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Method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells

A technology for insulin-secreting and mesenchymal stem cells, which is applied in a new field of high-efficiency induction and differentiation of mesenchymal stem cells to insulin-secreting cells, can solve problems such as prohibitive cost and limited number of organs, and achieve high-efficiency solutions to avoid cell growth Process instability, effect of excluding the risk of transmission of heterologous pathogens

Pending Publication Date: 2017-05-17
里程 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of organs available for transplantation is limited after all, and the cost of organ transplantation is often prohibitive, and organ transplantation often causes strong immune rejection. These problems have always plagued doctors and patients

Method used

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  • Method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells
  • Method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells
  • Method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Screening of medium composition

[0054] Basal medium: DMEM (glucose content 4.5g / L)+5%SR+1%NEAA+2%B27(50X)+1%N-2(100X)+10ng / ml HGF+10ng / ml EGF+10ng / ml b-FGF+10mmol / L Nicotinamide;

[0055] Screening components: Add 100ng / ml vinblastine III (Conophylline), 1 volume part of ITS, a final concentration of 1ug / ml heparin (Heparin), and a final concentration of 10ng / ml betacellulin (betacellulin) in the basal medium. ), sialin 4 (Exendi-4), insulin-like growth factor (IGF-1) and pentagastrin. The status of each group is shown in Table 3 below.

[0056] table 3

[0057]

[0058]

[0059] In the biosafety cabinet, the hUC-MSCs of the third passage isolated from the umbilical cord Huatong glue tissue of natural delivery newborns were collected and divided into 5×10 4 cells / cm 2 The density was inoculated in an ultra-low adsorption six-well plate, and 2ml of the test medium of each group shown in Table 3 was added to each well to observe the cell growth a...

Embodiment 2

[0061] Example 2 Screening of Content of Medium Component (Vinblastine Ⅲ)

[0062] Test medium: 89 parts by volume of high-sugar DMEM (glucose content 4.5g / L), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50X), 1 part by volume of ITS, 1 part by volume non-essential amino acid (NEAA), 1 volume part of N-2 (100X), heparin (Heparin) with a final concentration of 1ug / ml, nicotamide with a final concentration of 10mmol / L, and recombinant human base with a final concentration of 10ng / ml Fibroblast growth factor (b-FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), pentagastrin and concentration gradients of 1, 10, 50, 100, 200, 300ng / ml Vinblastine III (Conophylline).

[0063] Human umbilical cord mesenchymal stem cells were induced and differentiated in culture medium with different concentrations of vinblastine III (Conophylline).

[0064] Results: In the two groups of test media with concentrations of 1 and 10ng / ml vinblastine Ⅲ, f...

Embodiment 3

[0065] Example 3. Screening of media components (pentagastrin) content

[0066] Test medium: 89 parts by volume of high-sugar DMEM (glucose content 4.5g / L), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50X), 1 part by volume of ITS, 1 part by volume The non-essential amino acid (NEAA), the final concentration is 1ug / ml heparin (Heparin), 1 volume part of N-2 (100X), 100ng / ml vinblastine III (Conophylline), 10mmol / L nicotamide, Recombinant human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) with a final concentration of 10 ng / ml and a concentration gradient of 1, 2, 5, 10, 20 , 30, 50ng / ml of pentagastrin.

[0067] Human umbilical cord mesenchymal stem cells were induced and differentiated by using the medium with different concentrations of pentagastrin above, and then the sugar stimulation experiment was used to determine the unit cell (1×10 4 1) the insulin release amount of each group, and...

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Abstract

The invention discloses a method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells by adopting a novel serum-free medium. The medium contains DMEM (with the glucose content of 4.5 g / L) containing high glucose, B27, recombinant human basic fibroblast growth factors (b-FGF), Nicotinamide, N-2, Conophylline III (Conophylline), non-essential amino acids (NEAA), Heparin (Heparin), epidermal growth factors (EGF), hepatocyte growth factors (HGF), serum replacement (SR), insulin-transferrin-selenium compounds (ITS) and pentagastrin (Pentagastrin). The method using the medium can realize induced differentiation of mesenchymal stem cells to the insulin secretion cells in one step within six days.

Description

technical field [0001] The invention relates to the research field of stem cells, in particular to a novel and efficient method suitable for inducing differentiation of mesenchymal stem cells into insulin-secreting cells. Background technique [0002] Diabetes has become a worldwide epidemic in the 21st century. It is the third chronic non-communicable disease that seriously threatens human health after tumors and vascular diseases. It is characterized by high mortality, high disability and high medical expenses. . At present, the number of diabetic patients in China has reached 114 million, ranking first in the world, accounting for about one-third of the total number of diabetic patients in the world. Therefore, diabetes has become a severe public health problem faced by countries all over the world, especially China. [0003] After the middle of the 20th century, with the rise and development of human organ transplantation technology, pancreas transplantation was introd...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0775
CPCC12N5/0676C12N2500/25C12N2500/32C12N2500/34C12N2500/90C12N2501/06C12N2501/11C12N2501/115C12N2501/12C12N2501/345C12N2501/91C12N2501/999C12N2506/1392
Inventor 郭镭
Owner 里程
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