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Application of miR-125b in red blood cell maturation

A technology for red blood cells and mature red blood cells, which is applied to cells modified by introducing foreign genetic materials, DNA/RNA fragments, and vectors to introduce foreign genetic materials, etc. It can solve the problems of mature red blood cells that need to be improved, and achieve the effect of improving efficiency

Active Publication Date: 2013-05-01
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

[0003] However, the preparation of mature red blood cells still needs to be improved

Method used

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  • Application of miR-125b in red blood cell maturation
  • Application of miR-125b in red blood cell maturation
  • Application of miR-125b in red blood cell maturation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Expression of endogenous miR-125b is upregulated with erythrocyte maturation

[0046] 1. Isolate MNC cells from fresh umbilical cord blood (within 24 hours) according to standard operating procedures (use magnetic beads to separate CD34+ cells if necessary). The separation steps are as follows:

[0047] 1. to mix, settle

[0048] One part of fresh anticoagulated umbilical cord blood sample was mixed with PBS in an equal volume ratio of 1:1, and then 1 / 4 of the total volume of 0.5% methylcellulose was added. After mixing by inversion, let stand at room temperature for 30 minutes until the sedimentation is clear. Carefully suck out the supernatant into a 50ml centrifuge tube, and use a small pipette to absorb it when it is close to the interface to prevent the absorption of red blood cells. Then centrifuge at 1800 rpm for 5 minutes.

[0049] 2. Resuspension, gradient centrifugation

[0050] Discard the supernatant and add 5ml PBS to each tube to resuspend ...

Embodiment 2

[0070] Example 2: Exogenous overexpression of miR-125b promotes erythroid maturation and denucleation of erythroleukemia cells K562

[0071] 1. Cell transfection and stable strain screening:

[0072] 1. K562 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), and passaged every 3 days at a ratio of 1:5.

[0073] 2. During transfection, take 3×105K562 cells and resuspend them with 1.5ml medium, place them in a 6-well plate, mix 4μg plasmid and 10μl lipofectamin2000 with 250μl optiMEM respectively, incubate at room temperature for 5min, mix well, and let stand At room temperature for 20 minutes, dropwise added to the cell suspension. The medium was changed the next day after transfection, and G418 was added 48 hours later to select transfected positive cells at a screening concentration of 500 μg / ml. After 3 weeks, K562 cell lines stably transfected and expressing foreign genes were obtained.

[0074] 2. Perform erythroid induction and denuc...

Embodiment 3

[0115] Example 3: Overexpression of miR-125b in erythroblasts derived from umbilical cord blood can improve the maturation and denucleation efficiency of erythrocytes

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Abstract

The invention relates to an application of miR-125b in red blood cell maturation. A method for preparing mature red blood cell comprises the following steps: making precursor cells of the red blood cell to express miR-125b, wherein the miR-125b has a nucleotide sequence shown in a SEQ IDNO:1; and culturing the precursor cells of the red blood cell of the expressed miR-125b to obtain the mature red blood cell; wherein, the precursor cells of the red blood cell can be selected from original erythroblast, polychromatic erythroblast, orthoneutrophil erythroblast and reticulocyte. The method can be used for effectively preparing the mature red blood cell.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, the present invention relates to the use of miR-125b in red blood cell maturation. Background technique [0002] In the treatment of war wounds and conventional treatment, blood transfusion is an extremely critical link. The supply of blood in my country has been in short supply for a long time. In recent years, many cities in my country have even appeared "blood shortage". To solve the problem of lack of blood sources, the development of new blood sources has become an urgent need in current medical treatment. Stem cell research provides new hope for the preparation of red blood cells in vitro, including embryonic stem cells, induced pluripotent stem cells (iPSC) and umbilical cord, bone marrow, Hematopoietic stem and progenitor cells derived from peripheral blood may be used as seed cells, which can be induced and expanded in large quantities to generate red blood cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N5/10C12N15/113C12N15/11
Inventor 裴雪涛岳文谢小燕李艳华姚海雷习佳飞周军年曾泉房芳
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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