Induced differentiation of hemopoietic stem/progenitor cell into erythroid progenitor cells

A technology of hematopoietic progenitor cells and erythroid progenitor cells, applied in biochemical equipment and methods, medical preparations containing active ingredients, applications, etc., can solve problems such as loss of activity

Inactive Publication Date: 2007-06-13
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purified WNT3a protein is very prone to palmitoylation under external conditions, thereby losing its activity

Method used

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  • Induced differentiation of hemopoietic stem/progenitor cell into erythroid progenitor cells
  • Induced differentiation of hemopoietic stem/progenitor cell into erythroid progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, construction of adenovirus vector

[0028] 1. Acquisition of WNT3a gene

[0029] Using the mouse placenta cDNA as a template, according to the sequence of the WNT3a-cDNA coding region and introducing XbaI, HpaI and BamHI restriction sites to synthesize primers, the primer sequences and polymerase chain reaction (PCR) conditions are as follows:

[0030] P1 5′- ATGGCTCCTCTCGGATACCT-3′

[0031] P2 5′- CTACTTGCAGGTGTGCACGT-3′

[0032] The 20μl reaction system includes: 1ul cDNA (100ng / ul), 0.5μl each of primer1 and 2 (20μM), 1.5μl dNTP (2.5mM), 0.25μl La Taq DNA polymerase (TakaRa, 5u / μl), 2μl 5× GC Buffer I, 14.75 μl sterilized water. The reaction conditions are: denaturation at 94°C for 3 minutes: 45s at 94°C, 45s at 56°C, 2min at 72°C, 28 cycles; 7min at 72°C. The electrophoresis results are as shown in Figure 1. In Figure 1, swimming lane 1 is the target gene, and swimming lane 2 is the DNA molecular weight marker (DL2000). The arrow indicates the ...

Embodiment 2

[0040] Example 2, the effect of bone marrow stromal cells transfected with WNT3a gene on hematopoietic stem / progenitor cells

[0041] 1. Isolation and purity analysis of hematopoietic stem / progenitor cells

[0042] (1) Isolation of cord blood mononuclear cells (mononuclear cell, MNC)

[0043] Materials and Reagents

[0044] Umbilical cord blood is obtained from the umbilical cord of a healthy full-term pregnant fetus.

[0045] PBS: Dissolve 8.0g NaCl, 0.2g KCl, 1.44g Na per 1L deionized water 2 HPO 4 , 0.24g KH 2 PO 4 , adjusted to pH 7.4 with HCl or NaOH.

[0046]0.5% methylcellulose: Add 1g of methylcellulose to 200mL of normal saline, autoclave it and place it in a refrigerator at 4°C while it is hot to dissolve it, and shake well before use.

[0047] 1. The fresh umbilical cord blood anticoagulated with heparin is mixed with PBS at a ratio of 1:1, and then mixed with 0.5% methylcellulose at a volume ratio of 4:1. Let it stand at room temperature for 30 minutes until...

Embodiment 3

[0087] Example 3. Construction of recombinant retroviral plasmid pMSCV-WNT3a.

[0088] Using the mouse placenta cDNA as a template, according to the sequence of the WNT3a-cDNA coding region and introducing XbaI, HpaI and BamHI restriction sites to synthesize primers, the primer sequences and polymerase chain reaction (PCR) conditions are as follows:

[0089] P1 5′- ATGGCTCCTCTCGGATACCT-3′

[0090] P2 5′- CTACTTGCAGGTGTGCACGT-3′

[0091]The 20μl reaction system includes: 1ul cDNA (100ng / ul), 0.5μl each of primer1 and 2 (20μM), 1.5μl dNTP (2.5mM), 0.25μl La Taq DNA polymerase (TakaRa, 5u / μl), 2μl 5× GC Buffer I, 14.75 μl sterilized water. The reaction conditions are: denaturation at 94°C for 3min; 28 cycles at 94°C for 45s, 56°C for 45s, and 72°C for 2min; and 72°C for 7min. About 1000 bp after electrophoresis is the target gene WNT3a (see Figure 1). The reaction product was cloned into the pGEM-T eassy vector (Promega, USA) to obtain the cloning plasmid T-WNT3a.

[009...

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Abstract

The invention involves biomedical field, concretely speaking, it involves the method and application of inducing hematopoietic stem / progenitor cells to erythroid differentiation of hematopoietic progenitor cells in vitro. This invention combines the genetic engineering and stem cell engineering, using the multipotency of stem cells and the bone marrow stromal cells of WNT3a in protein secretion activity as trophoblast cells, hematopoietic stem / progenitor cells to erythroid differentiation of hematopoietic progenitor cells is induced. this in vitro inducing system not only provides corroboration for the 'adult stem cell plasticity' theory, but also provides rich source of the cells as clinical preparation for diseases such as anemia, leukemia and other blood diseases, cancer chemotherapy and radiotherapy in the treatment of hematopoietic support, immune system diseases and infectious diseases such as liver and kidney dysfunction cell disease.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the establishment of a method for inducing the differentiation of hematopoietic stem / progenitor cells to erythroid progenitor cells in vitro by bone marrow stromal cells transfected with WNT3a gene. Background technique [0002] Clinically, the treatment of anemia and leukemia requires a sufficient amount of red blood cells that meet the requirements. In order to obtain the amount of cells required for treatment, many scientists have attempted to obtain red blood cells through in vitro culture, mainly by adding various cytokines and the like. However, the current method used to expand the number of hematopoietic stem cells is limited, and the purity of red blood cells obtained is not high. [0003] Reya confirmed that the WNT signaling pathway plays a crucial role in the self-renewal of hematopoietic stem / progenitor cells. WNT3a is a very important gene in the WNT pathway. Experimen...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C12N5/08C12N5/06A61K35/12A61P35/02A61P7/06A61K35/28C12N5/10
Inventor 裴雪涛师伟韩姝李艳华闫舫王韫芳陈琳白慈贤南雪施双双
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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