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Method for induced differentiation of amnion epithelial stem cells into functional pancreatic beta cells as well as application of induced differentiation

A technology for inducing differentiation and stem cells, which can be used in cell culture active agents, biochemical equipment and methods, medical preparations containing active ingredients, etc. The effect of improving hyperglycemia state, abundant cell sources and improving quality of life

Pending Publication Date: 2019-11-08
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that frequent monitoring of blood sugar and insulin injection is required, and it cannot prevent the occurrence and development of complications such as renal failure, heart disease, fundus disease, and gangrene caused by diabetes.
However, the limited sources of menstrual blood and umbilical cord blood, the ethical issues and tumorigenicity of embryonic stem cells, and the tumorigenicity of induced pluripotent stem cells make it difficult for these cells to be used clinically.

Method used

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  • Method for induced differentiation of amnion epithelial stem cells into functional pancreatic beta cells as well as application of induced differentiation
  • Method for induced differentiation of amnion epithelial stem cells into functional pancreatic beta cells as well as application of induced differentiation
  • Method for induced differentiation of amnion epithelial stem cells into functional pancreatic beta cells as well as application of induced differentiation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Isolation, culture and expansion of amniotic epithelial stem cells (HAESCs):

[0060] Under the premise of obtaining the consent of the newborn's family members, the fresh amniotic membrane was isolated. After 2 hours of antibiotic treatment, microbiological detection and safety detection of infectious disease pathogens were carried out. After the amniotic membrane was digested by trypsin / EDTA (0.25%) in a 37°C water bath for 1 hour, a stop solution was added. After centrifugation, the supernatant was removed, and cultured in an incubator with 5% CO2, 95% humidity, and 37°C with amnion epithelial stem cell culture medium. Change the medium after 2-3 days to remove unattached cells, and then change the medium every 2 days. When the confluence of the cells reached 80%, trypsin / EDTA was used for passage. Amnion epithelial stem cells were identified by immunofluorescence and flow cytometry for OCT4, Nanog, SSEA-4, E-cadherin, CD29, CD73, CD90, CD105, HLA-ABC, HLA-DR, CD3...

Embodiment 2

[0137] In vivo and in vitro tumorigenicity detection of amniotic membrane epithelial stem cells

[0138] 1. In vivo and in vitro tumorigenicity detection of amniotic membrane epithelial stem cells

[0139] 1) Take the third-generation amnion epithelial cells in good growth condition, which are characterized by vigorous growth, large cell bodies, clear nuclei, rich cytoplasm, and strong refraction under the microscope, and digest them with trypsin-EDTA (0.25% ) digestion, under the microscope, when the cells became a single round shape, the digestion was terminated with DMEM medium containing 10% (volume concentration) fetal bovine serum, centrifuged after gentle blowing, and the cell pellet was obtained, washed with PBS (PBS without calcium and magnesium ions) solution) and washed twice (the purpose is to wash away trypsin, fetal bovine serum and other substances);

[0140] 2) Inoculate the amniotic epithelial stem cells in step 1) on soft agar, and after culturing for 30 day...

Embodiment 3

[0149] Induction and differentiation of amniotic epithelial stem cells into insulin-secreting cells and their identification

[0150] The specific operating procedures are as follows:

[0151] 1. Directed induction of amniotic epithelial stem cells to differentiate into insulin-secreting cells in vitro

[0152] 1) Take the well-growing cells of the third generation and digest them with trypsin-EDTA digestion solution (0.25%). The DMEM medium was digested, centrifuged after gently pipetting to obtain cell pellets, and washed twice with PBS (PBS washing solution without calcium and magnesium ions) (the purpose is to wash away trypsin, fetal bovine serum and other substances);

[0153] 2) Inoculate the cell pellet obtained in the above steps into a 10cm culture dish for induction culture, and the seeding cell density is 1×10 6 / dish, add 10ml differentiation induction medium I to each dish, and then place in 5% CO 2 , 95% humidity, and cultured in an incubator at 37°C; after 2...

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Abstract

The invention discloses a method for induced differentiation of amnion epithelial stem cells into pancreatic beta cells with biological functions. The method comprises the steps as follows: 1) separation, culture, amplification and identification of amnion epithelial stem cells; 2) induced differentiation in vitro: the second-third generation of amnion epithelial stem cells are selected for experiments and subjected to induced differentiation by a culture medium containing nicotinamide. Insulin secretion cells which can express specific markers of pancreatic beta cells and have functions of normal pancreatic beta cells are obtained by 14 days of differentiation in vitro; and 3) in vivo transplantation: insulin secretion cells produced by induction are transplanted into type-1 diabetes miceand can obviously relive the hyperglycemia state of mice.

Description

technical field [0001] The invention relates to a method for inducing and differentiating pancreatic islet β cells by using amniotic membrane epithelial stem cells. Background technique [0002] With the improvement of people's living standards and the reduction of exercise, the incidence of diabetes has been increasing in recent years. According to statistics in 2013, the number of people suffering from diabetes in the world has exceeded 382 million, and the number of diabetic patients in my country is as high as 114 million, ranking first in the world. In my country's urban population, the incidence of adult diabetes is close to 12%, and it is still increasing rapidly at a rate of 3%-5% per year. Therefore, diabetes, as a worldwide disease, is endangering the lives of many patients. The pathogenesis of diabetes is very complex, related to many factors such as environment and genetics, and its pathogenesis is still unclear. Diabetes mellitus is a metabolic disorder chara...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/073A61K35/39A61P3/10
CPCC12N5/0676C12N5/0605A61K35/39A61P3/10C12N2500/44C12N2500/38C12N2501/11C12N2506/025C12N2501/91C12N2500/32
Inventor 辛洪波李婧嫄柳全文刘倩玉
Owner NANCHANG UNIV
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