Method for inducing human umbilical cord mesenchymal stem cells to be differentiated into pancreatic beta cells

A technology of mesenchymal stem cells and human umbilical cords, applied in the direction of artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems of donor lack of immune rejection, insulin resistance, and inability to cure diabetes, etc., and achieve good differentiation effect and easy operation , the effect of the simple method

Inactive Publication Date: 2016-04-06
SHENZHEN BEIKE BIOTECH +1
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Problems solved by technology

However, although insulin therapy can control symptoms, delay and reduce the occurrence of complications, it cannot completely cure diabetes, and long-term use of insulin will cause serious complications such as blindness, renal failure, obesity, and insulin resistance. In addition, insulin is injected every day It also brings great suffering to patients and brings heavy economic burden to patients, families and society.
Although islet cell transplantation is an effective treatment strategy, the problems still face medical and ethical issues such as donor shortage and immune rejection

Method used

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  • Method for inducing human umbilical cord mesenchymal stem cells to be differentiated into pancreatic beta cells
  • Method for inducing human umbilical cord mesenchymal stem cells to be differentiated into pancreatic beta cells
  • Method for inducing human umbilical cord mesenchymal stem cells to be differentiated into pancreatic beta cells

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Embodiment 1

[0046] 1. Extraction of human umbilical cord mesenchymal stem cells.

[0047] Neonatal umbilical cords authorized with maternal consent were used as a source of mesenchymal stem cells.

[0048] Immediately after the fetus was delivered, the umbilical cord was ligated and cut according to the obstetrical routine, the umbilical cord was washed with normal saline, and then disinfected with medical alcohol, and the umbilical cord was stored in the umbilical cord preservation solution at a constant temperature of 2-8°C.

[0049] The obtained umbilical cord was washed with 0.9% sodium chloride injection, and repeated 2 to 3 times to remove blood stains. The whole umbilical cord was sterilized by immersing it in 75% ethanol. Sodium chloride injection was washed repeatedly to remove residual ethanol. Use sterile surgical scissors to cut the umbilical cord into several sections of about 2 to 5 cm, and remove the congestion and clots in the blood vessels of the small section of the um...

Embodiment 2

[0068] 1. Extraction of human umbilical cord mesenchymal stem cells.

[0069] With embodiment 1.

[0070] 2. Preparation of six adenoviruses containing transfection genes respectively PDX1, MAFA, NGN3, FOXA2, NEUROD1 and FGF21.

[0071] The adenovirus was prepared by outsourcing and stored at -80°C. The adenovirus titers were: PDX1: 2.96×10 8 pfu / mL, MAFA: 8.52×10 9 pfu / mL, NGN3: 5.24×10 9 pfu / mL, FOXA2: 4.35×10 9 pfu / mL, NEUROD1: 3.62×10 8 pfu / mL and FGF21: 4.15×10 8 pfu / mL.

[0072] 3. Induced differentiation of human umbilical cord mesenchymal stem cells.

[0073] 3.1 Resuscitate the cryopreserved human umbilical cord mesenchymal stem cells, add them to a centrifuge tube, then add pre-cooled 0.9% sodium chloride injection, blow and wash the pipette, constant volume, mix well, and centrifuge at 300g for 10min.

[0074] After washing once with 5×10 3 Cells / cm2 were cultured in a cell culture dish, and the medium was DMEM high-glucose medium, which was supplemented wi...

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Abstract

The invention discloses a method for obtaining pancreatic beta cells through induced differentiation of human umbilical cord mesenchymal stem cells through a gene transfer method. Pancreatic cell specific genes containing PDX1, MAFA, NGN3, FOXA2, NEUROD1 and FGF21 are used, adenovirus expression vectors of transmembrane singles are connected, various recombinant protein and small molecules are combined, and the human umbilical cord mesenchymal stem cells are induced to be directionally differentiated into the pancreatic beta cells. The inducing method is easy and convenient to implement, simple in operation and good in differentiation effect.

Description

technical field [0001] The invention relates to a method for inducing the formation of islet beta cells, in particular to a method for inducing human umbilical cord mesenchymal stem cells to differentiate into islet beta cells. Background technique [0002] Diabetes mellitus is an absolute or relative lack of insulin due to destruction of pancreatic β cells or insulin resistance. At present, the treatment of diabetes is still palliative, not the root cause. Although drug treatment can effectively lower blood sugar, it cannot effectively restore the function of islets of pancreas. Insulin supplementation is the traditional treatment for type 1 and some type 2 diabetes. However, although insulin therapy can control symptoms, delay and reduce the occurrence of complications, it cannot completely cure diabetes, and long-term use of insulin will cause serious complications such as blindness, renal failure, obesity, and insulin resistance. In addition, insulin is injected every d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 胡祥李陶刘沐芸李婵丁长才
Owner SHENZHEN BEIKE BIOTECH
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