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Method for inducing differentiation of mesenchyma stem cell into islet beta-like cells, and use thereof

A gene and double-gene technology, applied in the field of inducing cell differentiation, can solve the problems of low insulin expression level, failure to achieve clinical application, limit clinical application, etc., achieve huge economic and social benefits, good plasticity, and broad clinical application prospects Effect

Inactive Publication Date: 2011-03-02
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] my country is the second country with a high incidence of diabetes after India. In the past, taking drugs and injecting insulin were commonly used methods to treat diabetes, but taking drugs and injecting insulin often caused serious complications and brought heavy economic burdens. Therefore, looking for Therefore, a more effective and safer treatment plan is of great significance to diabetic patients, and also has great strategic significance to the national economy
Stem cells, especially embryonic stem cells, have the potential to differentiate into almost all adult tissue cells including pancreatic β cells, but their tumorigenicity limits their clinical application
In recent years, studies have shown that human fetal liver mesenchymal stem cells have the greatest potential to differentiate into pancreatic islet β cells, but the current domestic and foreign application of various induction factors and even combined partial gene transfection results in a very limited proportion of insulin-expressing cells. Moreover, the expression level of insulin is low and unstable, which is far from meeting the requirements of clinical application. Therefore, a more effective induction scheme is sought to induce human fetal liver mesenchymal stem cells to efficiently differentiate into islet β-like cells and secrete a higher level of insulin. , more stable insulin to meet clinical treatment requirements, so as to provide safe and effective cell adoptive therapy for clinical treatment of diabetes

Method used

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  • Method for inducing differentiation of mesenchyma stem cell into islet beta-like cells, and use thereof
  • Method for inducing differentiation of mesenchyma stem cell into islet beta-like cells, and use thereof
  • Method for inducing differentiation of mesenchyma stem cell into islet beta-like cells, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Construction of Adxsi-CMV-PDX1 / CMV-NKX6.1 adenoviral vector, the construction method is divided into four steps:

[0081] In the first step, NKX6.1 was excised and connected to the shuttle plasmid vector pShuttle-GFP-CMV (see figure 1 B) (China, Nuosai Genome Research Center Co., Ltd.), obtain pShuttle-GFP-CMV-NKX6.1 (see figure 1 C).

[0082] ①Treatment of shuttle plasmid vector: after combined enzyme digestion with Bgl II+EcoR I (pShuttle-GFP-CMV), dephosphorylation treatment with calf intestinal alkaline phosphatase (Calf Intestinal Alkaline Phosphatase, CIP), after enzyme digestion Add the sample to 1% agarose gel, electrophoresis, cut out the 5.1 kb target fragment of the vector under ultraviolet light, and use the DNA gel recovery and purification kit (column centrifugal type) (for specific steps, refer to the kit instructions, appendix 2. China, Weiglass Biotechnology (Beijing) Co., Ltd., DNA gel recovery and purification kit (column centrifugal type...

Embodiment 2

[0325] Example 2 Isolation, purification, identification and expansion of human fetal liver mesenchymal stem cells:

[0326] 1. Isolation of human fetal liver mesenchymal stem cells

[0327] (1) Isolation, purification and expansion of human fetal liver mesenchymal stem cells: rinse the fetus with sterile normal saline, cut off the umbilical cord, place it in a large beaker, soak it in 75% alcohol for 5 minutes, and remove it from the ultra-clean bench. Take out the fetal liver, wash the blood several times with PBS, cut off the capsule, cut the remaining liver tissue into small pieces, absorb Hank's solution containing 2% FCS with a pointed pipette to wash the liver tissue, collect the liver tissue suspension into 50ml or 15ml aseptic centrifugation In the tube, pipette repeatedly to separate the cells, centrifuge at 50×g for 5 minutes, the pellet is the hepatic parenchymal cells, take the supernatant and transfer it to another centrifuge tube (to remove the hepatic parenchym...

Embodiment 3

[0329] Example 3 Inducing human fetal liver mesenchymal stem cells to differentiate into islet β-like cells:

[0330] Take human fetal liver MSCs (hFL-MSCs) isolated and purified in step Example 2, after the third passage, and reach 80% to 90% fusion, and perform the following three-stage induction to obtain islet β-like cell clusters:

[0331] In the first phase, hFL-MSCs were infected with pAdxsi-CMV-PDX1 / CMV-NKX6.1.

[0332] Transfer cultured mesenchymal stem cells to 75cm 2 Plastic culture flasks to 5 x 10 7 cells / flask; add pAdxsi-CMV-PDX1 / CMV-NKX6.1 virus to the culture flask at an amount of 100 PFU / cell, and continue to culture for 4 days; cells transfected with pAdxsi-CMV-GFP empty adenoviral vector simultaneously are negative In the control group, the expression of EGFP in the cells was observed under a fluorescent microscope at regular intervals. After 24 hours, the cells of each group were lightly washed with PBS, and the cells were fixed with 4% paraformaldehyde...

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Abstract

The invention relates to a method for inducing mesenchymal stem cells to be differentiated into pancreatic island beta-like cells and application thereof. The method comprises the following three stages: firstly, a double-gene expression vector containing an NKX6.1 gene and a PDX1 gene is used to infect the mesenchymal stem cells to transfer double genes into the stem cells and express the doublegenes, the cells are cultivated, and the double genes are confirmed to be expressed for 4 to 7 days; secondly, a culture liquid containing a human epithelial growth factor, a human basic fibroblast growth factor, and B27 is used to continuously induce for 3 to 4 days; and finally a culture medium containing glucagon-like peptide-1, human beta cytosine, a hepatic cell growth factor, niacinamide, B27, beta-mercaptoethanol, and FCS is used to continuously induce for 7 to 10 days. The pancreatic island beta-like cells obtained by the method can be used to produce insulin, serve as seed cells for cellular transplantation to treat diabetes, and has huge economic and social significance.

Description

technical field [0001] The invention relates to the technical field of inducing cell differentiation, in particular to a method for efficiently inducing the differentiation of mesenchymal stem cells into islet β-like cells, related carriers and the application of the obtained islet β-like cells. Background technique [0002] my country is the second country with a high incidence of diabetes after India. In the past, taking drugs and injecting insulin were commonly used methods to treat diabetes, but taking drugs and injecting insulin often caused serious complications and brought heavy economic burdens. Therefore, looking for Therefore, a more effective and safe treatment plan is of great significance to diabetic patients, and also has great strategic significance to the national economy. Stem cells, especially embryonic stem cells, have the potential to differentiate into almost all adult tissue cells including pancreatic β cells, but their tumorigenicity limits their clinic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N15/86C12N15/867C12N15/12C12N5/10C12N5/08A61K35/12A61P3/10A61K35/39
Inventor 张洹唐小龙
Owner JINAN UNIVERSITY
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