Method for differentiating umbilical cord blood stem cells in cooperation with adipose-derived stem cells
A technology of umbilical cord blood stem cells and adipose stem cells, applied in chemical instruments and methods, biochemical equipment and methods, animal cells, etc., can solve the problem of umbilical cord blood stem cells not being available, achieve good application value, high-speed amplification, and promote proliferation Effect
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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 at concentrations 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 SDSSoftware softw...
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, cut the adipose tissue repeatedly until it becomes a paste, shake evenly at 37°C for 30 minutes, filter the digested adipose tissue into a centrifuge tube with 250ul nylon gauze, and centrifuge at 1000r / min for 30min , 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 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 100 ul 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: DME...
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