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Use of nucleic acid in improving differentiating efficiency of meganucleus of cell

A megakaryotic differentiation and cell technology, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, DNA/RNA fragments, etc. The problems of low differentiation efficiency and low cell viability can achieve the effect of improved efficiency, high efficiency of megakaryocytic induction and differentiation, and high cell purity.

Active Publication Date: 2017-05-31
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

Although studies on inducing stem cells to obtain megakaryocytes and mature platelets have been reported, the current megakaryotic differentiation efficiency and cell activity of stem cells are low, and the amount of functional cells and platelets produced is small, which cannot meet the needs of clinical applications.
Therefore, the current method of inducing megakaryotic differentiation of stem cells still needs to be improved

Method used

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  • Use of nucleic acid in improving differentiating efficiency of meganucleus of cell
  • Use of nucleic acid in improving differentiating efficiency of meganucleus of cell
  • Use of nucleic acid in improving differentiating efficiency of meganucleus of cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Separation of hematopoietic stem and progenitor cells

[0034] 1. Aseptically collect a portion of the umbilical cord blood of a full-term normal delivery fetus after the umbilical cord is cut, and mix the umbilical cord blood with the PBS solution at a ratio of 1:1, and then mix it with 2.3% ( w / v) of methyl cellulose, let it stand at room temperature for 30 minutes, wait for the red blood cells to settle naturally until the boundaries are clear, and settle the red blood cells.

[0035] 2. Aspirate the supernatant, put it in a 50mL centrifuge tube, and centrifuge at 20,000rpm at 25°C for 7 minutes. Add 5 mL of Ficoll Human Lymphocyte Separation Solution into a 15 mL centrifuge tube, then slowly add 5 mL of cell suspension along the tube wall, centrifuge at 25°C and 1800 rpm for 25 minutes to separate mononuclear cells.

[0036] 3. Collect interface mononuclear cells and wash with PBS. At this time, hematopoietic stem and progenitor cells are obtained, and t...

Embodiment 2

[0042] In vitro induction of embodiment 2 megakaryocytes

[0043] 1. Induction of primary cells

[0044] The mononuclear cells isolated in Example 1 were inoculated into a 6-well plate cell plate, and 2 ml was added to each well at a density of 1 × 10 7 mononuclear cells / ml, add 2 mL of megakaryotic induction medium, and then place at 37°C, 5% CO 2 cultured in an incubator.

[0045] Wherein, the megakaryotic induction medium is obtained by adding 100ng / mL recombinant human thrombopoietin (TPO), 100ng / mL stem cell growth factor (SCF), 20ng / mL interleukin 3 (IL-3) to Stemspan medium, 50ng / mL interleukin 6 (IL-6) and 20ng / mL interleukin 11 (IL-11). Depending on the growth level of the cells, half of the medium was changed every other day, and the culture continued for 20 days.

[0046] 2. Induction of human leukemia cell lines

[0047] Use 2ml of UT-7 cell culture medium (RPMI1640 medium adding 10% fetal bovine serum (FBS)) to take 3×10 5 UT-7 cells were resuspended and tra...

Embodiment 3

[0048] Overexpression of embodiment 3miR-1915-3p

[0049] 1. Transfection of miR-1915-3p was carried out in both isolated primary cells and human leukemia cell lines. Among them, the isolated primary cells were transfected with miR-1915-3p mimics (miRNA mimics are endogenous miRNAs that mimic organisms, synthesized by chemical synthesis, and have the function of enhancing endogenous miRNA) for miR -1915-3p overexpression, miR-1915-3p overexpression in leukemia cell lines by transfection of plasmids carrying miR-1915-3p sequences. Human leukemia cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), and passaged at a ratio of 1:5 every 3 days.

[0050] 2. For transfection, take 3×10 5 UT-7 cells were resuspended in 1.5ml culture medium, placed in a 6-well plate, mixed with 20pmol miRNA and 10μl lipofectamin2000 and 250μl optiMEM respectively, incubated at room temperature for 5min, then mixed well, left at room temperature for 20min, and added ...

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Abstract

The invention provides use of a nucleic acid in improving the differentiating efficiency of meganucleus of a cell and a method of improving the differentiating efficiency of meganucleus of the cell. The cell has a potential of differentiating meganucleus. The nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1. In the cell, the nucleic acid over-expresses the nucleotide sequence as shown in SEQ ID NO: 1, so that the differentiating efficiency of the cell with a meganucleus differentiating potential can be obviously improved.

Description

technical field [0001] The present invention relates to the field of biology, specifically, the present invention relates to the use of nucleic acid in improving the efficiency of cell megakaryotic differentiation and the method for improving the efficiency of cell megakaryotic differentiation Background technique [0002] Nuclear radiation, weapon injuries, high-dose chemotherapy, allogeneic tissue and organ transplantation, and some diseases of the immune system and blood system can all lead to a decrease in the number of platelets or poor function, causing internal bleeding and endangering life. Clinically, repeated platelet transfusions are often used to prevent the occurrence of internal bleeding in patients. Therefore, platelets are in great demand. At present, the machine-collected platelets commonly used have disadvantages such as low donor comfort, small supply, short storage time, immune response and pathogen contamination. To solve the problem of lack of blood so...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N5/10
Inventor 裴雪涛谢小燕曲洺逸岳文房芳陈琳何丽娟张博文
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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