonal antibody (ab93876, Abcam, 1:500).
  • Antigen visualization was performed using a mixture of DAB substrate and DAB chromogen. Slides were counterstained with hematoxylin and mounted with neutral gum. Images were captured and processed using the Motic Digital Slice Scanning System (VMV1 VMDPCS, Motic) and DSS scanner software.
  • VMV1 VMDPCS, Motic Motic Digital Slice Scanning System
  • the BM 1,000,000 nucleated cells were seeded into 6 well plates and cultured in complete medium MesenCult TM (Catalog 05512, StemCell Technology) in 10-14 days. BM cells were incubated in a humidified chamber at 37 ° C, 5% CO 2 and semi-media exchange was performed on day 7. Giemsa staining according to Yang D, Zhang X, Dong Y, et al. Enforced expression of Hoxa 5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo. Cell Cycle. 2015; 14(4): 612-620. (Giemsa staining).
  • Hematopoietic lineage analysis of peripheral blood (50 ⁇ l) and bone marrow such as Wang J, Liu Y, Li Z, et al. Endogenous oncogenic Nras mutation initiates hematopoietic malignancies in a dose-and cell type-dependent manner. Blood.2011; Flow cytometric analysis was performed as described in 118(2): 368-379.
  • Directly conjugated antibodies using the following surface antigens FITC-CD45.2 (104), APC-Thy1.2 (53-2.1), organisms -CD3 (145-2C11), biotin-CD3 (RM4-5), biotin-CD8 (53-6.7), PE-CD19 (1D3), PE-Cy7-Mac1 (M1/70) and APC-Gr
  • the -1 (RB6-8C5) antibody was purchased from eBiosciences.
  • Intracellular staining of cytokines, collection of cells and staining with surface antigens PerCP-Cy5.5-Mac1 (M1/70)/APC-Mac1 (M1/70), biotin-G1 (RB6-8C5), PE-Ly6C HK1.4), APC-eFluor 780-F4/80 (BM8) and streptavidin-PE antibody were purchased from eBiosciences.
  • the cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) and then intracellularly stained by using PerCP-Cy5.5-TGF- ⁇ 1 (TW7-16B4, Biolegend).
  • the cells were fixed with 4% PFA. Finally, the fixed cells were permeabilized with 0.1% saponin in PBS and stained with APC-Ki67 (SolA15, eBioscience) and then stained with DAPI (Invitrogen).
  • BM cells were isolated by flushing the tibia and femur with DPBS containing 2% FBS, then the bone was cut into small pieces and 1 mg/ml collagenase II was shaken in a shaking incubator at 37 °C. Digestion for 1-2 hours. The collected cells from the endosteal and bone marrow were mixed together and filtered using a 70 ⁇ m cell strainer.
  • APC-780F-CD45 (30-F11), APC-eFluor 780-Ter119 (TER-119), PE-Cy7-Sca-1 (D7), biotin - CD51 (RMV-7) and streptavidin-PE were purchased from eBiosciences, APC-CD31 (MEC 13.3) and PerCP-Cy5.5-CD146 (ME-9F1) were purchased from Biolegend.
  • Enforced expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo. Cell Cycle.
  • the staining scheme is described in 2015; 14(4): 612-620.
  • the stained cells were analyzed on a FACS Calibur or LSR Fortessa (BD Bioscience), and MSCs were sorted for RNA sequencing using Aria II (BD Bioscience), and then analyzed using Flowjo software (FlowJo).
  • Serum samples were collected from peripheral blood of NrasG12D receptor and control mice. Bone marrow extracellular fluid samples were collected from the bone marrow of the NrasG12D receptor and control mice.
  • concentration of cytokines was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Beijing 4A Biotech Co., Ltd., China) according to the manufacturer's instructions.
  • MSCs were seeded at a density of 2 x 10 4 cells/cm 2 in growth medium in 6-well plates pre-coated with gelatin solution, and cultured in a humidified incubator at 37 ° C, 5% CO 2 .
  • the growth medium was removed, and 2 mL of mouse mesenchymal stem cell osteogenic differentiation medium (MUBMX-90021, Cyagen) was added. The medium is changed every three days.
  • Peripheral serum (10 ⁇ L/mL) and 5 ng/mL TGF- ⁇ 1 cytokine from wild-type, untreated, MSC-treated NrasG12D leukemia mice were separately added to the induction medium during the whole process of osteogenic differentiation. After 3 weeks of induction, the cells were fixed with 4% neutral formaldehyde and stained with alizarin red. Images were captured and the area of calcium nodules formed was calculated by Image-Pro software.
  • GFP-labeled MSCs were seeded in a six-well plate at a density of 1 ⁇ 10 5 cells/well, CD45+BMNC was sorted from NrasG12D receptor mice, and seeded at 2 ⁇ 10 6 cells/ml in 2 ml of ⁇ -MEM+10. % FBS + 50 ng / ml in SCF medium. MSCs and CD45+ cells were cultured at 37 ° C, 5% CO 2 . 1 ml of fresh medium was added every other day. After 7 days of culture, the level of TGF- ⁇ 1 in the culture supernatant was measured by ELISA, and the cell source of TGF- ⁇ 1 was detected by flow cytometry.
  • Example 1 Establishing a mouse leukemia (CMML/JMML-like) model with progressive destruction of the bone marrow microenvironment
  • Leukocytes (with stromal cells removed) from NrasG12D mutant mice (LSL NrasG12D/+; Vav-Cre) or control mice (CD45.2) were transplanted through the retro-ocular vein to sublethal irradiation (6.5 Gy) CD45 .1 recipient mouse, thereby constructing a mouse leukemia model of chronic myelomonocytic leukemia (CMML/JMML-like).
  • CMML/JMML-like chronic myelomonocytic leukemia
  • Mac1 myeloid cells in the peripheral blood of mice are greater than 20% and less than 60%
  • PL phase leukemia latency phase
  • Mac1 > 60% it is the Leukemic phase.
  • Example 2 The histological analysis of Example 2 demonstrated that the endometrial osteoblasts gradually depleted in the bone marrow microenvironment during the latent stage of leukemia.
  • FIG. 1A shows that osteoblasts (osteoblasts, hereinafter sometimes referred to as OB) near the endosteal in NrasG12D receptor mice at the latent stage of leukemia have been depleted; osteocalcin histochemical staining (Fig. 1B) The osteoblasts near the endosteal in the NrasG12D receptor mice showing the latent phase of leukemia have been depleted. That is, histochemical staining of the tibia demonstrated that osteoblasts near the endosteal have almost disappeared during the latent stage of leukemia.
  • Example 3 The cytological analysis of Example 3 demonstrates that the mesenchymal stromal cells (MSC) of the bone marrow microenvironment are gradually depleted with the development of leukemia.
  • MSC mesenchymal stromal cells
  • CFU-F fibroblast colony forming unit experiment
  • Example 4 Flow cytometry showed that the bone marrow microenvironment gradually depleted with the development of leukemia.
  • the nucleated cells in the endosteoid were collected by cutting the blown bone into small bone pieces and digesting with 1 mg/ml type II collagenase in a shaking incubator at 37 ° C for 1-2 hours.
  • the nucleated cells from the bone marrow and endosteal were mixed and filtered through a 70 micron cell screen and 1 mL of ACK red blood cell lysate was added (the ACK red blood cell lysate was prepared by the following procedure: NH 4 Cl (150 mM) 8.02 g, KHCO 3 (10 mM) 1.00 g, Na 2 EDTA (0.1 mM) 0.37 g and H 2 O 800 ml were mixed, adjusted to pH 7.2-7.4 with 1N HCl, water was added to 1 L, and finally filtered through a 0.22 ⁇ m filter to remove bacteria.
  • Example 5 Increased systemic inflammation of the NrasG12D receptor during the latent phase of leukemia
  • TGF- ⁇ 1, TNF- ⁇ and GM-CSF proinflammatory cytokines TGF- ⁇ 1, TNF- ⁇ and GM-CSF in the serum of NrasG12D receptor mice at the latent stage of leukemia, and found that TGF- ⁇ 1, TNF- ⁇ and GM-CSF levels were significantly increased (Fig. 4A). -C).
  • Example 6 Isolation, in vitro culture and phenotypic identification of bone marrow mesenchymal stromal cells
  • mesenchymal stromal cells were isolated from the endosteal and bone marrow of 4 week old wild type mice. First, the bone marrow is blown out of the bone cavity, the bone is cut into small bone fragments and digested with 1 mg/ml type II collagenase at 37 ° C for 1-2 hours, and the digested bone fragments are washed and placed in a 6 Gm dish. The cells were cultured in 6 mL MSC complete medium ( ⁇ -MEM + 10% FBS) to obtain bone tissue culture-derived MSCs.
  • the bone tissue culture-derived MSCs and the bone marrow sorting-derived MSCs were mixed and cultured, and the culture was continued after washing and changing the medium on the third day. After 5 days of culture, the bone fragments were removed and washed, and adherent cells were collected by 0.25% (wt/vol) trypsinization, and passaged at 1:3, and the medium was changed every 48 hours.
  • isolated cultured MSCs Prior to injection, isolated cultured MSCs were harvested and phenotypically identified by flow cytometry (CD45, TER119, CD31, CD51, CD105, LepR, PDGFR ⁇ , PDGFR ⁇ , Sca-1, Figure 5).
  • the MSCs used for injection were all cells cultured to passage 3.
  • the MSC transplantation experiment was performed by intra-luminal injection according to the procedure shown in FIG.
  • Example 8 MSC intramedullary injection method for treating mouse model of leukemia induced by NrasG12D, successfully achieving osteoblast recovery in bone marrow microenvironment
  • Example 9 MSC intramedullary injection method for treatment of NrasG12D-induced leukemia mouse model, successful realization of mesenchymal stromal cell recovery in bone marrow microenvironment
  • NrasG12D leukemia mice after MSC treatment were sacrificed, and the bone marrow nucleated cells BMNC were first isolated and CFU-F was performed.
  • Experimental and statistical analysis ( Figures 9A and 9B). The results showed that BMNC formed more CFU-F colonies in the bone marrow of MSC-treated NrasG12D leukemia mice compared to untreated NrasG12D leukemia mice, indicating functionality in the bone marrow stroma microenvironment at the injection site. The MSC is partially restored.
  • Example 10 MSC intramedullary injection method successfully achieved normal hematopoietic recovery in NrasG12D-induced leukemia mice
  • Example 11 Local injection of MSC into the bone marrow cavity can successfully inhibit tumor growth and prolong survival.
  • TGF- ⁇ 1 has a negative regulatory effect on osteogenesis. Therefore, in order to investigate whether local injection of MSC into the bone marrow cavity promotes regeneration of endosteal osteoblasts by regulating TGF- ⁇ 1 levels, we performed the following experiments: First, by ELISA The levels of TGF- ⁇ 1 in the serum and bone marrow cavity of the treated mice were detected. At the same time, the bone marrow sections of NrasG12D leukemia mice were subjected to immunohistochemical staining for osteocalcin by histochemical methods. The results showed that: small untreated control The levels of TGF- ⁇ 1 in the serum and bone marrow cavities of MSC-treated NrasG12D leukemia mice were significantly reduced compared to the mice (see Figures 12A and 12B).
  • Example 13 Local Injection of MSCs in the Bone Marrow Cavity Activates Dormancy HSC by Decreasing the Level of TGF- ⁇ 1 to Promote Normal Hematopoietic Recovery
  • Example 14 MSC Regulates the Level of TGF- ⁇ 1 Secreted by Mac1 + F4/80 + NrasG12D Tumor Cells
  • GFP mouse bone marrow-derived MSC cells were isolated, seeded in a six-well plate at a density of 1 ⁇ 10 5 MSC/well, and then sorted into CD45+ cells in the bone marrow of NrasG12D receptor mice at a density of 1 ⁇ 10 6 cells/ml. Resuspend in the complete medium of co-culture ( ⁇ -MEM + 10% FBS + 50 ng / ⁇ l SCF), separately cultured, and co-cultured with MSC.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dermatology (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Oncology (AREA)
  • Diabetes (AREA)
  • Neurosurgery (AREA)
  • Rheumatology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

本发明提供了一种在骨髓微环境受损的受试者中重塑骨髓微环境的方法，其包括：将包含分离的间充质基质细胞(MSC)的组合物植入所述受试者的骨髓腔。本发明所述方法实现了骨髓微环境重塑，恢复正常骨髓造血功能，抑制/延缓了白血病病理进程，显著延长了生存期。此外，本发明的方法可用于治疗血液肿瘤如白血病以及再生障碍性贫血(Aplastic anemia，AA)，具有安全、有效、无副作用的特点。

Description

一种重塑骨髓微环境的方法 技术领域
本发明涉及骨髓微环境重塑领域，具体而言，涉及一种重塑骨髓微环境的方法及其在治疗癌症特别是白血病中的应用。
背景技术
慢性粒-单核细胞白血病(CMML)和幼年型粒-单核细胞白血病(JMML)是无性系血液恶性肿瘤，具有骨髓增生异常(MDS)和骨髓增殖性疾病(MPN)的特征。CMML和JMML与参与RAS/MAPK信号转导的基因的致癌点突变有关，25％至40％的CMML/JMML患者携带NRAS的体细胞突变。有报道显示，携带Nras等位基因的内源突变的小鼠发生CMML样疾病，其具有长的潜伏期。还有报道显示了在CMML/JMML的人类患者以及动物模型中，MDS/MPN疾病和骨髓(BM)微环境功能紊乱之间的频繁关联。再生障碍性贫血(Aplastic anemia，AA)简称再障，是由多种原因引起的骨髓造血干细胞缺陷、造血微环境损伤以及免疫机制改变，导致骨髓造血功能衰竭，出现以全血细胞减少为主要表现的疾病。骨髓微环境是一个具有多重功能的复杂网络结构，由数种细胞类型组成，包括间充质基质细胞(MSC)、骨内膜成骨细胞(OB)、内皮细胞和其他稀有的细胞类型。MSC可以分离并在体外进行增殖。MSC是骨髓基质细胞的前体细胞，占骨髓单个核细胞的0.01％至0.001％，相当于HSC数量的1％，在不同的诱导条件下可以向成骨细胞、软骨细胞、脂肪细胞、肌细胞、周细胞、网状成纤维细胞及神经细胞等分化。
目前，MSC普遍用于治疗全身性移植物抗宿主病(GVHD)，改善伤口愈合并促进肺损伤、肾损伤和心肌损伤的恢复。然而，对于MSC的响应并不总是成功的，例如，已知其受到患者的炎性状态的影响。此外，MSC是多能细胞，它们在注射进受体 宿主后可分化成脂肪细胞、成骨细胞、软骨细胞、成肌细胞和/或神经元样细胞。因此，难以预测在将MSC注射入BM微环境后的准确结果，如MSC注射在HSC池的正常化、正常造血功能的恢复以及疾病进展抑制等方面的作用。
目前，恶性血液肿瘤的治疗方法主要包括化疗、放疗以及联合造血干细胞移植。然而，上述治疗方法除了肿瘤容易复发外，还伴随不同程度的副作用，包括对非肿瘤健康细胞特别是生殖细胞的严重损害、移植物抗宿主病(GVHD)等，严重降低患者生活质量。急需一种对患者副作用小甚至无副作用的、能有效治疗血液肿瘤的治疗方法。恶性血液肿瘤患者的骨髓微环境通常会遭到肿瘤的破坏，继而出现骨髓正常造血减弱甚至衰竭的现象。反过来，骨髓间质微环境的功能异常/耗竭在白血病等血液肿瘤以及再生障碍性贫血的病理恶化进程中起着促进作用。迄今为止，尚没有一种有效的途径能解决病理骨髓微环境的重塑问题。因此，提供一种能有效地重塑骨髓微环境的方法对于病理状态下重塑骨髓微环境，缓解及治疗恶性血液肿瘤具有十分重要的意义。
发明内容
针对现有技术中存在的缺陷，本发明旨在提供一种重塑骨髓微环境的方法及其应用。本发明的方法首次实现了病理骨髓微环境的重塑，有效逆转骨髓微环境由于遭受肿瘤等破坏出现的骨髓间质细胞功能耗竭、骨内膜成骨细胞消失等问题引起的骨髓造血减弱及衰竭现象，成功恢复骨髓微环境正常造血，显著延长受试者的生存期。此外，本发明的方法可用于治疗血液肿瘤以及再生障碍性贫血(Aplastic anemia，AA)；优选地，所述血液肿瘤为白血病；进一步优选地，所述血液肿瘤为幼年型粒单核细胞白血病(Juvenile Myelomonocytic Leukemia)和慢粒单核细胞白血病(Chronic myelomonocytic leukemia)；具有安全、有效、无副作用的特点。
一方面，本发明提供了一种在骨髓微环境受损的受试者中重塑骨髓微环境的方法， 其包括：将包含分离的间充质基质细胞(MSC)的组合物植入所述受试者的骨髓腔。
在具体的优选实施方案中，所述间充质基质细胞从健康受试者的骨内膜和/或骨髓有核细胞获得。
优选地，所述受试者为哺乳动物。
进一步优选地，所述受试者为小鼠；当所述受试者为小鼠时，所述间充质基质细胞具有以下表型：TER119-CD45-CD31-Sca1+CD51+CD146+。
进一步优选地，所述受试者为人；当所述受试者为人时，所述间充质基质细胞具有以下表型：CD235ab-CD45-CD34-CD31-CD271+CD146+。
在具体的优选实施方案中，所述包含分离的间充质基质细胞的组合物还包含药学上可接受的载体、稀释剂或媒介物；优选地，所述药学上可接受的载体、稀释剂或媒介物为磷酸盐缓冲液或生理盐水。
在具体的优选实施方案中，所述植入为多次、局部注射。
在具体的优选实施方案中，按照1.0×105～3.0×107个MSC/kg体重、优选2.5×107个MSC/kg体重的剂量，进行所述植入。
在具体的优选实施方案中，每周一次、每两周一次、每三周一次或每个月一次地进行所述植入；优选地，每两周一次进行所述植入；优选地，在12～24周、优选16周的时间窗内，进行所述植入。
在具体的优选实施方案中，所述受试者患有选自血液肿瘤以及再生障碍性贫血(Aplastic anemia，AA)的疾病；优选地，所述血液肿瘤为白血病；进一步优选地，所述血液肿瘤为幼年型粒单核细胞白血病或慢粒单核细胞白血病。
在具体的优选实施方案中，在将包含分离的间充质基质细胞(MSC)的组合物植入所述受试者的骨髓腔后，骨髓微环境中呈现出选自以下的一种或多种情况：成骨细胞恢复、功能性MSC部分恢复。
在具体的优选实施方案中，在将包含分离的间充质基质细胞(MSC)的组合物植入所述受试者的骨髓腔后，所述受试者呈现出选自以下的一种或多种情况：正常造血恢复、肿瘤生长抑制和生存期延长。
第二方面，本发明提供了如第一方面所述的方法在治疗血液肿瘤以及再生障碍性贫血中的用途。
优选地，所述血液肿瘤为白血病；进一步优选地，所述血液肿瘤为幼年型粒单核细胞白血病或慢粒单核细胞白血病。
本发明从改善肿瘤负荷下的骨髓微环境入手，首次开发出骨髓腔内局部、多次注射供体骨髓间质细胞的方法，首次实现了病理骨髓微环境的重塑，有效逆转骨髓微环境遭受肿瘤破坏出现的骨髓间质细胞功能耗竭、骨内膜细胞消失等问题引起的骨髓造血减弱及衰竭现象，成功恢复骨髓微环境正常造血，显著延长白血病小鼠的生存期。本发明的干细胞治疗方法治疗白血病具有安全、有效、无副作用的特点。
本发明通过骨髓腔局部植入供体骨髓间质细胞、通过优化注射剂量、注射间隔时间，成功逆转了白血病小鼠模型中出现的骨髓微环境逐步耗竭现象，实现了骨髓微环境重塑，恢复正常骨髓造血功能，抑制/延缓了白血病病理进程，显著延长了生存期。
附图说明
图1显示代表性对照小鼠和NrasG12D受体小鼠的白血病潜伏期阶段(Pre-leukemic phase)的小鼠胫骨的苏木精-伊红染色(图1A)和骨钙素组化染色(图1B)。
图2为成纤维细胞集落形成单位实验(CFU-F)显示NrasG12D受体小鼠中的功能性MSC的耗竭情况；图2A，CFU-F实验的瑞氏吉姆萨染色；图2B，NrasG12D诱导的白血病发生发展过程中CFU-F集落的动态分析；WT-MSC集落来自接受野生型骨髓(WT BM)细胞移植的受体小鼠，NrasG12D-MSC集落来自接受带有NrasG12D突 变的骨髓细胞移植的受体小鼠。
图3为流式细胞学检测NrasG12D小鼠骨髓中TER119-CD45-CD31-CD51+Sca1+CD146+表型的MSC和TER119-CD45-CD31-CD51+Sca1-表型的成骨细胞(OB)的数量；图3A显示流式细胞学检测中，MSC和OB的圈门策略；图所示为来自一只代表性WT小鼠(上面，作为对照)和NrasG12D受体小鼠(下面)的流式图；图3B显示全部骨髓有核细胞中MSC细胞绝对数的统计结果；图3C显示全部骨髓有核细胞中OB细胞绝对数的统计结果。
图4显示白血病潜伏期阶段的NrasG12D受体小鼠血清中的促炎细胞因子水平上升；图4A：TGF-β1，图4B：TNF-α，图4C：GM-CSF。
图5显示分离培养的MSC的表面标志物鉴定。
图6显示实施例的MSC移植实验示意图。
图7显示苏木精-伊红染色(A和B)和骨钙素组化染色(C和D)显示MSC注射后NrasG12D受体小鼠靠近骨内膜的成骨细胞部分恢复。
图8显示苏木精-伊红染色(A)和骨钙素组化染色(B)显示MSC注射后第3周，NrasG12D受体小鼠靠近骨内膜的成骨细胞开始恢复。
图9为CFU-F实验(图9A)以及对于CFU-F克隆的统计分析(图9B，n＝4)显示MSC治疗后骨髓间质微环境中的功能性MSC部分恢复。
图10为统计分析显示MSC治疗后的NrasG12D受体小鼠外周血中白细胞总数显著下降(A)，血小板明显升高(B)，红细胞总数无显著变化(C)。
图11显示通过骨髓腔内原位注射MSC的早期干预，可以成功抑制白血病的发展(A)，并显著延长NrasG12D受体小鼠的存活时间(B)；MSC治疗组：n＝6，存活中值＝395.5天；MSC未治疗组：n＝6，存活中值＝238.5天，p＜0.0001。
图12为ELISA检测小鼠血清(图12A)和骨髓腔内TGF-β1水平(图12B)显示 与未治疗小鼠相比，MSC治疗小鼠血清和骨髓腔内的TGF-β1水平显著降低。
图13为体外成骨分化实验验证TGF-β1对成骨生成的影响；图13A显示MSC诱导成骨分化21天后茜素红染色的结果，图13B为单位面积内形成的钙结节面积统计分析(n＝3)。
图14显示流式细胞术分析未治疗和经MSC治疗的NrasG12D受体小鼠骨髓内HSC和MPP的细胞周期(图14A)、HSC的细胞周期统计结果(图14B)以及HSC和MPP绝对数量的统计分析(图14C)。
图15显示ELISA检测体外共培养上清中的TGF-β1水平(图15A)以及胞内流式细胞术染色检测共培养细胞中的TGF-β1水平(图15B)、Mac1+F4/80+NrasG12D肿瘤细胞分泌TGF-β1水平的统计分析(n＝3)(图15C)。
具体实施方式
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
术语定义
本文中使用的术语“骨髓微环境”是指骨髓中邻近造血干细胞的支持细胞所构成的参与造血干细胞的维持、自我更新和定向分化的细胞成分，本文中的骨髓微环境主要指骨髓间充质基质细胞和贴近骨内膜的成骨细胞。
本文中使用的术语“间充质基质细胞”是指骨髓中对造血干细胞具有支持作用的一种基质细胞，能够分泌多种因子来支持造血。
本文中使用的术语“骨内膜”，是指骨内侧贴壁生长的膜组织。
本文中使用的术语“NrasG12D受体小鼠”、“NrasG12D白血病小鼠”可互换使用，均是指通过引入NrasG12D突变构建的类慢粒单白血病(CMML/JMML-like)小鼠模型，其构建方法详见实施例1。
一般方法
骨切片的免疫组织化学染色
对于苏木精和伊红染色，将对照小鼠和NrasG12D受体的胫骨和股骨骨骼固定在4％多聚甲醛中，然后处理用于组织学苏木精和伊红染色(病理实验室，GIBH)。
对于骨钙素的染色，在30％蔗糖脱水后，在14％EDTA中对胫骨脱钙，并将其包埋在石蜡中用于切片(病理实验室，GIBH)。通过封闭缓冲液(5％正常山羊血清和0.3％Triton X-100在PBS中)淬灭和封闭内源性过氧化物酶活性。将载玻片与相关的一级兔抗小鼠多克隆抗体在4℃孵育过夜，随后是二级生物素化抗兔抗体(山羊抗兔IgG-HRP，sc-2004，Santa Cruz，1∶1000稀释)在室温下孵育1小时。使用以下一级抗体：抗骨钙素多克隆抗体(ab93876，Abcam，1∶500)。使用DAB底物和DAB色原体的混合物进行抗原可视化。载玻片用苏木精复染，并用中性树胶封片。使用Motic数字切片扫描系统(VMV1 VMDPCS，Motic)和DSS扫描仪软件捕获和处理图像。
成纤维细胞集落形成单位(CFU-F)测定
将100万个BM有核细胞接种到6孔板中，并在完全MesenCultTM培养基(Catalog 05512，StemCell Technology)中培养10-14天。将BM细胞在37℃、5％CO2下在加湿室中孵育，并在第7天进行半培养基更换。根据Yang D，Zhang X，Dong Y，et al.Enforced expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo.Cell Cycle.2015；14(4)：612-620.中所述，进行吉姆萨染色(Giemsa staining)。
流式细胞分析和分选
对于外周血(50微升)和骨髓的造血谱系分析：如Wang J，Liu Y，Li Z，et al.Endogenous oncogenic Nras mutation initiates hematopoietic malignancies in a dose-and cell type-dependent manner.Blood.2011；118(2)：368-379.中所述进行流式细胞分析。使用以下表面抗原的直接缀合的抗体：FITC-CD45.2(104)，APC-Thy1.2(53-2.1)，生物 素-CD3(145-2C11)，生物素-CD3(RM4-5)，生物素-CD8(53-6.7)，PE-CD19(1D3)，PE-Cy7-Mac1(M1/70)和APC-Gr-1(RB6-8C5)抗体购自eBiosciences。
对细胞因子的细胞内染色，收集细胞并用表面抗原染色：PerCP-Cy5.5-Mac1(M1/70)/APC-Mac1(M1/70)，生物素-G1(RB6-8C5)，PE-Ly6C HK1.4)，APC-eFluor 780-F4/80(BM8)和链霉亲和素-PE抗体购自eBiosciences。细胞固定并透化(Cytofix/Cytoperm，BD Biosciences)，然后通过用PerCP-Cy5.5-TGF-β1(TW7-16B4，Biolegend)进行细胞内染色。
如Wang J，Liu Y，Li Z，et al.Endogenous oncogenic Nras mutation initiates hematopoietic malignancies in a dose-and cell type-dependent manner.Blood.2011；118(2)：368-379.中所述，进行细胞周期分析。简言之，对于HSC/MPP细胞周期分析，用FITC缀合的抗体染色谱系标志物(CD3，CD4，CD8，CD19，B220，TER119，Gr1，Mac1，CD48)。还对于
Figure PCTCN2017108943-appb-000001
780-cKit(2B8)，PerCP-Cy5.5-Sca1(D7)，PE-Cy7-CD150(TC15-12F12.2)，PE-Flt3(A2F10)进行细胞染色。洗涤后，细胞用4％PFA固定。最后，将固定的细胞用PBS中的0.1％皂素透化，并用APC-Ki67(SolA15，eBioscience)染色，然后用DAPI(Invitrogen)染色。
为进行MSC鉴定和分选，通过使用含有2％FBS的DPBS冲洗胫骨和股骨来分离BM细胞，然后将骨切割成小片段，并在37℃下在震荡培养箱中以1mg/ml胶原酶II消化1-2小时。将来自骨内膜和骨髓的收集的细胞混合在一起，以使用70μm细胞过滤器过滤。为进行MSC鉴定和分选，细胞用以下抗体染色：APC-780F-CD45(30-F11)，APC-eFluor 780-Ter119(TER-119)，PE-Cy7-Sca-1(D7)，生物素-CD51(RMV-7)和链霉亲和素-PE购自eBiosciences，APC-CD31(MEC13.3)和PerCP-Cy5.5-CD146(ME-9F1)购自Biolegend。Yang D，Zhang X，Dong Y.et al.Enforced expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo.Cell Cycle. 2015；14(4)：612-620.中描述了染色方案。
在FACS Calibur或LSR Fortessa(BD Bioscience)上分析染色的细胞，并使用AriaII(BD Bioscience)对MSC进行分选以用于RNA测序，然后使用Flowjo软件(FlowJo)分析数据。
ELISA测定
从NrasG12D受体和对照小鼠的外周血中收集血清样品。从NrasG12D受体和对照小鼠的骨髓中收集骨髓细胞外液体样品。根据制造商的说明，使用酶联免疫吸附测定(ELISA)试剂盒(中国北京4A生物技术公司)测定细胞因子(TGF-β1，TNF-α，GM-CSF)的浓度。
成骨分化测定
将MSC以2×104个细胞/cm2的密度接种于用明胶溶液预先包被的6孔板中的生长培养基中，并在37℃，5％CO2的加湿培养箱中培养。当细胞约60-70％融合时，除去生长培养基，并加入2mL小鼠间充质干细胞成骨分化培养基(MUBMX-90021，Cyagen)。培养基每三天更换一次。在成骨分化的整个过程中，将来自野生型，未处理的，MSC处理的NrasG12D白血病小鼠的外周血清(10μL/mL)和5ng/mL TGF-β1细胞因子分别加入诱导培养基中。诱导3周后，将细胞用4％中性甲醛固定，并用茜素红染色。捕获图像，并通过Image-Pro软件计算形成的钙结节面积。
MSC-BMNC共培养
将GFP标记的MSC按照1x105细胞/孔的密度接种在六孔板中，从NrasG12D受体小鼠分选CD45+BMNC，并按照1x106细胞/ml的密度接种在2ml的α-MEM+10％FBS+50ng/ml SCF培养基中。将MSCs和CD45+细胞在37℃，5％CO2的条件下培养。隔天加入1ml新鲜培养基。培养7天后，通过ELISA测定培养上清中的TGF-β1水平，以及通过流式细胞术检测TGF-β1的细胞来源。
实施例
实施例1建立骨髓微环境逐步破坏的小鼠白血病(CMML/JMML-样)模型
将来自NrasG12D突变小鼠(LSL NrasG12D/+；Vav-Cre)或对照小鼠(CD45.2)的白细胞(去除了基质细胞的)通过眼球后静脉移植到亚致死辐照(6.5 Gy)的CD45.1受体鼠，以此构建类慢粒单白血病(CMML/JMML-like)的小鼠白血病模型。通过流式细胞术每月检测分析外周血中肿瘤细胞的负荷增生情况。在经NrasG12D诱导的白血病小鼠模型中，根据小鼠外周血中髓系细胞的比例定义白血病的不同发展阶段，当小鼠外周血中髓系细胞(Mac1)大于20％且小于60％时，为白血病潜伏期阶段(Pre-leukemic phase，下文有时简称PL阶段)，当Mac1＞60％时，为白血病阶段(Leukemic phase)。
实施例2组织学分析证明，在白血病潜伏期阶段，骨髓微环境出现骨内膜成骨细胞逐步耗竭
为了研究在NrasG12D诱导的白血病潜伏期阶段是否存在骨髓间质微环境逐渐耗竭的现象，我们首先将对照小鼠和白血病潜伏期阶段的NrasG12D受体小鼠牺牲后分离胫骨和股骨，并用4％多聚甲醛固定后进行苏木精-伊红染色和骨钙素(osteocalcin)的免疫组化染色。苏木精-伊红染色(图1A)显示白血病潜伏期阶段的NrasG12D受体小鼠中靠近骨内膜的成骨细胞(osteoblast，下文有时简称OB)已经耗竭；骨钙素组化染色(图1B)显示白血病潜伏期阶段的NrasG12D受体小鼠中靠近骨内膜的成骨细胞已经耗竭。也即，胫骨的组化染色证明：在白血病潜伏期阶段，靠近骨内膜的成骨细胞已经几乎消失。
实施例3细胞学分析证明，伴随白血病的发生发展，骨髓微环境的间充质基质细胞(MSC)逐步耗竭
为了评估NrasG12D受体小鼠中的功能性间充质基质细胞的耗竭情况，我们进行了成纤维细胞集落形成单位实验(CFU-F)。首先牺牲小鼠后无菌分离出骨髓有核细胞 (BMNC)并计数，分别将来自三个组中每个小鼠的1x106个BMNC用2mL MesenCultTM完全培养基(Catalog 05512，StemCell Technology)悬浮并接种在六孔板的单个孔。于细胞培养箱中在37℃，5％CO2条件下培养至第7天，在第7天更换一半培养基后继续培养3-7天，培养至第10-14天时将细胞用预冷的乙醇固定并进行瑞氏吉姆萨染色，统计形成的克隆数和克隆大小(图2A)。我们观察到，功能性MSC在白血病潜伏期阶段逐渐减少、耗竭(图2B)。
实施例4流式细胞学检测证明，伴随白血病的发生发展，骨髓微环境出现逐步耗竭
我们进一步通过流式细胞学分析了NrasG12D小鼠骨髓中的表型MSC(即，TER119-CD45-CD31-CD51+Sca1+CD146+)和表型OB(TER119-CD45-CD31-CD51+Sca1-)的数量。首先，用5mL含2％FBS的DPBS将骨髓细胞从胫骨和股骨中吹出，收集骨髓有核细胞。通过把吹净的骨头剪成小的骨片并用1mg/ml II型胶原酶在37℃的震荡培养箱中消化1-2小时，收集骨内膜中的有核细胞。将来自骨髓和骨内膜中的有核细胞混合并用70微米的细胞筛网过滤后加入1mL ACK红细胞裂解液(所述ACK红细胞裂解液通过以下步骤配制而得：将NH4Cl(150mM)8.02g，KHCO3(10mM)1.00g，Na2EDTA(0.1mM)0.37g和H2O 800ml混合，用1N HC1调节pH至7.2-7.4，加水至1L，最后通过0.22μm滤器过滤除菌，室温储存备用)裂解红细胞，离心(4℃，500g，5min)，加入5倍体积DPBS+2％FBS重悬细胞并离心，用500μL DPBS+2％FBS重悬细胞并加入CD16/32(1∶200)抗体冰上封闭15min，之后加入抗体抗CD51-生物素(RMV-7)冰上孵育15min，加入5x体积的DPBS+2％FBS离心，用500μL DPBS+2％FBS重悬细胞并加入以下抗体(1∶200)：抗
Figure PCTCN2017108943-appb-000002
780(30-F11)，抗
Figure PCTCN2017108943-appb-000003
780(TER-119)，抗Sca-1-PE/Cy7(D7)，抗CD31-APC(MEC13.3)，抗CD146-PerCP/Cy5.5(ME-9F1)，冰上孵育15min，加入5倍体积的DPBS+2％FBS 离心，用含DAPI(1∶400)的DPBS+2％FBS重悬细胞。染色的细胞用LSR Fortessa(BD Bioscience)进行上机分析，使用Flowjo软件(FlowJo)对数据进行分析(图3A)。我们的统计分析显示，伴随着白血病的发生发展，表型MSC和OB的数量逐渐减少(图3B和3C)。
实施例5在白血病潜伏期阶段，NrasG12D受体系统性炎症增加
我们对白血病潜伏期阶段的NrasG12D受体小鼠血清中的促炎细胞因子TGF-β1、TNF-α和GM-CSF进行检测发现，TGF-β1、TNF-α和GM-CSF水平显著增加(图4A-C)。
实施例6骨髓间充质基质细胞的分离、体外培养及表型鉴定
为调查MSC是否会帮助被破坏的骨髓间质微环境进行重建，我们尝试进行MSC移植实验。在我们的MSC移植实验中，使用的间充质基质细胞分离自4周龄野生型小鼠的骨内膜和骨髓。首先，将骨髓从骨腔中吹出，把骨头剪成小的骨碎片并用1mg/ml的II型胶原酶在37℃消化1-2小时，洗涤消化后的骨碎片，铺在6 Gm培养皿中加入6mL MSC完全培养基(α-MEM+10％FBS)培养，获得骨组织培养来源的MSC。将骨髓用FBS悬液吹打，获得骨髓有核细胞悬液，随后将其中的MSC进行染色标记(TER119-CD45-CD31-Sca1+CD51+CD146+)，然后直接分选到MSC培养基中，获得骨髓分选来源的MSC。将骨组织培养来源的MSC和骨髓分选来源的MSC混合在一起培养，在第三天洗涤并更换培养基后继续培养。在培养5天后，去除骨碎片并洗涤后通过0.25％(wt/vol)胰酶消化收集贴壁细胞，并按照1∶3进行传代，每48小时更换培养基。注射前，收集分离培养的MSC，用流式细胞术进行表型鉴定(CD45，TER119，CD31，CD51，CD105，LepR，PDGFRα，PDGFRβ，Sca-1，图5)。用于注射使用的MSC均是培养至第3代的细胞。
实施例7 MSC骨髓腔内局部注射方法
按照图6所示程序，通过骨腔内注射，进行MSC移植实验。我们按照2.5×107 个MSC/kg体重的剂量，将细胞悬浮在20μl的DPBS+2％FBS中，对NrasG12D诱导的白血病小鼠的胫骨通过骨髓腔原位注射的方式，每两周注射一次，共进行16周。为了使手术造成的创伤尽可能恢复，我们通过切换注射部位的方法，对每侧胫骨进行间隔治疗，并且每个月对治疗小鼠的外周血进行流式分析跟踪肿瘤负荷的变化情况。
实施例8 MSC骨髓腔内局部注射方法治疗NrasG12D诱导的白血病小鼠模型，成功实现骨髓微环境中成骨细胞恢复
为了弄清楚在MSC移植治疗的NrasG12D白血病小鼠中是否有再生的骨内膜成骨细胞，我们对MSC注射治疗后的NrasG12D白血病小鼠的腿骨进行了苏木精-伊红染色和骨钙素染色。结果显示，在经过MSC治疗后的小鼠胫骨中又观察到沿着骨内侧的骨内膜成骨细胞(图7A-D)。
我们进一步对MSC治疗后的NrasG12D白血病小鼠的胫骨进行了动态染色分析，结果显示，在第一次MSC注射后的第3周，骨内膜开始出现再生的成骨细胞，并且在第4周再生成骨细胞数量显著增加(图8A和8B)。
然而，发明人在进一步的实验中发现，将由发展阶段的NrasG12D白血病小鼠分离的MSC进行上述骨髓腔内注射，则不能降低肿瘤负担(数据未显示)。
实施例9 MSC骨髓腔内局部注射方法治疗NrasG12D诱导的白血病小鼠模型，成功实现骨髓微环境中的间充质基质细胞恢复
为了分析NrasG12D白血病小鼠经MSC治疗后的骨髓间质微环境中的功能性MSC，牺牲经原位MSC治疗后的NrasG12D白血病小鼠，首先分离其骨髓有核细胞BMNC，并进行了CFU-F实验及统计分析(图9A和9B)。结果显示，与未治疗的NrasG12D白血病小鼠相比，经过MSC治疗的NrasG12D白血病小鼠骨髓中BMNC形成了更多的CFU-F集落，这表明：注射部位的骨髓间质微环境中的功能性MSC部分恢复。
实施例10 MSC骨髓腔内局部注射方法在NrasG12D诱导的白血病小鼠中成功实现正常造血恢复
对上述MSC治疗后的NrasG12D白血病小鼠进行血细胞计数，结果显示：MSC治疗后的NrasG12D白血病小鼠外周血中WBC显著降低(p＜0.001)，血小板水平有显着升高(p＜0.001)(图10A-C)，这表明骨髓微环境的重塑可以部分恢复正常造血。
实施例11骨髓腔内局部注射MSC可成功抑制肿瘤生长，延长生存期
我们在白血病潜伏期阶段(外周血中Mac1+％＜60％)对小鼠进行MSC干预治疗，结果发现，在没有MSC治疗干预的情况下，对照组所有NrasG12D白血病小鼠的外周血中髓系细胞持续升高，并且都在36周内死亡。而经MSC治疗的NrasG12D白血病小鼠的外周血中髓系细胞没有继续升高(图11A)。并且，MSC早期干预治疗的NrasG12D白血病小鼠(存活中值＝395.5天)比无MSC干预的对照小鼠(存活中值＝238.5天)存活时间延长(图11B)。这表明，MSC的干预可以使NrasG12D白血病小鼠实现带瘤长期生存。
实施例12骨髓腔内局部注射MSC通过降低TGF-β1的水平促进骨髓间质微环境的重塑
TGF-β1对成骨生成具有负调控作用，因此，为研究MSC骨髓腔内局部注射是否通过调节TGF-β1水平促进骨内膜成骨细胞的再生，我们进行了如下实验：首先，通过ELISA方法检测了治疗小鼠血清和骨髓腔内的TGF-β1水平；同时，通过组化方法对NrasG12D白血病小鼠的骨髓切片进行骨钙素的免疫组化染色，结果均显示：与未治疗的对照小鼠相比，经MSC治疗的NrasG12D白血病小鼠的血清和骨髓腔内的TGF-β1水平显著降低(参见图12A和12B)。
然后，我们进行了体外成骨分化实验以验证TGF-β1对成骨生成的影响。在野生型MSCs诱导成骨分化过程中，分别持续添加来自未治疗的NrasG12D白血病小鼠血 清、经MSC治疗的NrasG12D白血病小鼠血清以及单独的TGF-β1因子，经过21天的成骨诱导分化，用4％中性甲醛固定细胞后经茜素红染液染色，比较计算单位面积内形成钙结节的面积。结果显示：未治疗的NrasG12D白血病小鼠血清对成骨分化的抑制作用比经MSC治疗的NrasG12D白血病小鼠血清的抑制作用强，而单独的TGF-β1因子也对成骨分化有很强的抑制作用(参见图13A和13B)。这表明，MSC骨髓腔内局部注射可能通过降低TGF-β1的水平来促进骨内膜成骨细胞的再生。
实施例13骨髓腔内局部注射MSC通过降低TGF-β1的水平激活休眠态HSC以促进正常造血恢复
高水平的TGF-β1会促进HSC进入休眠态，因此，为了研究MSC骨髓腔内局部注射是否通过调节TGF-β1水平促进正常造血恢复，我们通过流式细胞术对未治疗和经MSC治疗的NrasG12D白血病小鼠骨髓内HSC的细胞周期(Ki-67和DAPI)进行了分析，结果显示：与未治疗的NrasG12D受体小鼠相比，经MSC治疗的NrasG12D受体小鼠骨髓内进入分裂期的HSC比例明显增加，并且MPP细胞绝对数量显著增加(参见图14A、14B和14C)。这表明，骨髓腔内局部注射MSC可能通过降低TGF-β1的水平激活休眠态HSC进入细胞周期，从而促进正常造血恢复。
实施例14 MSC调节Mac1+F4/80+NrasG12D肿瘤细胞分泌TGF-β1的水平
为了研究MSC调控NrasG12D肿瘤细胞分泌TGF-β1的作用方式，我们设计了体外共培养实验。首先，分离GFP小鼠骨髓来源的MSC细胞，按照1x105个MSC/孔的密度接种在六孔板，然后分选NrasG12D受体小鼠骨髓内的CD45+细胞，按照1x106个细胞/ml的密度重悬在共培养的完全培养基中(α-MEM+10％FBS+50ng/μl SCF)，分别进行单独培养、与MSC共培养。每隔一天补充1ml完全培养基，在37℃细胞培养箱中培养7天后，通过ELISA检测培养上清中TGF-β1水平，以及通过流式细胞术检测共培养细胞的胞内TGF-β1水平。结果显示，MSC共培养组上清中的TGF-β1水平 显著低于NrasG12D肿瘤细胞单独培养组上清中的TGF-β1水平(图15A)。同时，胞内流式细胞术检测结果显示，主要为Mac1+F4/80+NrasG12D肿瘤细胞分泌TGF-β1(图15B和15C)。这表明，MSC调节Mac1+F4/80+NrasG12D肿瘤细胞分泌TGF-β1的水平。

Claims (10)

  1. 一种在骨髓微环境受损的受试者中重塑骨髓微环境的方法，其包括：将包含分离的间充质基质细胞(MSC)的组合物植入所述受试者的骨髓腔。
  2. 根据权利要求1所述的方法，其特征在于，所述间充质基质细胞从健康受试者的骨内膜和/或骨髓有核细胞获得；
    优选地，所述受试者为哺乳动物；
    优选地，所述受试者为小鼠，所述间充质基质细胞具有以下表型：TER119-CD45-CD31-Sca1+CD51+CD146+；
    优选地，所述受试者为人，所述间充质基质细胞具有以下表型：CD235ab-CD45-CD34-CD31-CD271+CD146+。
  3. 根据权利要求1所述的方法，其特征在于，所述包含分离的间充质基质细胞的组合物还包含药学上可接受的载体、稀释剂或媒介物；优选地，所述药学上可接受的载体、稀释剂或媒介物为磷酸盐缓冲液或生理盐水。
  4. 根据权利要求1所述的方法，其特征在于，所述植入为多次、局部注射。
  5. 根据权利要求1所述的方法，其特征在于，按照1.0×105～3.0×107个MSC/kg体重、优选2.5×107个MSC/kg体重的剂量，进行所述植入。
  6. 根据权利要求1-5任一项所述的方法，其特征在于，每周一次、每两周一次、每三周一次或每个月一次地进行所述植入；优选地，每两周一次进行所述植入；优选地，在12～24周、优选16周的时间窗内，进行所述植入。
  7. 根据权利要求1-6任一项所述的方法，其特征在于，所述受试者患有选自血液肿瘤以及再生障碍性贫血的疾病；优选地，所述血液肿瘤为白血病；进一步优选地，所述血液肿瘤为幼年型粒单核细胞白血病或慢粒单核细胞白血病。
  8. 根据权利要求1-7任一项所述的方法，其特征在于，在将包含分离的间充质基质细胞(MSC)的组合物植入所述受试者的骨髓腔后，骨髓微环境中呈现出选自以下 的一种或多种情况：成骨细胞恢复、功能性MSC部分恢复。
  9. 根据权利要求1-8任一项所述的方法，其特征在于，在将包含分离的间充质基质细胞(MSC)的组合物植入所述受试者的骨髓腔后，所述受试者呈现出选自以下的一种或多种情况：正常造血恢复、肿瘤生长抑制和生存期延长。
  10. 如权利要求1-9任一项所述的方法在治疗血液肿瘤以及再生障碍性贫血中的用途；
    优选地，所述血液肿瘤为白血病；
    进一步优选地，所述血液肿瘤为幼年型粒单核细胞白血病或慢粒单核细胞白血病。
PCT/CN2017/108943 2017-10-16 2017-11-01 一种重塑骨髓微环境的方法 WO2019075782A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201780027447.XA CN109069543B (zh) 2017-10-16 2017-11-01 一种重塑骨髓微环境的方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710958674.6 2017-10-16
CN201710958674.6A CN107714726A (zh) 2017-10-16 2017-10-16 一种重塑骨髓微环境的方法

Publications (1)

Publication Number Publication Date
WO2019075782A1 true WO2019075782A1 (zh) 2019-04-25

Family

ID=61211303

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/108943 WO2019075782A1 (zh) 2017-10-16 2017-11-01 一种重塑骨髓微环境的方法

Country Status (3)

Country Link
US (1) US10493106B2 (zh)
CN (1) CN107714726A (zh)
WO (1) WO2019075782A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2994159A1 (en) * 2015-07-30 2017-02-02 Optipulse Inc. Rigid high power and high speed lasing grid structures
WO2019033111A1 (en) 2017-08-11 2019-02-14 Optipulse Inc. LASER GRID STRUCTURES FOR HIGH-SPEED WIRELESS DATA TRANSFERS
US10374705B2 (en) 2017-09-06 2019-08-06 Optipulse Inc. Method and apparatus for alignment of a line-of-sight communications link
CN110398588A (zh) * 2019-08-14 2019-11-01 上海交通大学医学院附属新华医院 一种成骨检测试剂盒及促进骨修复的植入物

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1407088A (zh) * 2001-09-06 2003-04-02 周胜利 从胎盘组织中提取造血干细胞用于建立造血干细胞库的新方法
CN102016010A (zh) * 2008-05-07 2011-04-13 骨治疗公司 用于治疗免疫缺陷或免疫抑制相关之病症和骨疾病的人骨形成细胞
US20110207166A1 (en) * 2009-11-06 2011-08-25 Sarah Rivkah Vaiselbuh Human bone marrow microenvironments and uses thereof
CN104136034A (zh) * 2011-11-30 2014-11-05 先进细胞技术公司 间充质基质细胞及其相关用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8147824B2 (en) * 1999-08-05 2012-04-03 Athersys, Inc. Immunomodulatory properties of multipotent adult progenitor cells and uses thereof
WO2014018230A2 (en) * 2012-07-25 2014-01-30 Albert Einstein College Of Medicine Of Yeshiva University Methods to isolate human mesenchymal stem cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1407088A (zh) * 2001-09-06 2003-04-02 周胜利 从胎盘组织中提取造血干细胞用于建立造血干细胞库的新方法
CN102016010A (zh) * 2008-05-07 2011-04-13 骨治疗公司 用于治疗免疫缺陷或免疫抑制相关之病症和骨疾病的人骨形成细胞
US20110207166A1 (en) * 2009-11-06 2011-08-25 Sarah Rivkah Vaiselbuh Human bone marrow microenvironments and uses thereof
CN104136034A (zh) * 2011-11-30 2014-11-05 先进细胞技术公司 间充质基质细胞及其相关用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YUAN, YAHONG ET AL.: "Intrabone Marrow Injection Enhances Placental Mesenchymal Stem Cell Mediated Support of Hematopoiesis in Mice", TURK J MED SCI, vol. 46, 3 April 2015 (2015-04-03), pages 174 - 184 *

Also Published As

Publication number Publication date
US10493106B2 (en) 2019-12-03
CN107714726A (zh) 2018-02-23
US20190111084A1 (en) 2019-04-18

Similar Documents

Publication Publication Date Title
JP6755850B2 (ja) 間葉系幹細胞の使用
Phinney et al. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation
WO2019075782A1 (zh) 一种重塑骨髓微环境的方法
Raynaud et al. Comprehensive characterization of mesenchymal stem cells from human placenta and fetal membrane and their response to osteoactivin stimulation
Wang et al. Cell-to-cell contact induces mesenchymal stem cell to differentiate into cardiomyocyte and smooth muscle cell
Ochi et al. Use of isolated mature osteoblasts in abundance acts as desired‐shaped bone regeneration in combination with a modified poly‐DL‐lactic‐co‐glycolic acid (PLGA)‐collagen sponge
US10526581B2 (en) Modulation of cardiac stem-progenitor cell differentiation, assays and uses thereof
Maria et al. Human mesenchymal stem cells cultured with salivary gland biopsies adopt an epithelial phenotype
Xu et al. Isolation, characterization, and in vitro propagation of infantile hemangioma stem cells and an in vivo mouse model
Wan et al. Importance of the stem cell microenvironment for ophthalmological cell-based therapy
JP2022527998A (ja) 高機能な製造されたａｂｃｂ５＋間葉系幹細胞
WO2013146992A1 (ja) 歯髄由来の多能性幹細胞の製造方法
KR20140038236A (ko) 간엽줄기세포 응집체의 제조 방법
Zhou et al. Stimulation of hepatoma cell invasiveness and metastatic potential by proteins secreted from irradiated nonparenchymal cells
WO2001034775A1 (en) Mesenchymal stem cells and/or progenitor cells, their isolation and use
Pytlík et al. The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering
Wang et al. Interleukin-1β inhibits normal hematopoietic expansion and promotes acute myeloid leukemia progression via the bone marrow niche
Takeda et al. Osteogenic potential of human bone marrow–derived mesenchymal stromal cells cultured in autologous serum: A preliminary study
RU2530622C2 (ru) Биотрансплантат для восстановления объема костной ткани при дегенеративных заболеваниях и травматических пвореждениях костей и способ его получения
WO2005045008A1 (en) Method for inducing chondrogenesis on mesenchymal stem cells
Ryzhov et al. Adenosine A2B receptors on cardiac stem cell antigen (Sca)-1–positive stromal cells play a protective role in myocardial infarction
US11351119B2 (en) Stem cell-derived microvesicles with enhanced efficacy, use thereof, and method for enhancing efficacy
JPWO2017183676A1 (ja) 被検化合物の脈管新生阻害活性の評価方法
CN109069543B (zh) 一种重塑骨髓微环境的方法
WO2019098264A1 (ja) Ｃｄ３１陽性ｃｄ４５陰性ｃｄ２００陽性の哺乳動物細胞からなる細胞集団、およびその利用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17929374

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17929374

Country of ref document: EP

Kind code of ref document: A1