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Use of tnfsf15 protein in the in vitro expansion of human umbilical cord blood hematopoietic stem cells

A technology of hematopoietic stem cells and human umbilical cord blood, which is applied in the field of medicine, can solve the problems of differentiation, exhaustion, and unsatisfactory amplification effects, and achieve self-renewal promotion and good self-renewal effects

Active Publication Date: 2021-01-01
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the in vitro culture of hematopoietic stem / progenitor cells mainly relies on adding serum and SCF, TPO, Flt3L and other cytokines to the medium, but the expansion effect is still not ideal, and hematopoietic stem / progenitor cells will quickly differentiate and fail in vitro

Method used

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  • Use of tnfsf15 protein in the in vitro expansion of human umbilical cord blood hematopoietic stem cells
  • Use of tnfsf15 protein in the in vitro expansion of human umbilical cord blood hematopoietic stem cells
  • Use of tnfsf15 protein in the in vitro expansion of human umbilical cord blood hematopoietic stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0023] A medium for expanding human umbilical cord blood hematopoietic stem cells, the medium is composed of: IMDM+10%FBS+100ng / mL SCF+100ng / mL TPO+100ng / mL Flt3+100ng / mL IL-6+2μg / mL TNFSF15 protein.

Embodiment 2

[0025] A method for in vitro expansion of human umbilical cord blood hematopoietic stem cells, the method comprising the steps of: first adopting a magnetic bead sorting method to sort CD34+ hematopoietic stem cells in human umbilical cord blood, and using (IMDM+10%FBS+100ng / mLSCF+100ng / mL TPO+100ng / mL Flt3+100ng / mL IL-6+2μg / mL TNFSF15 protein) culture medium was resuspended, the cell concentration was adjusted to 50000 / mL, 200μL per well was added to a 96-well plate, and incubated In the box (37°C, 5% CO 2 ) for 5 days.

Embodiment 3

[0026] Example 3: Effect of TNFSF15 protein on expansion of hematopoietic stem cells

[0027] 1) Sorting of umbilical cord blood hematopoietic stem cells

[0028] (1) Take umbilical cord blood and precipitate red blood cells with hydroxyethyl starch. Cord blood and hydroxyethyl starch were added in a volume ratio of 5:1. After mixing, settle at room temperature for 40 minutes.

[0029] (2) Take the supernatant after sedimentation, add erythrocyte lysate to lyse the erythrocytes, and lyse the erythrocytes in a water bath at 37°C for 20 minutes;

[0030] (3) After centrifugation at 1500 rpm for 15 minutes, discard the supernatant. Add red blood cell lysate again, and lyse in a water bath at 37°C for 20 minutes;

[0031] (4) After centrifugation at 1500 rpm for 15 minutes, discard the supernatant. The pellet was resuspended in 30 mL PBS. Cells were washed by centrifugation and counted.

[0032] (5) The pellet was resuspended with 1 mL of PBS, every 10 8 Add 100 μL of magn...

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Abstract

The invention provides an application of TNFSF15 protein in in-vitro amplification of human umbilical cord blood hematopoietic stem cells. In the application, the concentration of the TNFSF15 protein in an amplification culture medium is 0 < concentration < 4 [mu]g / mL. The invention also provides a method of in-vitro amplification of the human umbilical cord blood hematopoietic stem cells. The TNFSF15 protein has excellent promotion effect on self-renewal of the human umbilical cord blood hematopoietic stem cells and provides a new method for clinical therapy in future.

Description

technical field [0001] The invention relates to the field of medicine, in particular to the application of TNFSF15 protein in the expansion of human umbilical cord blood hematopoietic stem cells in vitro. Background technique [0002] Bone marrow transplantation has been used to treat malignant blood diseases such as leukemia for more than 50 years. Hematopoietic stem cells have become one of the effective ways to treat various diseases clinically because of their ability of self-renewal and multi-directional differentiation. However, the number of hematopoietic stem cells in the bone marrow is low, and it is difficult to carry out hematopoietic reconstruction in patients after transplantation, which has become one of the bottlenecks in the clinical application of hematopoietic stem cells. Among the three sources of hematopoietic stem / progenitor cells currently available (normal human peripheral blood, bone marrow, and infant umbilical cord blood), the proportion of hematopo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2501/25
Inventor 李鲁远张智松丁亚辉高珊沈剑
Owner NANKAI UNIV
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