WO2003064636A1 - Separation, preparation et utilisation de la cellule souche hematopoietique medullaire - Google Patents

Separation, preparation et utilisation de la cellule souche hematopoietique medullaire Download PDF

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Publication number
WO2003064636A1
WO2003064636A1 PCT/CN2002/000805 CN0200805W WO03064636A1 WO 2003064636 A1 WO2003064636 A1 WO 2003064636A1 CN 0200805 W CN0200805 W CN 0200805W WO 03064636 A1 WO03064636 A1 WO 03064636A1
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WIPO (PCT)
Prior art keywords
cells
bone marrow
vascular
hematopoietic
blood
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PCT/CN2002/000805
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English (en)
Chinese (zh)
Inventor
Chunhua Zhao
Hong Guo
Jiewen Liu
Zhigang Zhao
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Institute Of Hematology & Hospital Of Blood Disease Chinese Academy Of Medical Science & Peking Union Medical College
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Application filed by Institute Of Hematology & Hospital Of Blood Disease Chinese Academy Of Medical Science & Peking Union Medical College filed Critical Institute Of Hematology & Hospital Of Blood Disease Chinese Academy Of Medical Science & Peking Union Medical College
Publication of WO2003064636A1 publication Critical patent/WO2003064636A1/fr

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    • 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
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0692—Stem cells; Progenitor cells; Precursor cells
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]

Definitions

  • the present invention relates to a method for isolating, preparing and in vitro inducing a phenotype cell population with blood vascular stem cells. Background technique
  • hematopoietic stem cell transplantation is used in the clinic to treat diseases such as blood and tumors, and vascular diseases caused by various causes have not been effectively treated.
  • the main difficulty is that sufficient vascular stem cells cannot be obtained.
  • many studies have shown that the presence of blood vascular stem cells in the yolk sac and embryonic stem cells can differentiate into hematopoietic and endothelial cells in both directions.
  • implementation is difficult due to ethical and technical reasons. Therefore, it is important to isolate and culture blood vascular stem cells from human tissues.
  • the present invention is to isolate, culture and identify a cell population with phenotypic characteristics of blood vessel stem cells from bone marrow cells, and provide a new therapeutic approach for certain diseases in the blood and blood vessels. Disclosure of invention
  • the purpose of the present invention is to establish a method for isolating and culturing blood vascular stem cells from bone marrow for the first time in the world, and to provide a new way for the treatment of clinical diseases.
  • the object of the present invention is achieved by the following methods:
  • the induced cells were fixed with 4% paraformaldehyde together with the gel, and Wright stained. The distance between two adjacent cells was measured with a light microscope with a ruler.
  • Lmg / ml of RNase A after cell cycle determination and cell growth mode After fetal bone marrow stromal cells were digested with trypsin, IX 10 5 cells / ml cells were permeated with 70% ethanol at 4 ° C for 10 minutes. Treat at 37 ° C for 20 minutes, then label the cell DNA with 3ug / ml propidium iodide for 5 minutes at room temperature, and place on ice for detection by flow cytometry.
  • cells were trypsinized and seeded in 24-well plates with IX 10 4 cells per well. The cells were continuously cultured, and trypsin-digested viable cells were continuously counted every day with fetal blue, and the cell growth curve was drawn.
  • Bone marrow stromal cells participate in angiogenesis after partial liver resection in rats To study whether bone marrow stromal cells participate in angiogenesis in vivo, we selected the characteristics of rat liver regeneration. Wister rats (purchased from the Fourth Academy of Military Medical Sciences), weighing about 250g, after intraperitoneal anesthesia with sodium pentobarbital, left liver resection was performed, about 40% of the liver was removed, and one day after liver resection, the tail vein was injected with 5 X 10 Five neonatal rat bone marrow stromal cells (cultured in the same way as fetal bone marrow stromal cells) at 1 day of birth were labeled with the fluorescent dye MPI. Three weeks after hepatectomy, frozen sections were taken from the liver and examined by fluorescence microscopy. '' Brief description of the drawings
  • Figure 1 (a) is a growth curve of human fetal bone marrow stromal cells
  • Figure 1 (b) is a cycle flow analysis diagram of human fetal bone marrow stromal cells
  • Figure 2 shows the vascular-like structure when Flkl + cells differentiate into vascular endothelial cells for 11 days;
  • Figure 3 is a blood vessel-like structure under an optical microscope
  • Figure 4 (a) shows the shape of a transmission electron microscope in which slender endothelial cells form a vascular-like structure, with a gap made of gel in the middle (magnification 10,000 times);
  • Figure 4 (b) shows the morphology of the ultrastructure of a single endothelial cell.
  • the nucleus is slender spindle-shaped, and heterochromatin is visible;
  • Figure 4 (c) shows the shape of a transmission electron microscope of a blood vessel-like structure where two adjacent cells are superimposedly connected
  • Figure 5 (a) Immunohistochemical staining of CD34 positive round cells.
  • FIG. 6 is a blood vessel-like structure diagram under a fluorescence microscope. The best way to implement the invention
  • Adherently grown fetal bone marrow stromal cells have a typical fibroblast-like morphology, and the cell growth pattern is shown in [la].
  • the growth curve shows that in the logarithmic growth phase, the cell doubling time is about 30 hours. 01
  • the DNA content was detected by flow cytometry, and the cell cycle was analyzed (see Figure [lb]). About 89. 90% of the cells were in the G0 / G1 phase, while the cells in the G2-M phase and the S phase were 3.01. % And 7. 08%. So what we get is a rapidly proliferating cell population.
  • Figure [3] shows a vascular-like structure composed of a plurality of elongated spindle-shaped cells arranged, and the nucleus is also elongated spindle-shaped.
  • the transmission electron microscopy results are shown in Figure [4]:
  • the tube-like structure is composed of elongated endothelial cells, and the gap is composed of gel in the middle. Two adjacent cells overlap and connect in the same manner as the endothelial cells that make up a blood vessel.
  • the immunohistochemistry (5) showed that the spherical-like structure composed of round cells was the center.
  • the tube-like structure was extended to the periphery, and the characteristic phenotype CD31 of cells constituting the tube-like structure was positive.
  • CD34 is a characteristic feature of progenitor cells of hematopoietic and endothelial cells, but in this differentiation culture system, about 20% of cells express CD34 and are round cells. This result suggests that these round cells are hematopoietic cells, and the cells that make up the tube-like structure are vascular endothelial cells that induce differentiation.
  • Rat livers have a strong ability to regenerate, and they can return to their original weight in 2-3 weeks after most of the liver has been removed. Three weeks after the liver was removed from the rat, the rat liver returned to its original weight and the liver lobes thickened.
  • DAPI mainly binds to DNA bases and tubulin in cells. Frozen sections were found under a fluorescent microscope ( Figure [6]) in the central vein of the liver Endothelial cells have blue fluorescence. This indicates that bone marrow stromal cells can participate in the repair or formation of blood vessels in the environment of liver regeneration after liver resection.
  • the isolated cells can form blood vessels in vitro and differentiate into hematopoietic cells.
  • the isolated cells can participate in the formation of blood vessels in the body.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hematology (AREA)
  • Vascular Medicine (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de séparation et de préparation de la cellule souche hématopoïétique médullaire. Ladite cellule souche se différencie en une cellule souche hématopoïétique et en une cellule endothéliale dans le système de stimulation in vitro. L'invention porte également sur un procédé d'incubation de la cellule souche hématopoïétique. Sont également décrits, le système de stimulation de la cellule souche hématopoïétique se différenciant en une cellule hématopoïétique et une cellule des vaisseaux sanguins, et son utilisation dans le traitement de maladies des vaisseaux sanguins et de tumeurs.
PCT/CN2002/000805 2002-01-28 2002-11-11 Separation, preparation et utilisation de la cellule souche hematopoietique medullaire WO2003064636A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN02102918.0 2002-01-28
CN02102918 2002-01-28

Publications (1)

Publication Number Publication Date
WO2003064636A1 true WO2003064636A1 (fr) 2003-08-07

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000325071A (ja) * 1999-05-21 2000-11-28 Asahi Medical Co Ltd 細胞分離回収方法
CN1280187A (zh) * 1999-07-13 2001-01-17 中国人民解放军第二军医大学 一种体外扩增造血干细胞的新方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000325071A (ja) * 1999-05-21 2000-11-28 Asahi Medical Co Ltd 細胞分離回収方法
CN1280187A (zh) * 1999-07-13 2001-01-17 中国人民解放军第二军医大学 一种体外扩增造血干细胞的新方法

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