In vitro preparation method of pancreas islet beta cell

A β-cell and pancreatic islet technology, applied in the field of in vitro induction of pancreatic islet β-cells, can solve problems such as gene mutation and affecting cell activity

Active Publication Date: 2018-08-24
JINING MEDICAL UNIV
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Problems solved by technology

At present, the method of directed differentiation of stem cells into islet β cells is mainly chemical reagent-induced differentiation method, which not only affects cell viability, but also has the risk of gene mutation

Method used

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  • In vitro preparation method of pancreas islet beta cell
  • In vitro preparation method of pancreas islet beta cell
  • In vitro preparation method of pancreas islet beta cell

Examples

Experimental program
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Embodiment Construction

[0069] Such as Figure 7 As shown, a method for preparing pancreatic beta cells in vitro, comprising the following steps:

[0070] 1. Exosome Extraction

[0071] Serum Replacement was used to replace the serum in the medium, and human β cells were cultured in vitro. During the cell culture, the culture supernatant was collected every two days, and the exosomes in the cell culture medium were separated by ultracentrifugation. Extract 10ml of cell culture supernatant, at 4°C, 300g for 10min, 2000g for 20min, discard the pellet, remove the cells, then 10,000g for 30min, discard the pellet, remove subcellular components, then use 10,000g for 60min, discard the upper The supernatant, and the final precipitate is both exosome.

[0072] 2. Electron microscope observation of exosomes

[0073] (1) Specimen fixation: Exosomes were fixed with 2.5% glutaraldehyde at 4°C for 2h, transferred into penicillin vials, and rinsed with PBS for 3 times at 4°C, 5min each time. Fix with tetrachl...

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Abstract

The invention discloses an in vitro preparation method of a pancreas islet beta cell. The method is that an exosome continuously incubates to induce stem cell differentiation, and comprises the following steps: (1) extracting the exosome; (2) observing the exosome by an electron microscope; (3) analyzing expression of an exosome marker by Western blotting; (4) inducing directional differentiationof iPSCs by the exosome; (5) verifying functions of the beta cell; and (5) verifying functions of miRNA of the exosome of the pancreas islet beta cell. Experiment results show that miRNA plays an important role in differentiating the iPSCs into the beta cell; according to experiment data analysis and miRNA function screening and verification, the condition that the miR-212 / 132 in the exosome has afunction of promoting directional differentiation of the iPSCs to the pancreas islet beta cell is found.

Description

technical field [0001] The invention relates to the technical field of in vitro induction of pancreatic beta cells, in particular to the use of exosomes derived from pancreatic beta cells to induce stem cells to differentiate into pancreatic beta cells. Background technique [0002] Diabetes Mellitus (DM) has become the third disease that threatens human health and affects people's quality of life after tumors and cardiovascular and cerebrovascular diseases, according to statistics released by the International Diabetes Federation (IDF) in December 2015 , about 8.8% of adults aged 20 to 79 suffer from diabetes worldwide, which means that there are about 415 million diabetics worldwide. According to the latest report of World Health Organization (WHO) on April 6, 2016, this number has risen to 422 million, and the number of diabetic patients in China exceeds 100 million, of which more than 1 million people with type 1 diabetes, ranking first in the world. [0003] At present...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/074
CPCC12N5/0676C12N2500/40C12N2500/80C12N2506/45
Inventor 白春雨高玉花
Owner JINING MEDICAL UNIV
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