WO2023241496A1 - Composition for promoting migration, homing and implantation of hematopoietic stem progenitor cells and use thereof - Google Patents
Composition for promoting migration, homing and implantation of hematopoietic stem progenitor cells and use thereof Download PDFInfo
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- WO2023241496A1 WO2023241496A1 PCT/CN2023/099606 CN2023099606W WO2023241496A1 WO 2023241496 A1 WO2023241496 A1 WO 2023241496A1 CN 2023099606 W CN2023099606 W CN 2023099606W WO 2023241496 A1 WO2023241496 A1 WO 2023241496A1
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- cell
- hematopoietic stem
- progenitor cells
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Definitions
- the present invention relates to a composition that promotes the migration, homing and implantation of hematopoietic stem and progenitor cells and its application.
- a composition containing the polypeptide hormone Corticotropin-releasing hormone (CRH) and its use in promoting hematopoiesis is of great significance for improving the efficiency of hematopoietic stem cell transplantation, especially for improving the slow engraftment of cord blood transplantation.
- the present invention discloses the role of polypeptide hormone-CRH in promoting the migration, homing and implantation of hematopoietic stem and progenitor cells.
- This hormone can promote the movement and migration of hematopoietic stem and progenitor cells, and significantly promote the homing and long-term colonization of hematopoietic stem and progenitor cells in the bone marrow in vivo, thereby greatly improving the delay in cord blood transplantation. Therefore, the present invention is of great significance for hematopoietic stem and progenitor cell transplantation, especially for promoting the efficacy of cord blood transplantation.
- the present invention provides the following technical solutions:
- the present invention provides a composition for promoting the migration, homing and implantation of hematopoietic stem and progenitor cells, characterized in that the composition includes: adrenocorticotropin-releasing hormone, stromal cell-derived factor, Fms-related tyrosine kinase 3 ligands and thrombopoietin.
- the content ratio of stromal cell-derived factor: Fms-related tyrosine kinase 3 ligand: thrombopoietin: corticotropin-releasing hormone in the composition is 1:1:1:(3-20 ).
- the content ratio of stromal cell-derived factor: Fms-related tyrosine kinase 3 ligand: thrombopoietin: corticotropin-releasing hormone in the composition is 1:1:1:3, 1: 1:1:4.8, 1:1:1:5, 1:1:1:6, 1:1:1:7, 1:1:1:8, 1:1:1:9, 1:1: 1:10, 1:1:1:11, 1:1:1:12, 1:1:1:13, 1:1:1:14, 1:1:1:15, 1:1:1: 16. 1:1:1:17, 1:1:1:18, 1:1:1:19, 1:1:1:20.
- the content of corticotropin-releasing hormone is 140ng/ml-950ng/ml, preferably 140ng/ml, 200ng/ml, 240ng/ml, 300ng/ml, 400ng/ml, 500ng/ml , 600ng/ml, 700ng/ml, 800ng/ml, 900ng/ml, 950ng/ml, more preferably 240ng/ml.
- the stromal cell-derived factor is present in an amount of 50 ng/ml to 100 ng/ml, preferably 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, and more Preferably 50ng/ml.
- the content of the Fms-related tyrosine kinase 3 ligand is 50ng/ml-100ng/ml, preferably 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, more preferably 50ng/ml.
- the content of thrombopoietin is 50ng/ml-100ng/ml, preferably 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, more preferably Ground 50ng/ml.
- the present invention provides a culture medium that promotes migration, homing and implantation of hematopoietic stem and progenitor cells, characterized in that the culture medium includes: basal culture medium, adrenocorticotropin-releasing hormone, stromal cell-derived factors, Fms Related tyrosine kinase 3 ligands and thrombopoietin.
- the basal medium is serum-free medium, preferably Stem Span SFEM serum-free medium.
- the present invention provides the use of adrenocorticotropin-releasing hormone in the preparation of a medicament for the treatment of hematological malignant tumors, hematological non-malignant tumors, solid tumors, immune system diseases, genetic or metabolic diseases.
- the hematologic malignancy is chronic myelogenous leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute promyelocytic leukemia, non-Hodgkin's lymphoma Hodgkin lymphoma, myeloma, multiple myeloma, myelofibrosis and myelodysplastic syndrome, Burkitt lymphoma, B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, T-cell Lymphoma, plasmablastic lymphoma, cutaneous T-cell lymphoma, primary macroglobulinemia, plasma cell leukemia, plasmablastic lymphoma, hairy cell leukemia, systemic mastocytosis, or blastoblastoma dendritic cell tumor;
- Non-malignant tumors of the hematological system are aplastic anemia, Fanconi anemia, thalassemia, sickle cell anemia, myelofibrosis, severe paroxysmal nocturnal hemoglobinuria or amegakaryocytic thrombocytopenia;
- Solid tumors are breast cancer, ovarian cancer, testicular cancer, renal cancer, neuroblastoma, small cell lung cancer, germ cell tumor, Ewing's sarcoma, soft tissue sarcoma, Wilms' tumor, osteosarcoma, medulloblastoma, or malignant brain tumors;
- Immune system diseases include severe combined immunodeficiency, severe autoimmune disease, primary central nervous system lymphoma, eczema and thrombocytopenia with immunodeficiency syndrome, chronic granulomatosis, IPEX syndrome, AL amylosis, POEMS syndrome, Hemophagocytic syndrome, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease, polymyositis, or dermatomyositis; and
- Genetic or metabolic diseases are mucopolysaccharidoses, dyskeratosis congenita, lysosomal metabolic diseases, spherocytoid leukoencephalopathy, metachromatic leukodystrophy, or X-linked adrenoleukodystrophy.
- the present invention provides a method for promoting the migration, homing and implantation of hematopoietic stem and progenitor cells, which is characterized by comprising applying the above composition to a subject for stimulation.
- the stimulation time is 12-18 hours, preferably 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, more preferably 16 hours.
- the present invention provides a method for promoting the implantation efficiency of hematopoietic stem and progenitor cells, which is characterized by including applying the above composition to a subject for stimulation.
- the present invention provides the use of corticotropin-releasing hormone in the preparation of a composition that promotes migration, homing and engraftment of hematopoietic stem and progenitor cells.
- Hematopoietic stem and progenitor cells express a specific molecule, CXCR4, which is the ligand of the chemokine SDF-1. Hematopoietic stem and progenitor cells migrate in response to the induction of SDF-1 through CXCR4. The migration ability of hematopoietic stem and progenitor cells plays a crucial role in their hematopoiesis. Abnormal migration ability of hematopoietic stem and progenitor cells often leads to impaired hematopoiesis.
- hematopoietic stem progenitor cells During traditional bone marrow transplantation, hematopoietic stem cells are infused through peripheral veins. The infused hematopoietic stem cells circulate through the body and enter other tissues such as bone marrow, liver, spleen, lungs, etc., but most of them remain in the lungs, with only a small amount remaining. Under the influence of cytokines, hematopoietic stem cells cross vascular endothelial cells and reach the bone marrow cavity. This process is called the "homing" of hematopoietic stem cells. Only hematopoietic stem cells that home and settle in the bone marrow microenvironment can further proliferate, differentiate, and reconstruct hematopoiesis.
- Hematopoietic stem and progenitor cell implantation Hematopoietic stem and progenitor cells reach the bone marrow cavity through homing to exert their hematopoietic function. However, not all hematopoietic stem and progenitor cells that reach the bone marrow cavity can exert their hematopoietic function. Only a small number of hematopoietic stem and progenitor cells can. They colonize in the bone marrow cavity and form a stem cell pool that can perform normal functions, which is called "Stem cell niche". In this microenvironment, hematopoietic stem and progenitor cells maintain totipotency by interacting with the extracellular matrix and cells in the microenvironment to ensure hematopoietic ability.
- Corticotropin-releasing hormone Stimulates the synthesis and secretion of adrenocorticotropic hormone from the anterior pituitary gland and is the main factor driving the body's response to stress. It is also present in diseases that cause inflammation and plays an important role in regulating the inflammatory response.
- the slowness of cord blood transplantation is related to a variety of factors.
- the main factors are the hematopoietic stem progenitor cells themselves and the number of functional cells. Hematopoietic stem and progenitor cells are transported to the bone marrow cavity through peripheral blood to perform hematopoietic function. However, not all hematopoietic stem and progenitor cells that reach the bone marrow cavity have long-term hematopoietic function. Only hematopoietic stem and progenitor cells with long-term hematopoietic function can continue to perform hematopoietic function to achieve therapeutic purposes.
- CD34 + hematopoietic stem and progenitor cells are a type of adult stem cells that have the characteristics of stem cell self-renewal and differentiation potential. They are the "seeds" in the blood system and can form all cells in the blood system during the hematopoietic process.
- the CD34 molecule is one of the most important markers of hematopoietic stem and progenitor cells. It was the first molecule to be extensively studied in the isolation and identification of hematopoietic stem and progenitor cells. The expression of CD34 in human bone marrow, peripheral blood and umbilical cord blood is 0.1-4.9%. Currently, some other surface marker molecules have been used in combination with CD34 to distinguish the original cell population.
- Stem Span SFEM Serum-Free Medium This medium is a serum-free expansion medium that has been developed and tested for the in vitro culture and expansion of human hematopoietic cells and is supplemented with appropriate growth factors and supplements. This allows the flexibility to prepare culture media that meets their requirements.
- SFEM When used in combination with appropriate cytokines, SFEM has been used to culture and expand hematopoietic cells isolated from other species, including mice, non-human primates, and dogs. SFEM is also used for the culture of various other hematopoietic and non-hematopoietic cell types.
- SFEM can be used to expand CD34 + cells isolated from human umbilical cord blood, mobilized peripheral blood, or bone marrow samples, or to expand and differentiate lineage-committed progenitor cells to generate erythrocytes, bone marrow, or megakaryocytes.
- Cell progenitor cell group cells can be used to expand CD34 + cells isolated from human umbilical cord blood, mobilized peripheral blood, or bone marrow samples, or to expand and differentiate lineage-committed progenitor cells to generate erythrocytes, bone marrow, or megakaryocytes.
- SCF Stromal cell-derived factor is a hematopoietic growth factor that regulates hematopoietic cells by binding to the c-Kit receptor and plays a vital role in the survival, proliferation and differentiation of hematopoietic stem cells.
- Flt3-L Fms-related tyrosine kinase 3 ligand, a hematopoietic growth factor. It regulates the apoptosis, proliferation and differentiation of hematopoietic progenitor cells by binding to Fms-related tyrosine kinase 3 ligand.
- TPO Thrombopoietin
- MPL myeloproliferative leukemia protein
- HSCs hematopoietic stem cells
- SDF-1 ⁇ Human stromal cell-derived factor 1 ⁇ plays an important role in the formation of pseudopods, migration, proliferation and intercellular adhesion of human hematopoietic cells.
- hCD45 + It is a human leukocyte antigen used to track and indicate the remodeling of implanted hematopoietic stem cells in NSG mice.
- NSG mice This mouse lacks mature T cells, B cells, and NK cells and can efficiently engraft human CD34+ hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), and cell line-derived xenografts (CDX) , patient-derived xenografts (PDX) or adult stem cells and tissues can realize the reconstruction of the human immune system.
- HSC hematopoietic stem cells
- PBMC peripheral blood mononuclear cells
- CDX cell line-derived xenografts
- PDX patient-derived xenografts
- adult stem cells and tissues can realize the reconstruction of the human immune system.
- HSC hematopoietic stem cells
- PBMC peripheral blood mononuclear cells
- CDX cell line-derived xenografts
- PDX patient-derived xenografts
- adult stem cells and tissues can realize the reconstruction of the human immune system.
- HSC hem
- GM-CSF mobilization uses granulocyte colony-stimulating factor to migrate hematopoietic stem cells in the bone marrow into the peripheral blood circulation, significantly increasing the number of CD34 + hematopoietic stem cells in the peripheral blood circulation, and then using a blood cell separator to separate and collect single nuclei from the donor's peripheral blood. cells, which are rich in a large number of hematopoietic stem cells, which can meet the needs of hematopoietic stem cell transplantation.
- Figure 1 shows that CRH can significantly promote the movement and migration of hematopoietic stem and progenitor cells in vitro.
- Figure 1a is the experimental model. Hematopoietic stem and progenitor cells are spread in the upper chamber of Transwell, and the lower layer contains SDF-1 ⁇ . After 4 hours, they are moved to the upper chamber. The lower chamber cells were collected for counting, and the collected lower chamber cells were subjected to colony formation experiments.
- the results in Figures 1b and 1c show that compared with the control group without CRH treatment, CRH treatment can significantly enhance the migration of hematopoietic stem and progenitor cells in response to SDF-1 ⁇ .
- Figure 2 shows that CRH can significantly promote the homing of hematopoietic stem and progenitor cells in vivo.
- the main sources of hematopoietic stem and progenitor cells include umbilical cord blood and peripheral blood mobilized with GM-CSF.
- the results in Figure 2a show that compared with the control group that was not treated with CRH, CRH treatment can significantly promote the growth of hematopoietic stem and progenitor cells derived from umbilical cord blood. Homing effect.
- the results in Figure 2b show that CRH treatment can also significantly promote the homing effect of mobilized hematopoietic stem and progenitor cells derived from peripheral blood.
- Figure 3 shows that CRH can significantly promote the initial long-term colonization of hematopoietic stem and progenitor cells in vivo.
- the peripheral blood of NSG mice was collected every 4 weeks, and the proportion of human leukocyte antigen hCD45 was measured by flow cytometry to evaluate the implantation effect.
- the results in Figures 3a and 3b show that compared with the control group without CRH treatment, CRH treatment can significantly Promotes human hematopoietic stem progenitor cell engraftment in primary transplanted NSG mice. Further, at 16 weeks, the mice were sacrificed, the bone marrow was isolated and prepared into a single cell suspension, and the proportion of hCD45 was detected by flow cytometry.
- Figure 4 shows that CRH can significantly promote secondary colonization of hematopoietic stem and progenitor cells in vivo.
- the transplantation effect of the secondary transplantation mice was evaluated using the same detection methods as the primary transplantation.
- the results in Figures 4a, 4b, and 4c show that compared with the control group without CRH treatment, CRH treatment can significantly promote the secondary colonization of hematopoietic stem and progenitor cells in NSG mice.
- the differentiation ability of hematopoietic stem progenitor cells in vivo was further measured by flow cytometry to detect the proportion of positive cells such as hCD3 + , hCD19 + , hCD33 + and so on.
- the results in Figures 4d and 4e show that CRH treatment can significantly regulate the differentiation of hematopoietic stem progenitor cells into the myeloid. differentiation of lineage cells.
- Example 1 can significantly promote the movement and migration of hematopoietic stem and progenitor cells in vitro
- Collect newborn umbilical cord blood (about 80mL each), dilute the blood sample with 1-2 times the volume of PBS solution, and mix thoroughly; first add 15ml Ficoll separation solution to each 50ml centrifuge tube, then slowly add 20-30ml PBS blood sample and mix liquid and centrifuged at room temperature. Adjust the centrifuge speed to 0, 20°C, 450g, and centrifuge for 30 minutes.
- Mononuclear cells will appear in the middle layer; carefully absorb them with a Pasteur pipette, then fill the centrifuge tube with 1 ⁇ PBS and fully wash the mononuclear cells (400g, Centrifuge for 10 minutes), count, and use MACS magnetic beads to sort cord blood CD34+ hematopoietic stem cells (Human CD34 Microbead Kit, Miltenyi Biotec, 130-046-702, according to the operating instructions of the magnetic beads).
- Stem Span SFEM serum-free medium Stem Span SFEM serum-free medium (Stem cell, Cat#09650) was resuspended and divided into two wells, 3x10 5 cells/well.
- One well was set as a blank control group: multi-cytokine factors were added to the culture medium: SCF (50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18).
- the other well was set as the CRH treatment group: multiple cytokines were added to the culture medium: SCF (50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18) and CRH (240ng/ml, MCE HY-P0086). Both groups were cultured at 5% CO2 and 37 °C for 16 h.
- RPMI 1640 suspension containing 1 ⁇ 10 5 cells was spread in the upper chamber of a prepared Transwell (Corning 14331) with a pore size of 5 ⁇ m.
- the lower chamber is 600 ⁇ l RPMI 1640 medium (containing SDF-1 ⁇ , 100ng/ml, Peprotech 300-28A), cultured at 5% CO 2 and 37°C, and the cells that migrated to the lower chamber were collected after 4 hours and counted.
- the cells were subjected to colony formation assay to compare the migration ability of hematopoietic stem and progenitor cells induced by SDF-1 ( Figure 1a).
- the results in Figure 1b show that compared with the control group, CRH treatment can increase the migration rate of CD34+ cells induced by SDF-1, and the migration rate is increased by 2.08 times.
- the results in Figure 1c show that the number of hematopoietic stem and progenitor cells that migrated to the lower chamber was 1.74 times that of the control group. The above results prove that CRH can promote the migration and movement of hematopoietic stem and progenitor cells in vitro.
- Example 2 CRH can significantly promote the homing of hematopoietic stem and progenitor cells in vivo
- CD34 + hematopoietic stem and progenitor cells were isolated and purified, resuspended in Stem Span SFEM serum-free medium, and two wells were set at 1.5x10 6 cells/well, and one well was set as a blank control group: multi-cytokine factor: SCF (50ng) was added to the culture medium /ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18).
- SCF 50ng
- Flt3-L 50ng/ml Peprotech, 300-19
- TPO 50ng/ml Peprotech, 300-18
- the other well was set as the CRH treatment group: multiple cytokines were added to the culture medium: SCF (50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18) and CRH (240ng/ml, MCE HY-P0086). Incubate at 5% CO2 and 37°C for 16h. After stimulation, cells were collected for counting, resuspended in 1 ⁇ PBS, and adjusted to a cell concentration of 1 ⁇ 10 6 /200 ⁇ l, and then injected through the tail vein into NSG mice (NM-NSG) irradiated with a sublethal dose (250Rad).
- SCF 50ng/ml Peprotech, 300-70
- Flt3-L 50ng/ml Peprotech, 300-19
- TPO 50ng/ml Peprotech, 300-18
- CRH 240ng/ml, MCE HY-P
- Lyse red blood cells in 1ml of red blood cell lysis solution (Biosharp EL503B) in ice bath for 5 to 10 minutes, then add 10ml to 15ml of 1 ⁇ PBS solution to terminate the lysis reaction, filter through a 200-mesh nylon mesh, 2000rpm, 10min, 4°C, centrifuge to collect cells, and add 1 ⁇ PBS solution again Wash 1 time. Adjust cell concentration.
- Add the currently prepared flow cytometry antibodies detect the proportion of human CD45-positive cells in mouse bone marrow through flow cytometry equipment, and evaluate the homing status of hematopoietic stem and progenitor cells.
- Figure 2 shows that compared with the control group, CRH treatment can increase the homing efficiency of cord blood-derived CD34 + hematopoietic stem progenitor cells in NSG by 2.17 times (Figure 2a). At the same time, hematopoietic stem and progenitor cells derived from peripheral blood were mobilized to conduct in vivo homing analysis in NSG mice ( Figure 2b). The results showed that compared with the control group, CRH treatment could increase 1.54 times. The above results indicate that CRH treatment can promote the homing effect of hematopoietic stem and progenitor cells in vivo.
- Example 3 CRH can significantly promote the initial long-term colonization of hematopoietic stem and progenitor cells in vivo
- CD34 + hematopoietic stem and progenitor cells were isolated and purified, resuspended in Stem Span SFEM serum-free medium, and two wells were set at 1.5 ⁇ 10 6 cells/well, and one well was set as a blank control group: multi-cytokine factors were added to the culture medium: SCF ( 50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18).
- the other well was set as the CRH treatment group: multiple cytokines were added to the culture medium: SCF (50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18) and CRH (240ng/ml, MCE HY-P0086), cultured at 5% CO2 and 37°C for 16h. After stimulation, cells were collected for counting, resuspended in 1 ⁇ PBS, and adjusted to a cell concentration of 1 ⁇ 10 5 /200 ⁇ l, and then injected through the tail vein into NSG mice irradiated with a sublethal dose (250Rad) (sublethal).
- SCF 50ng/ml Peprotech, 300-70
- Flt3-L 50ng/ml Peprotech, 300-19
- TPO 50ng/ml Peprotech, 300-18
- CRH 240ng/ml, MCE HY-P0086
- mice After a high dose of irradiation, it will cause the destruction of the original hematopoietic system of mice, thereby promoting the growth of hematopoietic stem and progenitor cells in the bone marrow. colonization and differentiation), the peripheral blood of mice was collected through the tail vein every 4 weeks, and flow cytometry was used to detect the proportion of hCD45 + cells in mononuclear cells (CD34 + hematopoietic stem and progenitor cells arrive at the bone cavity through homing, and only Only hematopoietic stem and progenitor cells that arrive in the bone cavity can differentiate and develop into human leukocytes and mobilize into the peripheral blood); the above-mentioned NSG is sacrificed at week 16 (at 12-16 weeks, the transplanted hematopoietic stem and progenitor cells already have a mature multi-cell lineage) Recipient mice, mouse bone marrow cells were aseptically isolated, part of which was used to detect the proportion of human CD45
- Example 4 can significantly promote secondary long-term colonization of hematopoietic stem and progenitor cells in vivo
- Aseptically isolated bone marrow from NSG mice that were first transplanted at 16 weeks was resuspended in 1 ⁇ PBS (pre-cooled), adjusted to a cell concentration of 5 ⁇ 10 6 /200 ⁇ l, and injected into the tail vein into NSG that had been irradiated with a sublethal dose (250Rad).
- a secondary transplantation model was constructed (used to detect the self-renewal and differentiation capabilities of hematopoietic stem and progenitor cells).
- the proportion of human hCD45 + cells in the peripheral blood mononuclear cells of NSG mice was detected by flow cytometry every 4 weeks; the NSG recipient mice with the above secondary transplantation were sacrificed at the 16th week, and the mouse bone marrow mononuclear cells were detected by flow cytometry.
- the proportion of nuclear cells, human hCD45 and other positive cells was used to evaluate the long-term colonization ability of hematopoietic stem and progenitor cells.
- the results of Figures 4a, 4b, and 4c show that compared with the control group, the enhanced engraftment effect of CRH treatment is also obvious in the transplanted secondary NSG recipient mice.
- the positive proportion of hCD33 in the CRH-treated group was 19.05 times that of the control group.
- the hCD33 positive proportion in the CRH-treated group was 5.71 times that of the control group.
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Abstract
Provided in the present invention are a composition for promoting the migration, homing and implantation of hematopoietic stem progenitor cells and the use thereof. The composition contains a corticotropin-releasing hormone. The composition has important significance for improving the hematopoietic stem cell transplantation efficiency, especially for improving the slow implantation of umbilical cord blood transplantation.
Description
本发明涉及促进造血干祖细胞迁移、归巢和植入的组合物及其应用，尤其涉及包含多肽激素—促肾上腺皮质激素释放激素(Corticotropin-releasing hormone，CRH)的组合物及其在促进造血干祖细胞迁移、归巢和植入中的应用，这对于提高造血干细胞移植效率，尤其对于改善脐血移植植入缓慢具有重要意义。The present invention relates to a composition that promotes the migration, homing and implantation of hematopoietic stem and progenitor cells and its application. In particular, it relates to a composition containing the polypeptide hormone Corticotropin-releasing hormone (CRH) and its use in promoting hematopoiesis. The application of stem and progenitor cell migration, homing and implantation is of great significance for improving the efficiency of hematopoietic stem cell transplantation, especially for improving the slow engraftment of cord blood transplantation.
植入延缓依然是造血干细胞移植，尤其是脐血造血干细胞移植，当前临床面临的瓶颈难题。目前，改善脐血移植植入延缓的方法有很多，但大多机制复杂，临床应用的可实施性及简便性较低，很难满足临床应用。例如：在2003年由Minnesota团队首先使用氟达拉滨、环磷酰胺和亚致死剂量全身辐射的方案，并注入两份匹配的脐血单元用于移植治疗，他们发现这种方式能够将单份脐血移植治疗后病人的康复率由30％提升到50％1。但是，最近的研究数据表明这种方法只能够针对特定的恶性血液疾病，并不具有普适性，且这种方式可能只适用于孩童的移植治疗，对于成年人的数据还待进一步确认。此外，脐血样本的选择也是提升移植效率的常用方式。Barker JN在对1061名成人和儿童脐血移植后的分析中发现，无论细胞剂量如何，和供者人类白细胞抗原(HLA)完全相匹配的受者，都体现出良好的预后2-4。然而，这种完全相匹配的难度较高，往往会错过最佳治疗时间导致移植失败。Delay in engraftment is still the bottleneck problem faced by hematopoietic stem cell transplantation, especially cord blood hematopoietic stem cell transplantation. At present, there are many methods to improve the delay in cord blood transplantation, but most of them have complex mechanisms, low feasibility and simplicity of clinical application, and are difficult to meet clinical application. For example: In 2003, the Minnesota team first used a regimen of fludarabine, cyclophosphamide, and sublethal doses of whole-body radiation, and infused two matched cord blood units for transplantation treatment. They found that this method could convert a single unit into The recovery rate of patients after cord blood transplantation has increased from 30% to 50% 1 . However, recent research data shows that this method can only target specific malignant hematological diseases and is not universal. Moreover, this method may only be suitable for transplantation treatment of children, and the data for adults need to be further confirmed. In addition, cord blood sample selection is also a common way to improve transplantation efficiency. In an analysis of 1061 adults and children after cord blood transplantation, Barker JN found that recipients who were fully matched to the donor's human leukocyte antigen (HLA) showed a good prognosis regardless of the cell dose2-4 . However, this exact matching is difficult, and the optimal treatment time is often missed, resulting in transplant failure.
因此，还需要切实可行、简单有效的方法，以提高造血干祖细胞移植效率，解决脐血移植植入延缓。Therefore, practical, simple and effective methods are needed to improve the efficiency of hematopoietic stem and progenitor cell transplantation and solve the delay in cord blood transplantation.
发明内容Contents of the invention
为了寻找切实可行、简单有效的方法，以提高造血干祖细胞移植效率，解决脐血移植植入延缓，本发明公开了多肽类激素-CRH在促进造血干祖细胞迁移，归巢以及植入中的应用。该激素能够促进造血干祖细胞的运动和迁移，并在体内显著促进造血干祖细胞定向骨髓的归巢和长期定植，从而大大改善脐血移植植入延缓。因此，本发明对造血干祖细胞移植，尤其对促进脐血移植疗效意义重大。In order to find a feasible, simple and effective method to improve the transplantation efficiency of hematopoietic stem and progenitor cells and solve the delay of cord blood transplantation and implantation, the present invention discloses the role of polypeptide hormone-CRH in promoting the migration, homing and implantation of hematopoietic stem and progenitor cells. Applications. This hormone can promote the movement and migration of hematopoietic stem and progenitor cells, and significantly promote the homing and long-term colonization of hematopoietic stem and progenitor cells in the bone marrow in vivo, thereby greatly improving the delay in cord blood transplantation. Therefore, the present invention is of great significance for hematopoietic stem and progenitor cell transplantation, especially for promoting the efficacy of cord blood transplantation.
具体来说，本发明提供以下技术方案：Specifically, the present invention provides the following technical solutions:
一方面，本发明提供促进造血干祖细胞迁移、归巢和植入的组合物，其特征在于，所述组合物包含：促肾上腺皮质激素释放激素、基质细胞衍生因子、Fms相关酪氨酸激酶3配体和血小板生成素。In one aspect, the present invention provides a composition for promoting the migration, homing and implantation of hematopoietic stem and progenitor cells, characterized in that the composition includes: adrenocorticotropin-releasing hormone, stromal cell-derived factor, Fms-related tyrosine kinase 3 ligands and thrombopoietin.
在一些实施方案中，所述组合物中基质细胞衍生因子：Fms相关酪氨酸激酶3配体：血小板生成素：促肾上腺皮质激素释放激素的含量比为1:1:1:(3-20)。In some embodiments, the content ratio of stromal cell-derived factor: Fms-related tyrosine kinase 3 ligand: thrombopoietin: corticotropin-releasing hormone in the composition is 1:1:1:(3-20 ).
在一些实施方案中，所述组合物中基质细胞衍生因子：Fms相关酪氨酸激酶3配体：血小板生成素：促肾上腺皮质激素释放激素的含量比为1:1:1:3、1:1:1:4.8、1:1:1:5、1:1:1:6、1:1:1:7、1:1:1:8、1:1:1:9、1:1:1:10、1:1:1:11、1:1:1:12、1:1:1:13、1:1:1:14、1:1:1:15、1:1:1:16、1:1:1:17、1:1:1:18、1:1:1:19、1:1:1:20。In some embodiments, the content ratio of stromal cell-derived factor: Fms-related tyrosine kinase 3 ligand: thrombopoietin: corticotropin-releasing hormone in the composition is 1:1:1:3, 1: 1:1:4.8, 1:1:1:5, 1:1:1:6, 1:1:1:7, 1:1:1:8, 1:1:1:9, 1:1: 1:10, 1:1:1:11, 1:1:1:12, 1:1:1:13, 1:1:1:14, 1:1:1:15, 1:1:1: 16. 1:1:1:17, 1:1:1:18, 1:1:1:19, 1:1:1:20.
在一些实施方案中，所述促肾上腺皮质激素释放激素的含量为140ng/ml-950ng/ml，优选地140ng/ml、200ng/ml、240ng/ml、300ng/ml、400ng/ml、500ng/ml、600ng/ml、700ng/ml、800ng/ml、900ng/ml、950ng/ml，更优选地240ng/ml。
In some embodiments, the content of corticotropin-releasing hormone is 140ng/ml-950ng/ml, preferably 140ng/ml, 200ng/ml, 240ng/ml, 300ng/ml, 400ng/ml, 500ng/ml , 600ng/ml, 700ng/ml, 800ng/ml, 900ng/ml, 950ng/ml, more preferably 240ng/ml.
在一些实施方案中，所述基质细胞衍生因子的含量为50ng/ml-100ng/ml，优选地50ng/ml、60ng/ml、70ng/ml、80ng/ml、90ng/ml、100ng/ml，更优选地50ng/ml。In some embodiments, the stromal cell-derived factor is present in an amount of 50 ng/ml to 100 ng/ml, preferably 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, and more Preferably 50ng/ml.
在一些实施方案中，所述Fms相关酪氨酸激酶3配体的含量为50ng/ml-100ng/ml，优选地50ng/ml、60ng/ml、70ng/ml、80ng/ml、90ng/ml、100ng/ml，更优选地50ng/ml。In some embodiments, the content of the Fms-related tyrosine kinase 3 ligand is 50ng/ml-100ng/ml, preferably 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, more preferably 50ng/ml.
在一些实施方案中，所述血小板生成素的含量为50ng/ml-100ng/ml，优选地50ng/ml、60ng/ml、70ng/ml、80ng/ml、90ng/ml、100ng/ml，更优选地50ng/ml。In some embodiments, the content of thrombopoietin is 50ng/ml-100ng/ml, preferably 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, more preferably Ground 50ng/ml.
另一方面，本发明提供促进造血干祖细胞迁移、归巢和植入的培养基，其特征在于，所述培养基包含：基础培养基、促肾上腺皮质激素释放激素、基质细胞衍生因子、Fms相关酪氨酸激酶3配体和血小板生成素。On the other hand, the present invention provides a culture medium that promotes migration, homing and implantation of hematopoietic stem and progenitor cells, characterized in that the culture medium includes: basal culture medium, adrenocorticotropin-releasing hormone, stromal cell-derived factors, Fms Related tyrosine kinase 3 ligands and thrombopoietin.
在一些实施方案中，所述基础培养基为无血清培养基，优选地Stem Span SFEM无血清培养基。In some embodiments, the basal medium is serum-free medium, preferably Stem Span SFEM serum-free medium.
另一方面，本发明提供促肾上腺皮质激素释放激素在用于制备治疗血液系统恶性肿瘤、血液系统非恶性肿瘤、实体瘤、免疫系统疾病、遗传或代谢性疾病的药物中的用途。In another aspect, the present invention provides the use of adrenocorticotropin-releasing hormone in the preparation of a medicament for the treatment of hematological malignant tumors, hematological non-malignant tumors, solid tumors, immune system diseases, genetic or metabolic diseases.
在一些实施方案中，所述血液系统恶性肿瘤为慢性粒细胞白血病、急性髓细胞白血病、急性淋巴细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病、急性早幼粒细胞白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、骨髓瘤、多发性骨髓瘤、骨髓纤维化及骨髓增生异常综合征、伯基特淋巴瘤、B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、T细胞淋巴瘤、浆母细胞性淋巴瘤、皮肤T细胞淋巴瘤、原发性巨球蛋白血症、浆细胞白血病、浆母细胞性淋巴瘤、毛细胞白血病、系统性肥大细胞增多症或成胚浆样树突状细胞瘤；In some embodiments, the hematologic malignancy is chronic myelogenous leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute promyelocytic leukemia, non-Hodgkin's lymphoma Hodgkin lymphoma, myeloma, multiple myeloma, myelofibrosis and myelodysplastic syndrome, Burkitt lymphoma, B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, T-cell Lymphoma, plasmablastic lymphoma, cutaneous T-cell lymphoma, primary macroglobulinemia, plasma cell leukemia, plasmablastic lymphoma, hairy cell leukemia, systemic mastocytosis, or blastoblastoma dendritic cell tumor;
血液系统非恶性肿瘤为再生障碍性贫血、范可尼贫血、地中海贫血、镰状细胞贫血、骨髓纤维化、重型阵发性睡眠性血红蛋白尿症或无巨核细胞性血小板减少症；Non-malignant tumors of the hematological system are aplastic anemia, Fanconi anemia, thalassemia, sickle cell anemia, myelofibrosis, severe paroxysmal nocturnal hemoglobinuria or amegakaryocytic thrombocytopenia;
实体瘤为乳腺癌、卵巢癌、睾丸癌、肾癌、神经母细胞瘤、小细胞肺癌、生殖细胞瘤、尤因氏肉瘤、软组织肉瘤、维尔姆斯瘤、骨肉瘤、成神经管细胞瘤或恶性脑瘤；Solid tumors are breast cancer, ovarian cancer, testicular cancer, renal cancer, neuroblastoma, small cell lung cancer, germ cell tumor, Ewing's sarcoma, soft tissue sarcoma, Wilms' tumor, osteosarcoma, medulloblastoma, or malignant brain tumors;
免疫系统疾病为重症联合免疫缺陷症、严重自身免疫性疾病、原发性中枢神经系统淋巴瘤、湿疹血小板减少伴免疫缺陷综合征、慢性肉芽肿，IPEX综合征，AL淀粉变性、POEMS综合征、嗜血细胞综合征、类风湿性关节炎、多发性硬化、系统性硬化、系统性红斑狼疮、克罗恩病、多肌炎或皮肌炎；以及Immune system diseases include severe combined immunodeficiency, severe autoimmune disease, primary central nervous system lymphoma, eczema and thrombocytopenia with immunodeficiency syndrome, chronic granulomatosis, IPEX syndrome, AL amylosis, POEMS syndrome, Hemophagocytic syndrome, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease, polymyositis, or dermatomyositis; and
遗传或代谢性疾病为粘多糖病、先天性角化不良、溶酶体代谢病、球形细胞样脑白质病、异染性脑白质营养不良或X连锁的肾上腺脑白质营养不良。Genetic or metabolic diseases are mucopolysaccharidoses, dyskeratosis congenita, lysosomal metabolic diseases, spherocytoid leukoencephalopathy, metachromatic leukodystrophy, or X-linked adrenoleukodystrophy.
另一方面，本发明提供促进造血干祖细胞迁移、归巢和植入的方法，其特征在于，包括将上述组合物施用至对象进行刺激。On the other hand, the present invention provides a method for promoting the migration, homing and implantation of hematopoietic stem and progenitor cells, which is characterized by comprising applying the above composition to a subject for stimulation.
在一些实施方案中，所述刺激时间为12-18小时，优选地12小时、13小时、14小时、15小时、16小时、17小时、18小时，更优选地16小时。In some embodiments, the stimulation time is 12-18 hours, preferably 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, more preferably 16 hours.
另一方面，本发明提供促进造血干祖细胞植入效率的方法，其特征在于，包括将上述组合物施用至对象进行刺激。On the other hand, the present invention provides a method for promoting the implantation efficiency of hematopoietic stem and progenitor cells, which is characterized by including applying the above composition to a subject for stimulation.
另一方面，本发明提供促肾上腺皮质激素释放激素在制备促进造血干祖细胞迁移、归巢和植入的组合物中的用途。In another aspect, the present invention provides the use of corticotropin-releasing hormone in the preparation of a composition that promotes migration, homing and engraftment of hematopoietic stem and progenitor cells.
定义
definition
造血干祖细胞迁移：造血干祖细胞上表达特定分子CXCR4，为趋化因子SDF-1的配体，造血干祖细胞通过CXCR4响应SDF-1的诱导进行迁移运动。造血干祖细胞这种迁移能力对于其发挥造血具有至关重要的作用，造血干祖细胞迁移能力发生异常，往往会导致造血受损。Migration of hematopoietic stem and progenitor cells: Hematopoietic stem and progenitor cells express a specific molecule, CXCR4, which is the ligand of the chemokine SDF-1. Hematopoietic stem and progenitor cells migrate in response to the induction of SDF-1 through CXCR4. The migration ability of hematopoietic stem and progenitor cells plays a crucial role in their hematopoiesis. Abnormal migration ability of hematopoietic stem and progenitor cells often leads to impaired hematopoiesis.
造血干祖细胞归巢：传统骨髓移植时造血干细胞经外周静脉输注，输注的造血干细胞随体循环进入骨髓、肝、脾、肺等其他组织，但绝大部分滞留于肺部，仅有少量造血干细胞在细胞因子的影响下穿越血管内皮细胞到达骨髓腔，这一过程称造血干细胞的“归巢”。只有归巢并定居于骨髓微环境的造血干细胞才能进一步增殖、分化并重建造血。Homing of hematopoietic stem progenitor cells: During traditional bone marrow transplantation, hematopoietic stem cells are infused through peripheral veins. The infused hematopoietic stem cells circulate through the body and enter other tissues such as bone marrow, liver, spleen, lungs, etc., but most of them remain in the lungs, with only a small amount remaining. Under the influence of cytokines, hematopoietic stem cells cross vascular endothelial cells and reach the bone marrow cavity. This process is called the "homing" of hematopoietic stem cells. Only hematopoietic stem cells that home and settle in the bone marrow microenvironment can further proliferate, differentiate, and reconstruct hematopoiesis.
造血干祖细胞植入：造血干祖细胞通过归巢作用到达骨髓腔发挥其造血功能，然而并不是所有到达骨髓腔的造血干祖细胞均能发挥造血功能，仅有少部分造血干祖细胞能够定植在骨髓腔中，组成能发挥正常功能的干细胞池，被称为“Stem cell niche”。造血干祖细胞在这种微环境通过和细胞外基质以及微环境细胞相互作用共同维持全能性，保证造血能力。Hematopoietic stem and progenitor cell implantation: Hematopoietic stem and progenitor cells reach the bone marrow cavity through homing to exert their hematopoietic function. However, not all hematopoietic stem and progenitor cells that reach the bone marrow cavity can exert their hematopoietic function. Only a small number of hematopoietic stem and progenitor cells can. They colonize in the bone marrow cavity and form a stem cell pool that can perform normal functions, which is called "Stem cell niche". In this microenvironment, hematopoietic stem and progenitor cells maintain totipotency by interacting with the extracellular matrix and cells in the microenvironment to ensure hematopoietic ability.
促肾上腺皮质激素释放激素：能够刺激垂体前叶的促肾上腺皮质激素的合成及分泌，是驱动身体对压力做出反应的主要因素。它也存在于引起炎症的疾病中，对于调控炎症反应也具有重要的作用。Corticotropin-releasing hormone: Stimulates the synthesis and secretion of adrenocorticotropic hormone from the anterior pituitary gland and is the main factor driving the body's response to stress. It is also present in diseases that cause inflammation and plays an important role in regulating the inflammatory response.
脐血移植植入缓慢：脐血移植缓慢与多种因素相关，造血干祖细胞本身以及能发挥功能的数量为主要因素。造血干祖细胞经外周血输送到骨髓腔中发挥造血功能，然而并不是所有到达骨髓腔的造血干祖细胞均具有长期造血的功能。只有具有长期造血功能的造血干祖细胞才能一直发挥造血功能从而达到治疗目的。Slow engraftment of cord blood transplantation: The slowness of cord blood transplantation is related to a variety of factors. The main factors are the hematopoietic stem progenitor cells themselves and the number of functional cells. Hematopoietic stem and progenitor cells are transported to the bone marrow cavity through peripheral blood to perform hematopoietic function. However, not all hematopoietic stem and progenitor cells that reach the bone marrow cavity have long-term hematopoietic function. Only hematopoietic stem and progenitor cells with long-term hematopoietic function can continue to perform hematopoietic function to achieve therapeutic purposes.
CD34+造血干祖细胞：造血干祖细胞是一类成体干细胞，具有干细胞自我更新以及分化潜能的特性，它是血液系统中的“种子”，在造血过程中能够形成血液系统中的所有细胞。CD34分子是造血干祖细胞最重要的标志之一，在造血干祖细胞的分离以及鉴定中，该分子是第一个被广泛研究的分子。CD34在人骨髓、外周血以及脐带血中的表达为0.1-4.9％，目前一些其他表面标记分子已经和CD34结合使用用来区分原始细胞群体。CD34 + hematopoietic stem and progenitor cells: Hematopoietic stem and progenitor cells are a type of adult stem cells that have the characteristics of stem cell self-renewal and differentiation potential. They are the "seeds" in the blood system and can form all cells in the blood system during the hematopoietic process. The CD34 molecule is one of the most important markers of hematopoietic stem and progenitor cells. It was the first molecule to be extensively studied in the isolation and identification of hematopoietic stem and progenitor cells. The expression of CD34 in human bone marrow, peripheral blood and umbilical cord blood is 0.1-4.9%. Currently, some other surface marker molecules have been used in combination with CD34 to distinguish the original cell population.
Stem Span SFEM无血清培养基：该培养基为无血清扩增培养基，已被开发并测试用于人类造血细胞的体外培养和扩增，并添加了适当的生长因子和补充剂。从而可以灵活地制备满足其要求的培养基。当与适当的细胞因子结合使用时，SFEM已用于培养和扩增从其他物种(包括小鼠、非人类灵长类动物和狗)中分离的造血细胞。SFEM也被用于各种其他造血和非造血细胞类型的培养。使用适当的Stem Span公司扩增补充剂，SFEM可用于扩增从人脐带血、动员外周血或骨髓样本中分离的CD34+细胞，或扩增和分化谱系定型祖细胞以产生红细胞、骨髓或巨核细胞祖细胞群细胞。Stem Span SFEM Serum-Free Medium: This medium is a serum-free expansion medium that has been developed and tested for the in vitro culture and expansion of human hematopoietic cells and is supplemented with appropriate growth factors and supplements. This allows the flexibility to prepare culture media that meets their requirements. When used in combination with appropriate cytokines, SFEM has been used to culture and expand hematopoietic cells isolated from other species, including mice, non-human primates, and dogs. SFEM is also used for the culture of various other hematopoietic and non-hematopoietic cell types. Using appropriate Stem Span expansion supplements, SFEM can be used to expand CD34 + cells isolated from human umbilical cord blood, mobilized peripheral blood, or bone marrow samples, or to expand and differentiate lineage-committed progenitor cells to generate erythrocytes, bone marrow, or megakaryocytes. Cell progenitor cell group cells.
SCF：基质细胞衍生因子，是一种造血生长因子，通过和c-Kit受体相互结合调控造血细胞，对于造血干细胞的存活、增殖和分化具有至关重要的作用。SCF: Stromal cell-derived factor is a hematopoietic growth factor that regulates hematopoietic cells by binding to the c-Kit receptor and plays a vital role in the survival, proliferation and differentiation of hematopoietic stem cells.
Flt3-L：Fms相关酪氨酸激酶3配体，是一种造血生长因子。通过和Fms相关酪氨酸激酶3配体相互结合调控造血祖细胞的凋亡、增殖以及分化。Flt3-L: Fms-related tyrosine kinase 3 ligand, a hematopoietic growth factor. It regulates the apoptosis, proliferation and differentiation of hematopoietic progenitor cells by binding to Fms-related tyrosine kinase 3 ligand.
TPO：血小板生成素(TPO)是哺乳动物巨核细胞发育和血小板生成的主要调节因子。人类血小板生成素通过与其同源受体骨髓增殖性白血病蛋白(MPL)的相互作用组成性循环并维持血小板生成。血小板生成素还在造血干细胞(HSC)的维持和调节中发挥重要作用。TPO: Thrombopoietin (TPO) is a major regulator of mammalian megakaryocyte development and platelet production. Human thrombopoietin constitutively circulates and maintains platelet production through interaction with its cognate receptor myeloproliferative leukemia protein (MPL). Thrombopoietin also plays an important role in the maintenance and regulation of hematopoietic stem cells (HSCs).
SDF-1α：人基质细胞衍生因子1α，对于人造血组细胞在形成伪足、迁移、增殖和细胞间的粘附具有重要的作用。
SDF-1α: Human stromal cell-derived factor 1α plays an important role in the formation of pseudopods, migration, proliferation and intercellular adhesion of human hematopoietic cells.
hCD45+：为人源白细胞抗原，用于追踪及表明植入的造血干细胞在NSG小鼠体内重塑情况。hCD45 + : It is a human leukocyte antigen used to track and indicate the remodeling of implanted hematopoietic stem cells in NSG mice.
NSG小鼠：该小鼠缺失成熟T细胞、B细胞和NK细胞，可高效地植入人CD34+造血干细胞(HSC)、外周血单核细胞(PBMC)、细胞系来源的异体移植物(CDX)、病人来源异种移植物(PDX)或成体干细胞及组织，可以实现人类免疫系统的重建，是研究人体免疫功能、传染病、糖尿病、肿瘤学和干细胞生物学的重要免疫缺陷小鼠，是目前国际公认的免疫缺陷程度较高、较适合人源细胞或组织移植的工具小鼠。NSG mice: This mouse lacks mature T cells, B cells, and NK cells and can efficiently engraft human CD34+ hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), and cell line-derived xenografts (CDX) , patient-derived xenografts (PDX) or adult stem cells and tissues can realize the reconstruction of the human immune system. They are important immunodeficiency mice for studying human immune function, infectious diseases, diabetes, oncology and stem cell biology. They are currently the international It is recognized as a tool mouse with a higher degree of immunodeficiency and more suitable for human cell or tissue transplantation.
GM-CSF动员：通过粒细胞集落刺激因子使骨髓中的造血干细胞迁移到外周血液循环中，使外周血液循环中造血干细胞CD34+数显著增加，再用血细胞分离机分离采集供者外周血单个核细胞，其中富含有大量的造血干细胞，可满足造血干细胞移植的需要。GM-CSF mobilization: uses granulocyte colony-stimulating factor to migrate hematopoietic stem cells in the bone marrow into the peripheral blood circulation, significantly increasing the number of CD34 + hematopoietic stem cells in the peripheral blood circulation, and then using a blood cell separator to separate and collect single nuclei from the donor's peripheral blood. cells, which are rich in a large number of hematopoietic stem cells, which can meet the needs of hematopoietic stem cell transplantation.
从下面结合附图的详细描述中，本发明的上述特征和优点将更明显，其中：The above features and advantages of the present invention will be more apparent from the following detailed description in conjunction with the accompanying drawings, in which:
图1示出CRH能够显著促进体外造血干祖细胞的运动和迁移，图1a为实验模式图，将造血干祖细胞铺在Transwell上室中，下层含有SDF-1α，4小时后移走上室收集下室细胞计数，并将收集的下室细胞进行集落形成实验。图1b、1c结果显示，相较于未使用CRH处理的对照组，CRH的处理能够明显增强造血干祖细胞对于SDF-1α响应的迁移。Figure 1 shows that CRH can significantly promote the movement and migration of hematopoietic stem and progenitor cells in vitro. Figure 1a is the experimental model. Hematopoietic stem and progenitor cells are spread in the upper chamber of Transwell, and the lower layer contains SDF-1α. After 4 hours, they are moved to the upper chamber. The lower chamber cells were collected for counting, and the collected lower chamber cells were subjected to colony formation experiments. The results in Figures 1b and 1c show that compared with the control group without CRH treatment, CRH treatment can significantly enhance the migration of hematopoietic stem and progenitor cells in response to SDF-1α.
图2示出CRH能够显著促进体内造血干祖细胞的归巢。造血干祖细胞来源主要有脐带血以及使用GM-CSF动员的外周血，图2a结果显示，相较于未使用CRH处理的对照组，CRH的处理能够显著促进脐带血来源的造血干祖细胞的归巢作用，图2b结果显示，CRH的处理也能够显著促进动员外周血来源的造血干祖细胞的归巢作用。Figure 2 shows that CRH can significantly promote the homing of hematopoietic stem and progenitor cells in vivo. The main sources of hematopoietic stem and progenitor cells include umbilical cord blood and peripheral blood mobilized with GM-CSF. The results in Figure 2a show that compared with the control group that was not treated with CRH, CRH treatment can significantly promote the growth of hematopoietic stem and progenitor cells derived from umbilical cord blood. Homing effect. The results in Figure 2b show that CRH treatment can also significantly promote the homing effect of mobilized hematopoietic stem and progenitor cells derived from peripheral blood.
图3示出CRH能够显著促进体内造血干祖细胞初次长期定植。通过每4周采集NSG小鼠外周血，流式细胞仪检测人白细胞抗原hCD45的比例评估植入效果，图3a、3b结果显示，和未使用CRH处理的对照组相比，CRH的处理能够显著促进人造血干祖细胞在初次移植的NSG小鼠植入。进一步在16周时，牺牲小鼠，分离骨髓制备成单细胞悬液，流式细胞仪检测hCD45的比例，图3c、3d结果显示，和未使用CRH处理的对照组相比，CRH的处理显著促进了人造血干祖细胞在初次移植的NSG小鼠骨髓中定植。Figure 3 shows that CRH can significantly promote the initial long-term colonization of hematopoietic stem and progenitor cells in vivo. The peripheral blood of NSG mice was collected every 4 weeks, and the proportion of human leukocyte antigen hCD45 was measured by flow cytometry to evaluate the implantation effect. The results in Figures 3a and 3b show that compared with the control group without CRH treatment, CRH treatment can significantly Promotes human hematopoietic stem progenitor cell engraftment in primary transplanted NSG mice. Further, at 16 weeks, the mice were sacrificed, the bone marrow was isolated and prepared into a single cell suspension, and the proportion of hCD45 was detected by flow cytometry. The results in Figures 3c and 3d show that compared with the control group without CRH treatment, CRH treatment significantly Promotes the colonization of human hematopoietic stem progenitor cells in the bone marrow of primary transplanted NSG mice.
图4示出CRH能够显著促进体内造血干祖细胞二次定植。通过和初次移植相同的检测方式来评估二次移植小鼠移植效果。图4a、4b、4c结果显示，和未使用CRH处理的对照组相比，CRH的处理能够显著促进造血干祖细胞在NSG小鼠中的二次定植。进一步通过流式细胞仪检测hCD3+、hCD19+、hCD33+等阳性细胞比例来评估造血干祖细胞在体内的分化能力，图4d、4e结果显示，CRH的处理能够明显调控造血干祖细胞向髓系细胞的分化。Figure 4 shows that CRH can significantly promote secondary colonization of hematopoietic stem and progenitor cells in vivo. The transplantation effect of the secondary transplantation mice was evaluated using the same detection methods as the primary transplantation. The results in Figures 4a, 4b, and 4c show that compared with the control group without CRH treatment, CRH treatment can significantly promote the secondary colonization of hematopoietic stem and progenitor cells in NSG mice. The differentiation ability of hematopoietic stem progenitor cells in vivo was further measured by flow cytometry to detect the proportion of positive cells such as hCD3 + , hCD19 + , hCD33 + and so on. The results in Figures 4d and 4e show that CRH treatment can significantly regulate the differentiation of hematopoietic stem progenitor cells into the myeloid. differentiation of lineage cells.
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.
实施例1 CRH能够显著促进体外造血干祖细胞的运动和迁移Example 1 CRH can significantly promote the movement and migration of hematopoietic stem and progenitor cells in vitro
收集新生儿脐带血(每份约80mL)，1-2倍体积PBS溶液稀释血样，充分混匀后；每个50ml离心管中先加入15ml Ficoll分离液，然后再缓慢加入20-30ml PBS血样混合液，室温离心。离心机升降速均调为0，20℃、450g，离心30min，中间层为单个核细胞；用巴斯德吸管小心吸取，之后用1×PBS加满离心管，充分洗涤单个核细胞(400g，离心10min)，计数，采用MACS磁珠分选脐带血CD34+造血干细胞(Human CD34 Microbead Kit，Miltenyi Biotec,
130-046-702，根据磁珠说明书操作方法)。Stem Span SFEM无血清培养基(Stem cell，Cat#09650)重悬，分为两孔，3x105细胞/孔，一孔设置为空白对照组：培养液中加入多细胞因子：SCF(50ng/ml Peprotech，300-70)，Flt3-L(50ng/ml Peprotech，300-19)，TPO(50ng/ml Peprotech，300-18)。另一孔设置为CRH处理组：培养液中加入多细胞因子：SCF(50ng/ml Peprotech，300-70)，Flt3-L(50ng/ml Peprotech，300-19)，TPO(50ng/ml Peprotech，300-18)以及CRH(240ng/ml，MCE HY-P0086)。将两组在5％CO2和37℃下培养16h。刺激结束后，收集细胞进行计数调整细胞浓度为1×106个/ml，将100μl RPMI 1640含有1x105个细胞的悬液铺在预先准备好的5μm孔径的Transwell(Corning 14331)上室中，下室为600μl RPMI 1640培养基(含SDF-1α，100ng/ml，Peprotech 300-28A)，在5％CO2和37℃下培养，4小时后收集迁移到下室的细胞计数并将收集的细胞进行集落形成实验用于比较造血干祖细胞对于SDF-1诱导的迁移能力(图1a)。图1b结果显示与对照组相比，CRH的处理能够提高CD34+细胞对于SDF-1诱导迁移率，迁移率上调2.08倍。图1c结果显示，迁移至下室的造血干祖细胞数目是对照组的1.74倍。以上结果证明，CRH能够促进造血干祖细胞在体外的迁移以及运动。Collect newborn umbilical cord blood (about 80mL each), dilute the blood sample with 1-2 times the volume of PBS solution, and mix thoroughly; first add 15ml Ficoll separation solution to each 50ml centrifuge tube, then slowly add 20-30ml PBS blood sample and mix liquid and centrifuged at room temperature. Adjust the centrifuge speed to 0, 20°C, 450g, and centrifuge for 30 minutes. Mononuclear cells will appear in the middle layer; carefully absorb them with a Pasteur pipette, then fill the centrifuge tube with 1×PBS and fully wash the mononuclear cells (400g, Centrifuge for 10 minutes), count, and use MACS magnetic beads to sort cord blood CD34+ hematopoietic stem cells (Human CD34 Microbead Kit, Miltenyi Biotec, 130-046-702, according to the operating instructions of the magnetic beads). Stem Span SFEM serum-free medium (Stem cell, Cat#09650) was resuspended and divided into two wells, 3x10 5 cells/well. One well was set as a blank control group: multi-cytokine factors were added to the culture medium: SCF (50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18). The other well was set as the CRH treatment group: multiple cytokines were added to the culture medium: SCF (50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18) and CRH (240ng/ml, MCE HY-P0086). Both groups were cultured at 5% CO2 and 37 °C for 16 h. After stimulation, cells were collected for counting and the cell concentration was adjusted to 1×10 6 cells/ml. 100 μl of RPMI 1640 suspension containing 1×10 5 cells was spread in the upper chamber of a prepared Transwell (Corning 14331) with a pore size of 5 μm. The lower chamber is 600 μl RPMI 1640 medium (containing SDF-1α, 100ng/ml, Peprotech 300-28A), cultured at 5% CO 2 and 37°C, and the cells that migrated to the lower chamber were collected after 4 hours and counted. The cells were subjected to colony formation assay to compare the migration ability of hematopoietic stem and progenitor cells induced by SDF-1 (Figure 1a). The results in Figure 1b show that compared with the control group, CRH treatment can increase the migration rate of CD34+ cells induced by SDF-1, and the migration rate is increased by 2.08 times. The results in Figure 1c show that the number of hematopoietic stem and progenitor cells that migrated to the lower chamber was 1.74 times that of the control group. The above results prove that CRH can promote the migration and movement of hematopoietic stem and progenitor cells in vitro.
实施例2 CRH能够显著促进体内造血干祖细胞的归巢Example 2 CRH can significantly promote the homing of hematopoietic stem and progenitor cells in vivo
分离纯化得到CD34+造血干祖细胞，Stem Span SFEM无血清培养基重悬，设置两孔，1.5x106细胞/孔，一孔设置为空白对照组：培养液中加入多细胞因子：SCF(50ng/ml Peprotech，300-70)，Flt3-L(50ng/ml Peprotech，300-19)，TPO(50ng/ml Peprotech，300-18)。另一孔设置为CRH处理组：培养液中加入多细胞因子：SCF(50ng/ml Peprotech，300-70)，Flt3-L(50ng/ml Peprotech，300-19)，TPO(50ng/ml Peprotech，300-18)以及CRH(240ng/ml，MCE HY-P0086)。在5％CO2和37℃下培养16h。刺激结束后，收集细胞进行计数，1×PBS重悬，并调整细胞浓度为1×106/200μl，随后通过尾静脉注射至经过亚致死剂量(250Rad)辐照的NSG小鼠(NM-NSG-001)中，20h后脱颈牺牲小鼠，喷潵75％酒精移入生物安全柜中，使用无菌剪镊解剖摘取股骨，1ml注射器吸取1×PBS溶液冲洗髓腔，收集冲洗液于15ml离心管中，2000rpm，10min，4℃，离心收集细胞。1ml红细胞裂解液(Biosharp EL503B)冰浴裂解红细胞5min～10min后，10ml～15ml 1×PBS溶液终止裂解反应，200目尼龙网过滤，2000rpm，10min，4℃，离心收集细胞，1×PBS溶液再洗1次。调整细胞浓度。加入现配现用的流式抗体，通过流式细胞仪器检测小鼠骨髓中人CD45阳性细胞比例，评价造血干祖细胞归巢情况。图2显示，和对照组相比，CRH处理能够将脐血来源的C D34+造血干祖细胞在NSG体内归巢效率提升2.17倍(图2a)。同时使用动员外周血来源的造血干祖细胞进行NSG小鼠体内归巢分析(图2b)，结果显示，相较于对照组，CRH的处理能够提升1.54倍。以上结果表明，CRH的处理能够促进造血干祖细胞在体内的归巢效应。CD34 + hematopoietic stem and progenitor cells were isolated and purified, resuspended in Stem Span SFEM serum-free medium, and two wells were set at 1.5x10 6 cells/well, and one well was set as a blank control group: multi-cytokine factor: SCF (50ng) was added to the culture medium /ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18). The other well was set as the CRH treatment group: multiple cytokines were added to the culture medium: SCF (50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18) and CRH (240ng/ml, MCE HY-P0086). Incubate at 5% CO2 and 37°C for 16h. After stimulation, cells were collected for counting, resuspended in 1×PBS, and adjusted to a cell concentration of 1×10 6 /200 μl, and then injected through the tail vein into NSG mice (NM-NSG) irradiated with a sublethal dose (250Rad). -001), sacrifice the mouse after 20 hours by cervical dislocation, spray 75% alcohol and move it into a biological safety cabinet. Use sterile forceps to dissect and remove the femur. Use a 1ml syringe to absorb 1×PBS solution to flush the medullary cavity, and collect the flushing fluid in 15ml. Collect cells by centrifugation in a centrifuge tube at 2000 rpm, 10 min, 4°C. Lyse red blood cells in 1ml of red blood cell lysis solution (Biosharp EL503B) in ice bath for 5 to 10 minutes, then add 10ml to 15ml of 1×PBS solution to terminate the lysis reaction, filter through a 200-mesh nylon mesh, 2000rpm, 10min, 4°C, centrifuge to collect cells, and add 1×PBS solution again Wash 1 time. Adjust cell concentration. Add the currently prepared flow cytometry antibodies, detect the proportion of human CD45-positive cells in mouse bone marrow through flow cytometry equipment, and evaluate the homing status of hematopoietic stem and progenitor cells. Figure 2 shows that compared with the control group, CRH treatment can increase the homing efficiency of cord blood-derived CD34 + hematopoietic stem progenitor cells in NSG by 2.17 times (Figure 2a). At the same time, hematopoietic stem and progenitor cells derived from peripheral blood were mobilized to conduct in vivo homing analysis in NSG mice (Figure 2b). The results showed that compared with the control group, CRH treatment could increase 1.54 times. The above results indicate that CRH treatment can promote the homing effect of hematopoietic stem and progenitor cells in vivo.
实施例3 CRH能够显著促进体内造血干祖细胞初次长期定植Example 3 CRH can significantly promote the initial long-term colonization of hematopoietic stem and progenitor cells in vivo
分离纯化得到CD34+造血干祖细胞，Stem Span SFEM无血清培养基重悬，设置两孔，1.5×106细胞/孔，一孔设置为空白对照组：培养液中加入多细胞因子：SCF(50ng/ml Peprotech，300-70)，Flt3-L(50ng/ml Peprotech，300-19)，TPO(50ng/ml Peprotech，300-18)。另一孔设置为CRH处理组：培养液中加入多细胞因子：SCF(50ng/ml Peprotech，300-70)，Flt3-L(50ng/ml Peprotech，300-19)，TPO(50ng/ml Peprotech，300-18)以及CRH(240ng/ml，MCE HY-P0086)，在5％CO2和37℃下培养16h。刺激结束后，收集细胞进行计数，1×PBS重悬，并调整细胞浓度为1×105/200μl，随后通过尾静脉注射至经过亚致死剂量(250Rad)辐照的NSG小鼠中(亚致死剂量辐照之后，会引发小鼠原有的造血系统毁坏，进而促进造血干祖细胞在骨髓中
的定植以及分化)，每4周通过尾静脉采集小鼠外周血，使用流式细胞仪检测hCD45+细胞在单个核细胞中的比例(CD34+造血干祖细胞通过归巢作用抵达骨腔，只有抵达骨腔的造血干祖细胞才能够分化发育为人白细胞并动员至外周血中)；第16周(12-16周时，移植的造血干祖细胞已经具有成熟的多细胞谱系)时牺牲上述NSG受体鼠，无菌分离小鼠骨髓细胞，部分用来检测小鼠骨髓中人CD45等阳性细胞比例，另一部分重悬用于后续二次移植实验。图3a、3b结果显示，在移植4周后，通过流式检测hCD45在NSG小鼠外周血单个核细胞的比例，在CRH处理组中平均比例为26.06％，而在对照组外周血hCD45平均比例为7.33％。进一步在初次移植后16周，通过流式检测，在CRH处理组的NSG小鼠外周血hCD45平均比例高达46.09％，在对照组NSG小鼠外周血hCD45平均比例为13.53％。随后分析hCD45在16周时所占骨髓单个核细胞的比例，图3c、3d显示，hCD45所占骨髓单个核细胞的平均比例在对照组中为44.84％，在CRH处理组中为83.1％。以上结果显示，无论在外周还是在骨髓中均能够在CRH组检测到更高的人白细胞抗原，表明CRH的处理能够显著促进人造血干祖细胞在NSG小鼠的定植。CD34 + hematopoietic stem and progenitor cells were isolated and purified, resuspended in Stem Span SFEM serum-free medium, and two wells were set at 1.5×10 6 cells/well, and one well was set as a blank control group: multi-cytokine factors were added to the culture medium: SCF ( 50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18). The other well was set as the CRH treatment group: multiple cytokines were added to the culture medium: SCF (50ng/ml Peprotech, 300-70), Flt3-L (50ng/ml Peprotech, 300-19), TPO (50ng/ml Peprotech, 300-18) and CRH (240ng/ml, MCE HY-P0086), cultured at 5% CO2 and 37°C for 16h. After stimulation, cells were collected for counting, resuspended in 1×PBS, and adjusted to a cell concentration of 1×10 5 /200 μl, and then injected through the tail vein into NSG mice irradiated with a sublethal dose (250Rad) (sublethal). After a high dose of irradiation, it will cause the destruction of the original hematopoietic system of mice, thereby promoting the growth of hematopoietic stem and progenitor cells in the bone marrow. colonization and differentiation), the peripheral blood of mice was collected through the tail vein every 4 weeks, and flow cytometry was used to detect the proportion of hCD45 + cells in mononuclear cells (CD34 + hematopoietic stem and progenitor cells arrive at the bone cavity through homing, and only Only hematopoietic stem and progenitor cells that arrive in the bone cavity can differentiate and develop into human leukocytes and mobilize into the peripheral blood); the above-mentioned NSG is sacrificed at week 16 (at 12-16 weeks, the transplanted hematopoietic stem and progenitor cells already have a mature multi-cell lineage) Recipient mice, mouse bone marrow cells were aseptically isolated, part of which was used to detect the proportion of human CD45 and other positive cells in the mouse bone marrow, and the other part was resuspended for subsequent secondary transplantation experiments. The results in Figure 3a and 3b show that 4 weeks after transplantation, the proportion of hCD45 in peripheral blood mononuclear cells of NSG mice was detected by flow cytometry. The average proportion of hCD45 in the peripheral blood of NSG mice was 26.06%, while the average proportion of hCD45 in the peripheral blood of the control group was 26.06%. is 7.33%. Further, 16 weeks after the initial transplantation, flow cytometry showed that the average proportion of hCD45 in the peripheral blood of NSG mice in the CRH-treated group was as high as 46.09%, and the average proportion of hCD45 in the peripheral blood of NSG mice in the control group was 13.53%. The proportion of hCD45 in bone marrow mononuclear cells at 16 weeks was then analyzed. Figures 3c and 3d show that the average proportion of hCD45 in bone marrow mononuclear cells was 44.84% in the control group and 83.1% in the CRH-treated group. The above results show that higher human leukocyte antigens can be detected in the CRH group both in the periphery and in the bone marrow, indicating that CRH treatment can significantly promote the colonization of human hematopoietic stem and progenitor cells in NSG mice.
实施例4 CRH能够显著促进体内造血干祖细胞二次长期定植Example 4 CRH can significantly promote secondary long-term colonization of hematopoietic stem and progenitor cells in vivo
将无菌分离的初次移植16周NSG小鼠骨髓使用1×PBS(预冷)重悬，调整细胞浓度为5×106/200μl，尾静脉注射至经过亚致死剂量(250Rad)辐照的NSG小鼠中，构建二次移植模型(用于检测造血干祖细胞自我更新能力及分化能力的强弱)。每4周通过流式细胞仪检测人hCD45+细胞在NSG小鼠外周血单个核细胞中的比例；第16周牺牲上述二次移植的NSG受体小鼠，流式细胞术检测小鼠骨髓单个核细胞人hCD45等阳性细胞比例，评价造血干祖细胞长期定植能力。图4a、4b、4c结果显示，和对照组相比，CRH处理的增强植入效应在移植的二次NSG受体小鼠中也很明显，即通过流式检测发现人hCD45在对照组骨髓占单个核的平均比例为4.76％，而在CRH处理组平均比例为32.30％，为对照组的6.79倍。在外周血中也显示相同的趋势，即hCD45阳性比例，CRH组为对照组的3.28倍。同时使用流式细胞仪检测各个谱系在初次以及二次移植受体小鼠中的比例，图4d、4e结果显示，CRH处理组能够在体内使HSPC向髓系方向分化发育，hCD33为人髓系细胞的代表细胞标志分子，在初次移植NSG受体小鼠中，CRH处理组的hCD33阳性比例为对照组的19.05倍。在二次移植NSG受体小鼠中，CRH处理组的hCD33阳性比例为对照组的5.71倍。以上结果表明，CRH的处理能够明显促进造血干祖细胞在NSG小鼠体内长期定植，提示CRH对于促进体内造血干祖细胞移植具有显著的促进作用。Aseptically isolated bone marrow from NSG mice that were first transplanted at 16 weeks was resuspended in 1×PBS (pre-cooled), adjusted to a cell concentration of 5×10 6 /200 μl, and injected into the tail vein into NSG that had been irradiated with a sublethal dose (250Rad). In mice, a secondary transplantation model was constructed (used to detect the self-renewal and differentiation capabilities of hematopoietic stem and progenitor cells). The proportion of human hCD45 + cells in the peripheral blood mononuclear cells of NSG mice was detected by flow cytometry every 4 weeks; the NSG recipient mice with the above secondary transplantation were sacrificed at the 16th week, and the mouse bone marrow mononuclear cells were detected by flow cytometry. The proportion of nuclear cells, human hCD45 and other positive cells was used to evaluate the long-term colonization ability of hematopoietic stem and progenitor cells. The results of Figures 4a, 4b, and 4c show that compared with the control group, the enhanced engraftment effect of CRH treatment is also obvious in the transplanted secondary NSG recipient mice. That is, through flow cytometry, it was found that human hCD45 accounted for 10% of the bone marrow in the control group. The average proportion of single nuclei was 4.76%, while the average proportion in the CRH-treated group was 32.30%, which was 6.79 times that of the control group. The same trend was also shown in peripheral blood, that is, the proportion of hCD45 positive in the CRH group was 3.28 times that of the control group. At the same time, flow cytometry was used to detect the proportion of each lineage in primary and secondary transplant recipient mice. The results in Figure 4d and 4e show that the CRH treatment group can differentiate and develop HSPCs toward the myeloid lineage in vivo. hCD33 is a human myeloid lineage cell. As a representative cell marker molecule, in first-time transplanted NSG recipient mice, the positive proportion of hCD33 in the CRH-treated group was 19.05 times that of the control group. In the secondary NSG recipient mice, the hCD33 positive proportion in the CRH-treated group was 5.71 times that of the control group. The above results show that CRH treatment can significantly promote the long-term colonization of hematopoietic stem and progenitor cells in NSG mice, suggesting that CRH has a significant promoting effect on promoting hematopoietic stem and progenitor cell transplantation in vivo.
参考文献references
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以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent substitutions, improvements, etc. shall be included in the protection scope of the present invention.
Claims (9)
- 促进造血干祖细胞迁移、归巢和植入的组合物，其特征在于，所述组合物包含：促肾上腺皮质激素释放激素、基质细胞衍生因子、Fms相关酪氨酸激酶3配体和血小板生成素。A composition that promotes migration, homing and implantation of hematopoietic stem and progenitor cells, characterized in that the composition contains: corticotropin-releasing hormone, stromal cell-derived factor, Fms-related tyrosine kinase 3 ligand and platelet production white.
- 促进造血干祖细胞迁移、归巢和植入的培养基，其特征在于，所述培养基包含：基础培养基、促肾上腺皮质激素释放激素、基质细胞衍生因子、Fms相关酪氨酸激酶3配体和血小板生成素，优选地所述基础培养基为无血清培养基，更优选地Stem Span SFEM无血清培养基。A culture medium that promotes migration, homing and implantation of hematopoietic stem and progenitor cells, characterized in that the culture medium contains: basal culture medium, corticotropin-releasing hormone, stromal cell-derived factors, Fms-related tyrosine kinase 3 complex body and thrombopoietin, preferably the basal medium is a serum-free medium, more preferably Stem Span SFEM serum-free medium.
- 根据权利要求1所述的组合物或根据权利要求2所述的培养基，其特征在于，基质细胞衍生因子：Fms相关酪氨酸激酶3配体：血小板生成素：促肾上腺皮质激素释放激素的含量比为1:1:1:(3-20)，优选地，基质细胞衍生因子：Fms相关酪氨酸激酶3配体：血小板生成素：促肾上腺皮质激素释放激素的含量比为1:1:1:3、1:1:1:4.8、1:1:1:5、1:1:1:6、1:1:1:7、1:1:1:8、1:1:1:9、1:1:1:10、1:1:1:11、1:1:1:12、1:1:1:13、1:1:1:14、1:1:1:15、1:1:1:16、1:1:1:17、1:1:1:18、1:1:1:19、1:1:1:20，更优选地，基质细胞衍生因子：Fms相关酪氨酸激酶3配体：血小板生成素：促肾上腺皮质激素释放激素的含量比为1:1:1:4.8。The composition according to claim 1 or the culture medium according to claim 2, characterized in that: stromal cell-derived factor: Fms-related tyrosine kinase 3 ligand: thrombopoietin: adrenocorticotropin-releasing hormone The content ratio is 1:1:1:(3-20). Preferably, the content ratio of stromal cell-derived factor: Fms-related tyrosine kinase 3 ligand: thrombopoietin: adrenocorticotropin-releasing hormone is 1:1. :1:3, 1:1:1:4.8, 1:1:1:5, 1:1:1:6, 1:1:1:7, 1:1:1:8, 1:1:1 :9, 1:1:1:10, 1:1:1:11, 1:1:1:12, 1:1:1:13, 1:1:1:14, 1:1:1:15 , 1:1:1:16, 1:1:1:17, 1:1:1:18, 1:1:1:19, 1:1:1:20, more preferably, stromal cell-derived factor: The content ratio of Fms-related tyrosine kinase 3 ligand: thrombopoietin: corticotropin-releasing hormone is 1:1:1:4.8.
- 根据权利要求1所述的组合物或根据权利要求2所述的培养基，其特征在于，所述促肾上腺皮质激素释放激素的含量为140ng/ml-950ng/ml，更优选地140ng/ml、200ng/ml、240ng/ml、300ng/ml、400ng/ml、500ng/ml、600ng/ml、700ng/ml、800ng/ml、900ng/ml、950ng/ml，甚至更优选地240ng/ml。The composition according to claim 1 or the culture medium according to claim 2, characterized in that the content of the corticotropin-releasing hormone is 140ng/ml-950ng/ml, more preferably 140ng/ml, 200ng/ml, 240ng/ml, 300ng/ml, 400ng/ml, 500ng/ml, 600ng/ml, 700ng/ml, 800ng/ml, 900ng/ml, 950ng/ml, even more preferably 240ng/ml.
- 根据权利要求1所述的组合物或根据权利要求2所述的培养基，其特征在于，所述基质细胞衍生因子的含量为50ng/ml-100ng/ml，优选地50ng/ml、60ng/ml、70ng/ml、80ng/ml、90ng/ml、100ng/ml，更优选地50ng/ml。The composition according to claim 1 or the culture medium according to claim 2, characterized in that the content of the stromal cell-derived factor is 50ng/ml-100ng/ml, preferably 50ng/ml, 60ng/ml , 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, more preferably 50ng/ml.
- 根据权利要求1所述的组合物或根据权利要求2所述的培养基，其特征在于，所述Fms相关酪氨酸激酶3配体的含量为50ng/ml-100ng/ml，优选地50ng/ml、60ng/ml、70ng/ml、80ng/ml、90ng/ml、100ng/ml，更优选地50ng/ml。The composition according to claim 1 or the culture medium according to claim 2, characterized in that the content of the Fms-related tyrosine kinase 3 ligand is 50ng/ml-100ng/ml, preferably 50ng/ml. ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, more preferably 50ng/ml.
- 根据权利要求1所述的组合物或根据权利要求2所述的培养基，其特征在于，所述血小板生成素的含量为50ng/ml-100ng/ml，优选地50ng/ml、60ng/ml、70ng/ml、80ng/ml、90ng/ml、100ng/ml，更优选地50ng/ml。The composition according to claim 1 or the culture medium according to claim 2, characterized in that the content of thrombopoietin is 50ng/ml-100ng/ml, preferably 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, more preferably 50ng/ml.
- 促肾上腺皮质激素释放激素在用于制备治疗血液系统恶性肿瘤、血液系统非恶性肿瘤、实体瘤、免疫系统疾病、遗传或代谢性疾病的药物中的用途，任选地，所述血液系统恶性肿瘤为慢性粒细胞白血病、急性髓细胞白血病、急性淋巴细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病、急性早幼粒细胞白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、骨髓瘤、多发性骨髓瘤、骨髓纤维化及骨髓增生异常综合征、伯基特淋巴瘤、B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、T细胞淋巴瘤、浆母细胞性淋巴瘤、皮肤T细胞淋巴瘤、原发性巨球蛋白血症、浆细胞白血病、浆母细胞性淋巴瘤、毛细胞白血病、系统性肥大细胞增多症或成胚浆样树突状细胞瘤；血液系统非恶性肿瘤为再生障碍性贫血、范可尼贫血、地中海贫血、镰状细胞贫血、骨髓纤维化、重型阵发性睡眠性血红蛋白尿症或无巨核细胞性血小板减少症；实体瘤为乳腺癌、卵巢癌、睾丸癌、肾癌、神经母细胞瘤、小细胞肺癌、生殖细胞瘤、尤因氏肉瘤、软组织肉瘤、维尔姆斯瘤、骨肉瘤、成神经管细胞瘤或恶性脑瘤；免疫系统疾病为重症联合免疫缺陷症、严重自身免疫性疾病、原发性中枢神经系统淋巴瘤、湿疹血小板减少 伴免疫缺陷综合征、慢性肉芽肿，IPEX综合征，AL淀粉变性、POEMS综合征、嗜血细胞综合征、类风湿性关节炎、多发性硬化、系统性硬化、系统性红斑狼疮、克罗恩病、多肌炎或皮肌炎；以及遗传或代谢性疾病为粘多糖病、先天性角化不良、溶酶体代谢病、球形细胞样脑白质病、异染性脑白质营养不良或X连锁的肾上腺脑白质营养不良。Use of corticotropin-releasing hormone in the preparation of a medicament for the treatment of hematological malignancies, hematological non-malignant tumors, solid tumors, immune system diseases, genetic or metabolic diseases, optionally, the hematological malignant tumors For chronic myelogenous leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute promyelocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, myeloma, multiple Myeloma, myelofibrosis and myelodysplastic syndromes, Burkitt lymphoma, B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, T-cell lymphoma, plasmablastic lymphoma, cutaneous T Cellular lymphoma, primary macroglobulinemia, plasma cell leukemia, plasmablastic lymphoma, hairy cell leukemia, systemic mastocytosis, or blastoplasmoid dendritic cell tumor; nonmalignant hematologic neoplasms Aplastic anemia, Fanconi anemia, thalassemia, sickle cell anemia, myelofibrosis, severe paroxysmal nocturnal hemoglobinuria, or amegakaryocytic thrombocytopenia; solid tumors are breast cancer, ovarian cancer, Testicular cancer, renal cancer, neuroblastoma, small cell lung cancer, germ cell tumor, Ewing's sarcoma, soft tissue sarcoma, Wilms' tumor, osteosarcoma, medulloblastoma, or malignant brain tumor; immune system disease is severe Combined immunodeficiency, severe autoimmune disease, primary central nervous system lymphoma, eczema thrombocytopenia With immunodeficiency syndrome, chronic granulomatosis, IPEX syndrome, AL amylosis, POEMS syndrome, hemophagocytic syndrome, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease , polymyositis or dermatomyositis; and genetic or metabolic diseases such as mucopolysaccharidosis, dyskeratosis congenita, lysosomal metabolism disease, spherocytoid leukoencephalopathy, metachromatic leukodystrophy or X-linked Adrenoleukodystrophy.
- 促肾上腺皮质激素释放激素在制备促进造血干祖细胞迁移、归巢和植入的组合物或培养基中的用途。 Use of corticotropin-releasing hormone in the preparation of a composition or culture medium that promotes migration, homing and engraftment of hematopoietic stem and progenitor cells.
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| CN114246948A (en) * | 2020-09-24 | 2022-03-29 | 广州辑因医疗科技有限公司 | Use of compounds for increasing the efficiency of human hematopoietic stem cell transplantation |
| CN114990063A (en) * | 2022-06-14 | 2022-09-02 | 中国科学技术大学 | Compositions for promoting migration, homing and engraftment of hematopoietic stem and progenitor cells and uses thereof |
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| CN101310007A (en) * | 2005-04-19 | 2008-11-19 | 约翰·霍普金斯大学 | Method of using stroma cells from cord blood to expand and engraft nucleated cells from cord blood |
| CN105106938A (en) * | 2015-08-07 | 2015-12-02 | 中国人民解放军军事医学科学院野战输血研究所 | Application of thrombopoietin on homing promotion of hemopoietic stem cells |
| CN110997904A (en) * | 2017-03-21 | 2020-04-10 | 法国血液机构 | Method of improving hematopoietic grafts |
| KR102121348B1 (en) * | 2017-12-20 | 2020-06-10 | 이화여자대학교 산학협력단 | A composition for promoting bone marrow engraftment |
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| CN1361180A (en) * | 2000-12-26 | 2002-07-31 | 上海博德基因开发有限公司 | New polypeptide corticotrophin releasing factor 8.8 and polynucleotides encoding this polypeptide |
| CN1382700A (en) * | 2001-04-26 | 2002-12-04 | 上海博德基因开发有限公司 | Polypeptide-corticotrophin releasing factor-12.87 and polynucleotide for coding it |
| CN114246948A (en) * | 2020-09-24 | 2022-03-29 | 广州辑因医疗科技有限公司 | Use of compounds for increasing the efficiency of human hematopoietic stem cell transplantation |
| CN114990063A (en) * | 2022-06-14 | 2022-09-02 | 中国科学技术大学 | Compositions for promoting migration, homing and engraftment of hematopoietic stem and progenitor cells and uses thereof |
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