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Application of t1 peptide in promoting proliferation of umbilical cord blood hematopoietic stem cells in vitro

A technology for hematopoietic stem cells and umbilical cord blood, applied in the field of stem cells, can solve the problems of difficult separation of hematopoietic stem cells, insufficient nutrition and high cost, and achieve the effects of improving differentiation potential and promoting expansion.

Active Publication Date: 2022-03-25
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the shortcomings of this method are also obvious, such as high cost, complicated culture components, and difficulty in separating hematopoietic stem cells in the later stage, which limit the promotion of this expansion method.
Another method is to expand hematopoietic stem cells in vitro by adding cytokines or small molecular compounds and fetal bovine serum, but this method is prone to hypoxia, nutritional deficiencies, complex serum components, and more influencing factors, which may not be able to reach Expected Amplification Effect

Method used

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  • Application of t1 peptide in promoting proliferation of umbilical cord blood hematopoietic stem cells in vitro
  • Application of t1 peptide in promoting proliferation of umbilical cord blood hematopoietic stem cells in vitro
  • Application of t1 peptide in promoting proliferation of umbilical cord blood hematopoietic stem cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Effects of stem cell factor, thrombopoietin, interleukin-6 combined with FLT3-L or different concentrations of T1 peptide on the proliferation of hematopoietic stem cells in vitro

[0052] 1.1 Reagent materials:

[0053] Umbilical cord blood (UCB) was collected from the First Affiliated Hospital of Jinan University; lymphocyte separation fluid was purchased from Guangzhou Squirrel Biotechnology Co., Ltd. (Product No.: 07861, STEMCELL); umbilical cord blood dilution buffer was purchased from Guangzhou Yuwei Biotechnology Instrument Co., Ltd. (Cat. No.: 130-091-221, Miltenyi Biotec).

[0054] 1.2 Experimental method:

[0055] 1.2.1 Cord blood CD34 + Cell Sorting and Purity Verification

[0056] Dilute the collected umbilical cord blood with umbilical cord blood dilution buffer, and then separate mononuclear cells with lymphocyte separation medium to obtain umbilical cord blood mononuclear cells, and then use immunomagnetic bead cell sorting to separate CD34 ...

Embodiment 2

[0065] Example 2, Colony Formation Experiment of Umbilical Cord Blood CD34+ Cells and Cultures thereof

[0066] 2.1 Reagent materials:

[0067] Sodium methylcellulose semi-solid medium (MethoCult H4434 Classic); IMDM medium.

[0068] 2.2 Experimental method:

[0069] Sorted cord blood CD34 + CD34 after cells (sorting method is the same as in Example 1) and different factor combinations + The cells were adjusted to a cell density of 1x10 with IMDM medium (containing 2% (v / v) fetal bovine serum (FBS)) 4 cells / ml, then mix according to the ratio of 100ul cell suspension + 1ml methylcellulose sodium semi-solid medium, mix well on a vortexer, let stand for 10-15min, then transfer to a 35mm culture dish and flatten Culture cultures were placed on 100mm petri dishes, and each experimental group was repeated three times, and a 35mm petri dish filled with sterile water was added to maintain high humidity throughout the culture process to prevent the culture from drying up; place th...

Embodiment 3

[0072] Example 3. Effects of stem cell factor, thrombopoietin, interleukin-6 combined with FLT3-L and different concentrations of T1 peptide on the proliferation of hematopoietic stem cells in vitro

[0073] 3.1 Reagent materials: Same as Example 1.

[0074] 3.2 Experimental method:

[0075] 3.2.1 Sorting and purity identification of cord blood CD34+ cells: same as in Example 1.

[0076] 3.2.2 In vitro expansion of cord blood CD34+ hematopoietic stem cells

[0077] (1) Centrifuge the cell suspension separated by immunomagnetic beads at 300 g for 8 minutes, and discard the supernatant.

[0078](2) Resuspend the cell pellet with StemSpan SFEM+100ng / ml SCF+100ng / ml TPO+20ng / ml IL-6 medium (both are the final concentration of the system), and adjust the cell concentration to 10 5 cells / ml, 500 μL per well, added to a 24-well plate, and then 500 μL of the same medium was added to the wells to culture, a total of 6 columns, numbered A to F, and 3 replicate wells in each column. ...

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Abstract

The invention discloses the application of a T1 peptide in promoting the proliferation of umbilical cord blood hematopoietic stem cells in vitro. The present invention cultivates CD34+ hematopoietic stem cells in human umbilical cord blood under serum-free conditions, and simultaneously tests the effect of T1 (GPANVET) on the in vitro expansion of human umbilical cord blood hematopoietic stem cells, and finds that T1 peptide has an effect on the self-proliferation of human umbilical cord blood hematopoietic stem cells. The update has a better promotion effect. The present invention also found that the combination medium of stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), interleukin-6 (IL-6) and T1 peptide has the effect of promoting hematopoiesis Stem cell expansion and maintenance of the proportion of stem cells can also improve the differentiation potential of hematopoietic stem cells, which can provide a new method for clinical treatment in the future.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and particularly relates to the application of a T1 peptide in promoting the proliferation of umbilical cord blood hematopoietic stem cells in vitro. Background technique [0002] Hematopietic stem cells (HSCs) are stem cells that generate other blood cells in the blood system, have long-term self-renewal ability and the potential to differentiate into various mature blood cells. Autologous or allogeneic transplantation has been successfully used in the treatment of hematological diseases, malignant tumors, primary immunodeficiency diseases and metabolic abnormalities in children and adults that endanger human health. However, hematopoietic stem cell transplantation is also limited by many factors, such as bone marrow hematopoietic stem cells need highly similar HLA matching, G-CSF mobilization of peripheral blood hematopoietic stem cells may cause donor spleen rupture. [0003] Umbilical cor...

Claims

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Application Information

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IPC IPC(8): C12N5/0789A61K35/28
CPCC12N5/0647A61K35/28C12N2500/90C12N2501/999C12N2501/125C12N2501/145C12N2501/2306C12N2501/26
Inventor 陈小佳洪岸张碧辉
Owner JINAN UNIVERSITY
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