Method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro

A technology of insulin secretion and mesenchymal stem cells, which is applied in the field of stem cell induced differentiation research to achieve high transfection efficiency and good safety

Inactive Publication Date: 2012-08-01
TIANJIN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The latest research found that the insulin gene promoter contains the binding site CArG box of SRF, and SRF and PDX-1 can synergistically promote the transcription of insulin gene, but the combination of SRF and growth factors to induce the differentiation of mesenchymal stem cells into smooth muscle cells has not yet See the report

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  • Method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro
  • Method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro
  • Method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro

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Embodiment 1

[0047] A method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro. In this embodiment, human bone marrow mesenchymal stem cells are used. The steps are as follows:

[0048] 1. Isolation and culture of human bone marrow mesenchymal stem cells

[0049] Human bone marrow mesenchymal stem cells were isolated from the hip bones of healthy volunteers, and then the cells were collected for culture. When the cells reached 70-80% confluence, they were washed with PBS, digested with trypsin, and then added to DMEM containing 10% fetal bovine serum in low sugar Medium (5.6mM glucose) terminates the digestion, pipettes into a single cell suspension, discards the supernatant after centrifugation, and then resuspends the cells with DMEM low-sugar medium (5.6mM glucose) containing 10% fetal bovine serum; then subcultures;

[0050] 2. Construction of recombinant eukaryotic expression plasmid containing SRF gene

[0051] (1) Extraction of the mou...

Embodiment 2

[0070] A method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro. This embodiment uses pCMV-Tag2B as a carrier. The specific method is as follows:

[0071] 1. The isolation and cultivation of human bone marrow mesenchymal stem cells are the same as in Example 1.

[0072] 2. Construction of pCMV-SRF eukaryotic expression plasmid

[0073] (1) Using the pcDNA3.1-SRF plasmid constructed in Example 1 as a template, using pCMV-Tag2B as a carrier, using PCR to amplify the SRF gene fragment from the pcDNA3.1-SRF plasmid, according to the full length of SRF Sequence information and the pCMV vector information used to design primers, the primer sequences are as follows:

[0074] Upstream primer 2: 5'-ATCGGGATCCATGTTACCGACCCAAGCT-3'

[0075] Downstream primer 2: 5'-GGTCTCGAGGTTCACCACCTGTAGCTCGG-3'

[0076] Perform PCR amplification, detect the amplified PCR product by agarose gel electrophoresis, and then purify and recover the SRF gene ...

Embodiment 3

[0088] A method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro. Human umbilical cord blood mesenchymal stem cells are used in this embodiment. The specific steps are as follows:

[0089] 1. The isolation and cultivation of human umbilical cord blood mesenchymal stem cells are the same as in Example 1.

[0090] Take anticoagulated umbilical cord blood and mix it with an equal volume of PBS, separate the mononuclear cell layer with Ficoll separation medium, wash with PBS, and count the cells at 2×10 7 Cell density inoculation, placed at 37 ° C, volume fraction of 5% CO 2 , in a saturated humidity incubator. After 2 hours of adherence, replace the fresh culture medium, and when the cells reach 80% confluence, mix with 0.25% trypsin and 0.02% EDTA 1:1, and carry out subculture.

[0091] 2. Construction of recombinant eukaryotic expression plasmid containing SRF gene, same as in Example 1.

[0092] 3. Inducing human umbilical cor...

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Abstract

The invention relates to the field of biomedicine, in particular to a method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro, which includes, according to the technical scheme, constructing recombinant eukaryotic expression plasmids of SRF (serum response factor) gene; preliminarily inducing human mesenchymal stem cells into nestin cells first; further inducing the nestin cells into pancreatic progenitor cells; transfecting an eukaryotic expression vector containing the SRF gene to the cells above; and finally inducing the cells into insulin-secreting cells, wherein the eukaryotic expression vector comprises pEGFP (plasmid enhanced green florescence protein), pcDNA or pCMV. By the method, new seed cell sources for cell therapy of diabetes mellitus are provided, new theoretical and experimental basis is provided for directed induction of mesenchymal stem cells differentiating into insulin-secreting cells and clinical application of the insulin-secreting cells, and new models for drug development and screening related to the insulin-secreting cells are provided.

Description

technical field [0001] The invention belongs to the research field of stem cell induction and differentiation, and relates to a method for inducing stem cell differentiation, in particular to a method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro. Background technique [0002] Diabetes mellitus is a group of metabolic diseases characterized by elevated blood glucose levels, mainly due to relative or absolute deficiency of insulin secretion or insulin receptor defects caused by disorders of sugar, fat and protein metabolism. The mortality rate of diabetes is second only to cardiovascular and cerebrovascular diseases and tumors, which seriously threatens human life and health. As its incidence rate increases year by year, the harm of diabetes to human health will be further aggravated. The World Health Organization estimates that 180 million people worldwide suffer from diabetes, a number that is likely to more than double by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
Inventor 王楠张同存俞如发
Owner TIANJIN UNIV OF SCI & TECH
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