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Application of cepharanthine and culture medium and method for amplifying hemopoietic stem cell of placenta

A technology of hematopoietic stem cells and fenugreek, which is applied in the directions of cell culture active agents, blood/immune system cells, biochemical equipment and methods, etc. Enhanced amplification effect, high activity effect

Inactive Publication Date: 2017-03-15
GUANGZHOU KOHEALA BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after research, it is found that the above-mentioned medium is still insufficient for the proliferation of the number and content of hematopoietic stem cells, and needs to be further improved.

Method used

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  • Application of cepharanthine and culture medium and method for amplifying hemopoietic stem cell of placenta
  • Application of cepharanthine and culture medium and method for amplifying hemopoietic stem cell of placenta
  • Application of cepharanthine and culture medium and method for amplifying hemopoietic stem cell of placenta

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Extraction and separation of placental hematopoietic stem cells

[0031] With the consent of the obstetrics and gynecology department of Huaqiao Hospital in advance, the placenta was collected through the formal collection method of Huaqiao Hospital in the form of voluntary donation.

[0032] Wash the surface of the placenta with PBS and discard all the cleaning solution; separate the two arteries of the umbilical cord with tweezers, and fix the infusion set in the lumen of the artery; perfuse PBS into the umbilical cord artery and repeat at least 1 LPBS; collect the perfused solution into a centrifuge tube In the medium, centrifuge at 1500rpm for 5-10min; add 6% hydroxyethyl starch solution to the centrifuged precipitate, mix well and let stand for 20-30min; That is placental hematopoietic stem cells.

Embodiment 2

[0033] Example 2: Safety test of stephenine on placental hematopoietic stem cells

[0034] Stephanine was diluted to 0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μmol / ml drug concentration with DMEM basal medium.

[0035] The cells isolated in Example 1 were mixed with 1×10 4 Inoculate in a 96-well plate at a density of 1 / mL, add different concentrations of stephine, 100ul per well, take the basal medium of placental hematopoietic stem cells without stephine as the normal cell group, and place at 37°C, 5% CO 2 Cultivate under the condition for 48h and 72h.

[0036]According to the instructions of the MTT kit, the OD value was detected by a microplate reader, and the survival rate of the cells was calculated. The results are shown in figure 1 .

[0037] Depend on figure 1 It can be seen that whether it is cultured for 48h or 72h, when the concentration of stephine is 50-100 μmol / ml, the survival rate of hematopoietic stem cells is the highest, so 50-100 μmol / ml is used as the sa...

Embodiment 3

[0038] Embodiment 3: the comparative test of the amplification effect of different expansion medium

[0039] 1. Medium

[0040] Control medium 1: DMEM / F12+10% FBS;

[0041] Control medium 2: expansion medium of CN105112374A Example 3;

[0042] Positive control medium: DMEM / F12+10%FBS+15ng / ml thrombopoietin+100ng / ml stem cell growth factor+33ng / ml human FMS-like tyrosine kinase 1 ligand+0.2ng / ml interleukin-1+0.2 ng / ml interleukin-6+1uMSR-1 (Note: SR-1 is a puromycin derivative that has been obtained through screening technology, and it has been confirmed so far that it can promote the massive expansion and self-renewal of human CD34+ hematopoietic stem cells Small molecules can generally increase the number of cells by 50 times, so they can be used as positive control drugs, but their cost is high and they are not easy to purchase);

[0043] Culture medium 1 of the present invention: DMEM+15% FBS+15ng / ml GM-CSF+150ng / ml stem cell growth factor+45ng / ml human FMS-like tyrosin...

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Abstract

The invention relates to the technical field of biologics, and discloses application of cepharanthine in preparing an in-vitro amplification culture medium of hemopoietic stem cells of placentae and / or in in-vitro amplification of the hemopoietic stem cells of the placentae, and a culture medium containing cepharanthine amplified hemopoietic stem cells of placentae, and a method. As multiple appropriate components are combined with the cepharanthine to prepare the in-vitro amplification culture medium, the amplification effect of the in-vitro amplification culture medium can be remarkably improved in in-vitro amplification of the hemopoietic stem cells of the placentae, and the in-vitro amplification culture medium has the characteristics of quick in dryness and high in activity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of stephine and a culture medium and method for expanding placental hematopoietic stem cells. Background technique [0002] Hematopoietic stem cells are a group of primitive hematopoietic cells that exist in hematopoietic tissues. They are adult stem cells in the blood system. They have the ability of long-term self-renewal and the potential to differentiate into various mature blood cells. It is a type of adult stem cell with the longest and most in-depth research history, and it has important guiding significance for the study of various stem cells, including tumor stem cells. Hematopoietic stem cells are an effective method for the treatment of leukemia, lymphoma and other malignant tumors, autoimmune diseases and congenital immune defects and other diseases that seriously endanger human health, and can effectively alleviate the suffering of patients. [0003] The...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/30C12N2500/84C12N2501/125C12N2501/22C12N2501/2303C12N2501/2306C12N2501/727
Inventor 王一飞陈海佳葛啸虎马岩岩王小燕
Owner GUANGZHOU KOHEALA BIOTECHNOLOGY CO LTD
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