Method for inducing differentiation of human embryo mesenchymal stem cells into pancreatic islet beta-like cell

A bone marrow mesenchymal and cell differentiation technology, applied in the field of inducing human fetal bone marrow mesenchymal stem cells to differentiate into islet β-like cells, can solve the problems of severe complications, limited clinical application, and low insulin secretion of differentiated cells, etc. Achieve great economic and social benefits, broad clinical application prospects, and low immunogenicity

Inactive Publication Date: 2008-05-28
JINAN UNIVERSITY
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AI Technical Summary

Problems solved by technology

In the past, taking drugs and injecting insulin were commonly used methods to treat diabetes, but taking drugs and injecting insulin would not only bring a heavy economic burden, but also cause serious complications; Insufficient tissue sources and immune rejection are two major problems
Stem cell research has brought new hope to diabetic patients. Embryonic stem cells have the strongest multidirectional differentiation potential and can differentiate into almost all adult tissue cells including pancreatic β cells. Tumor and strong immunogenicity, these factors

Method used

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  • Method for inducing differentiation of human embryo mesenchymal stem cells into pancreatic islet beta-like cell
  • Method for inducing differentiation of human embryo mesenchymal stem cells into pancreatic islet beta-like cell
  • Method for inducing differentiation of human embryo mesenchymal stem cells into pancreatic islet beta-like cell

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Method for Inducing Human Fetal Bone Marrow Mesenchymal Stem Cells to Differentiate into Islet β-like Cells

[0025] (1) Isolation, culture and expansion of human fetal bone marrow mesenchymal stem cells

[0026] Rinse the fetus with sterile normal saline, cut off the umbilical cord and place it in a large beaker, soak it in alcohol with a volume fraction of 75% for 5 minutes, take out the fetal tibia and femur aseptically in the ultra-clean table, remove the muscles attached to it, and cut off both ends Epiphysis, suck out Hank’s solution containing 2% FCS with a 2ml syringe to rinse the bone marrow cavity until the marrow cavity turns white, collect the rinsed liquid into a 15ml sterile centrifuge tube, pipette the pipette repeatedly to separate the cells, centrifuge at 100×g for 5min, and sediment Mostly red blood cells, take the supernatant and transfer it to another centrifuge tube (remove red blood cells), centrifuge at 500×g for 5 minutes, discard the s...

Embodiment 2

[0029] Example 2, identification of islet-like cell clusters

[0030] (1) General morphological changes: Figure 1 is a morphological diagram of uninduced MSCs. Figure 2 is a morphological diagram of the islet-like cell mass obtained after 6 days of induction. It can be seen from Figure 1 that the uninduced MSCs are long spindle-shaped and arranged in parallel; it can be seen from Figure 2 that the MSCs after 6 days of induction aggregated into islet-like cell clusters.

[0031] (2) Observation of insulin expression by immunocytochemical method: Figure 3 is the negative expression of insulin in uninduced MSCs; Figure A is the positive expression of islet β-like cell cytoplasm; Figure B is the positive expression of islet β-like cell cytoplasm; Kernel map; Figure C is the combined figure of Figure A and Figure B. Immunofluorescence staining results showed that insulin was stained red by the fluorescent dye Cy3 and localized in the cytoplasm (Figure A); the nucleus was stained ...

Embodiment 3

[0045] Embodiment 3, mice intraperitoneal injection experiment

[0046] Take 300 μL of normal saline, simple induction solution, 6-day culture supernatant (insulin level detected by chemiluminescence method is 3 U / mL) and standard insulin solution (4 U / mL), and intraperitoneally inject them into normal mice and diabetic mice In vivo, Table 1. Effects of different solutions on blood glucose levels in normal mice. Table two, the influence of different solutions on the blood sugar level of diabetic mice. The results showed that the standard insulin solution had a rapid and obvious hypoglycemic effect on both normal mice and diabetic mice, while the culture supernatant induced for 6 days had no hypoglycemic effect on normal mice, but had a rapid effect on diabetic mice. And obvious hypoglycemic effect, the effect trend is similar to that of standard insulin solution. Injection of the same volume of normal saline and simple induction solution had no hypoglycemic effect on normal ...

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Abstract

The invention provides a process for inducing mescenchymal stem cell of human fetal bone marrow to differentiate to be islet beta cells. The process provided by the invention comprises the following steps including separating, purifying and augmenting mescenchymal stem cell of human fetal bone marrow, and then inducing mescenchymal stem cell of human fetal bone marrow to differentiate to islet beta cells, and beta cell mass is achieved after six to twelve days of inducement. The process provided by the invention induces mescenchymal stem cell of human fetal bone marrow to be islet beta cells which can secrete insulin in vitro. The achieved islet beta cells can be further used for preparing medicament which is used for treating diabetes. The study of the field has wide clinical application prospect and can bring relatively great economic and social benefit.

Description

technical field [0001] The invention relates to cell differentiation, in particular to a method for inducing human fetal bone marrow mesenchymal stem cells to differentiate into pancreatic 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 would not only bring a heavy economic burden, but also cause serious complications; There are two major problems of insufficient tissue source and immune rejection. Stem cell research has brought new hope to diabetic patients. Embryonic stem cells have the strongest multidirectional differentiation potential and can differentiate into almost all adult tissue cells including pancreatic β cells. These factors limit its clinical application; pancreatic stem cells are adult stem cells with the most potential to differentiate into β c...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/071
Inventor 张洹王跃春
Owner JINAN UNIVERSITY
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