Method for promoting proliferation of umbilical cord blood hematopoietic stem cells by adipose-derived stem cells

A technology of umbilical cord blood stem cells and adipose stem cells, which is applied in the field of biomedicine, can solve the problem of umbilical cord blood stem cells, and achieve good application value, promote proliferation, and high-speed expansion

Active Publication Date: 2021-03-12
广州市拜沃思生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] However, at present, there is no research on the use of adipose-derived stem cells to improve the proliferation and red blood cell differentiation of umbilical cord blood stem cells.

Method used

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  • Method for promoting proliferation of umbilical cord blood hematopoietic stem cells by adipose-derived stem cells
  • Method for promoting proliferation of umbilical cord blood hematopoietic stem cells by adipose-derived stem cells
  • Method for promoting proliferation of umbilical cord blood hematopoietic stem cells by adipose-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0026] The effect of embodiment 2 polypeptide in promoting the expression of FOXO3

[0027] The polypeptide of SEQ ID NO: 1 was added to the StemSpan SFEM medium according to the concentration dosage of 0, 1ug / mL, 10ug / mL, 50ug / mL and 100ug / mL respectively. The cells separated in Example 1 were divided into 5×10 5 The density of cells / ml was inoculated in 1 ml medium for suspension culture for 48 hours to detect the expression of FOXO3 gene. The same amount of RNA was extracted, and the TaKaRa kit was used to reverse transcribe the RNA into cDNA. RT-PCR detection was performed with the following primers. The primer sequence of FOXO3 is: upstream 5′-CGGACAAACGGCTCACTCT-3′, downstream 5′-GGACCCGCATGAATCGACTAT-3′; the sequence of GAPDH primer is: upstream 5′-ACAACTTTGGTATCGTGGAAGG-3′, downstream 5′-GCCATCACGCCACAGTTTC-3′. Placed in 7300HTRT-PCR instrument for amplification. The reaction conditions were set according to the conditions of the TaKaRa kit. Utilize ABI7300 system...

Embodiment 3

[0029] Example 3 Separation of Adipose Stem Cells

[0030] Abdominal fat from healthy people was cut into tissues with a size of 0.1cm*0.1cm*0.1cm, and washed 3-5 times with PBS buffer. Digest with collagenase I 5 times the volume of fat, repeatedly cut the fat tissue into a paste, shake evenly at 37°C for 30 minutes, filter the digested fat tissue into a centrifuge tube with 250ul nylon gauze, and centrifuge at 1000r / min for 30 minutes , discard the supernatant and suspended fat fragments, resuspend the pellet in 00.075mmol / L potassium chloride, centrifuge after standing for 10min, discard the wound, and resuspend the pellet in DMEM culture medium containing 10% fetal bovine serum in a culture dish, placed in a 37°C, 5% CO2 incubator, and cultured at 100% humidity. The culture medium was replaced every other day. After the cells covered 90% of the bottom area of ​​the culture dish, they were digested with 0.25% trypsin. Subculture was performed at a ratio of 1:3. The third-...

Embodiment 4

[0031] Example 4 The promoting effect of fat stem cells on umbilical cord hematopoietic stem cells

[0032] (1) Co-cultivation experiment group: Take a 24-well Transwell culture plate, carefully clip out the upper chamber containing the semi-permeable membrane with sterile tweezers, and add 600ul of Example 3 resuspended with adipose-derived stem cell culture medium to the lower chamber The separated adipose stem cell suspension was mixed by pipetting. Carefully add 100ul of the umbilical cord blood stem cell suspension isolated in Example 1 into the upper chamber with a pipette gun. so that the upper chamber of each well contains 10 cells 4 , the lower chamber contains 5×10 cells 4 indivual. The total liquid volume in each well is 700ul, and 3 duplicate wells are set. Wherein the umbilical cord hematopoietic stem cell culture medium: DMEM-F12 medium 90%+FBS 10%+SEQ ID NO: 1 polypeptide 50μg / mL+SCF15ng / ml+IL-330ng / ml+hyaluronic acid 10μg / mL. Adipose stem cell medium: DMEM...

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Abstract

The invention relates to a method for promoting proliferation of umbilical cord blood hematopoietic stem cells by using adipose-derived stem cells. The bioactive peptide capable of specifically promoting high expression of FOXO3 in umbilical cord blood stem cells is obtained through screening and optimization, and the polypeptide can promote proliferation of the umbilical cord blood stem cells anddifferentiation of the umbilical cord blood stem cells into red blood cells by improving expression of FOXO3. The invention further provides an improved method for improving umbilical cord blood stemcell proliferation by adopting the adipose-derived stem cells. The method comprises the step of culturing the adipose-derived stem cells and the umbilical cord blood stem cells together. The amplified hematopoietic stem cells are further cultured in a special induction medium to obtain differentiated red blood cells. According to the method, the hematopoietic stem cells can be amplified at a highspeed, erythrocytes with good erythroid characteristics can be obtained, so that the method can be used for subsequent clinical research on a large scale and has good application value.

Description

technical field [0001] The invention relates to the field of biomedicine, and more specifically, relates to a method for promoting the proliferation of umbilical cord blood hematopoietic stem cells by fat stem cells. Background technique [0002] As a new donor for hematopoietic stem cell transplantation, umbilical cord blood has the advantages of wide sources, easy collection, no harm to the donor, low requirements for HLA matching, and high content of hematopoietic stem / progenitor cells (HSPC). Furthermore, cord blood preparation takes 4-5 weeks less than the time required to obtain hematopoietic stem cells from a matched unrelated donor. In addition, the incidence of disease recurrence and graft-versus-host disease after umbilical cord blood transplantation (UCBT) is also lower compared with other sources of hematopoietic stem cell transplantation. However, the biggest limitation of UCBT in clinical application is that the number of total nucleated cells (TNC) that can b...

Claims

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Application Information

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IPC IPC(8): C12N5/078C12N5/0789
CPCC12N5/0641C12N5/0647C12N2502/1382
Inventor 刘欢李蒙
Owner 广州市拜沃思生物科技有限公司
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