Method for proliferating human hemapoietic stem cell and progenitor cell by GST-hDSL recombinant protein

A technology of recombinant protein and stem cells, applied in the field of bioengineering

Inactive Publication Date: 2008-01-02
SHANGHAI JIAO TONG UNIV
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  • Abstract
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Problems solved by technology

[0004] Find through literature retrieval to prior art, Han W etc. have published " A soluble form of human Delta-like-1 inhibitions differentiation of hematopoietic progenitor " in " Blood " (" blood ", 2000 95 volumes 1616-1625 pages) cells" ("Inhibition of human hematopoietic progenitor cell differentiation by soluble human Delta-like-1 protein"), the purified GST-hDSL fusion protein (GST is glutathione-S-transferase) combined with cytokine -The combination of macrophage colony-stimulating factor, IL-1 (interleukin 1), and IL-3 (interleukin 3) can promote the expansion of mouse hematopoietic stem and progenitor cells, but it has not been used for human hematopoietic stem and progenitor cells Amplification of

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  • Method for proliferating human hemapoietic stem cell and progenitor cell by GST-hDSL recombinant protein

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Embodiment 1

[0016] This embodiment 1 is implemented under the following conditions of implementation and technical requirements:

[0017] (1) Preparation of umbilical cord blood mononuclear cells and CD34 + Separation and purification of hematopoietic stem and progenitor cells

[0018] Under routine aseptic conditions, take about 100ml of umbilical cord blood from a normal full-term fetus, mix and dilute it with phosphate buffer at a volume ratio of 1:2, take ten 50ml sterile centrifuge tubes, and add 15ml of lymphocyte separation solution to each tube (density is 1.077±0.002g / ml), then slowly add diluted cord blood along the tube wall to form a clear layer, centrifuge the centrifuge tube at 2300rpm for 30min, after centrifugation carefully use a straw to absorb the white misty cell layer in the middle of the centrifuge tube Into another 50mL sterile centrifuge tube, add 20ml of phosphate buffer and mix well, then centrifuge at 2000rpm for 10min, discard the supernatant, then suspend the...

Embodiment 2

[0023] This embodiment 2 is implemented under the following conditions of implementation and technical requirements:

[0024] (1) Preparation of bone marrow mononuclear cells and CD34 + Separation and purification of hematopoietic stem and progenitor cells

[0025] Under routine aseptic conditions, take about 100ml of bone marrow fluid, mix and dilute it with phosphate buffer at a volume ratio of 1:2, take 10 50ml sterile centrifuge tubes, and add 15ml of lymphocyte separation solution (density 1.077± 0.002g / ml), then slowly add diluted cord blood along the tube wall to form a clear layer, centrifuge the centrifuge tube at 2300rpm for 30min, after centrifugation, carefully use a straw to draw the white misty cell layer in the middle of the centrifuge tube to another 50mL free Bacteria centrifuge tube, add 20ml of phosphate buffer and mix well, then centrifuge at 2000rpm for 10min, discard the supernatant, then suspend the precipitated cells with phosphate buffer and combine t...

Embodiment 3

[0029] This embodiment 3 is implemented under the following conditions of implementation and technical requirements:

[0030] (1) Preparation of peripheral blood mononuclear cells and CD34 + Separation and purification of hematopoietic stem and progenitor cells

[0031] Under routine aseptic conditions, take about 100ml of bone marrow fluid, mix and dilute it with phosphate buffer at a volume ratio of 1:2, take 10 50ml sterile centrifuge tubes, and add 15ml of lymphocyte separation solution (density 1.077± 0.002g / ml), then slowly add diluted umbilical cord blood along the tube wall to form a clear layer, centrifuge the centrifuge tube at 2300rpm for 30min, after centrifugation carefully use a straw to draw the white misty cell layer in the middle of the centrifuge tube to another 50mL free Bacteria centrifuge tube, add 20ml of phosphate buffer and mix well, then centrifuge at 2000rpm for 10min, discard the supernatant, then suspend the precipitated cells with phosphate buffer...

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Abstract

The invention discloses a breeding method of human hemapoietic stem cell and master cell through GST-hDSL recombinant protein in the biological engineering technical domain, which comprises the following steps: (1)using density gradient centrifugation method to make single nuclear cell from bone marrow, peripheral blood or umbilical cord blood; marking the CD34 cell; purifying the CD34 cell; obtaining the CD34+; (2)adding CD34+ cell in the cell culture medium to train; adding cytokine and GST-hDSL recombinant protein; rolling in the fresh cell culture medium to add cytokine and GST-hDSL recombinant protein again to train continuously; obtaining the product. The invention can accelerate the breeding of human hemapoietic stem cell and master cell to be detected from CD34+ cell and main colony number and high-breeding potential colony number, which can be widely applied in the biological medical domain.

Description

technical field [0001] The invention relates to a cell proliferation method in the technical field of bioengineering, in particular to a method for proliferating human hematopoietic stem cells and progenitor cells with GST-hDSL recombinant protein. Background technique [0002] Hematopoietic stem cell transplantation has become the clinical treatment choice for an increasing number of patients with leukemia, malignant tumors and certain genetic diseases. Hematopoietic stem cells are derived from bone marrow, umbilical cord blood and mobilized peripheral blood. However, due to the limited source and the number of available cells, the clinical application is limited, so the in vitro expansion of hematopoietic stem and progenitor cells is a research hotspot. [0003] Notch (Notch is a widely used, evolutionarily highly conserved cell differentiation regulatory signaling pathway) pathway plays an important role in regulating the fate of cells in many developmental systems, and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08A61K38/16C12N5/0789
Inventor 韩伟吴明媛林小娟姜俊芬
Owner SHANGHAI JIAO TONG UNIV
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