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Method for inducing the differentiation of mesenchymal stem cells of human embryo livers into islet beta-like cells and stably expressing insulin and special induced liquid thereof

A technology of mesenchymal stem cells and cell differentiation, applied in the field of induced stem cell differentiation, can solve safety problems and other problems, and achieve the effect of avoiding biological safety problems

Inactive Publication Date: 2009-11-18
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are safety problems in the clinical application of virus-mediated mediation, so there is an urgent need for a safe and efficient mediation method

Method used

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  • Method for inducing the differentiation of mesenchymal stem cells of human embryo livers into islet beta-like cells and stably expressing insulin and special induced liquid thereof
  • Method for inducing the differentiation of mesenchymal stem cells of human embryo livers into islet beta-like cells and stably expressing insulin and special induced liquid thereof
  • Method for inducing the differentiation of mesenchymal stem cells of human embryo livers into islet beta-like cells and stably expressing insulin and special induced liquid thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Inducing human fetal liver mesenchymal stem cells to differentiate into islet β-like cells

[0029] (1) Isolation, purification and expansion of human fetal liver mesenchymal stem cells: rinse the fetus with sterile normal saline, cut off the umbilical cord and place it in a large beaker, soak it in 75% alcohol for 5 minutes, and place it in an ultra-clean Taichung Take out the fetal liver aseptically, wash the blood several times with PBS, cut off the capsule, cut the remaining liver tissue into small pieces, absorb the Hank solution containing 2% (volume fraction) FCS with a sharp-nosed pipette, wash the liver tissue, and collect the liver tissue suspension into 50ml Or in a 15ml sterile centrifuge tube, blow the 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 parenchymal cells), and centrifuge at 500×...

Embodiment 2 Embodiment 1

[0031] Example 2 Identification of the islet-like cell mass obtained in Example 1

[0032] (1) Morphological changes of cells before and after induction: figure 1 Is the morphology of uninduced hFL-MSCs seen under an inverted microscope. figure 2 It is the morphological diagram of islet β-like cell clusters seen under an inverted microscope after 10 days of induction. From figure 1 It can be seen that the uninduced MSCs are long fusiform and arranged in parallel; from figure 2 It can be seen that hFLMSCs induced for 10 days aggregated into islet-like cell clusters.

[0033] (2) Dithizone (DTZ) staining:

[0034] Take the islet-like cell mass obtained in Example 1, wash it twice with PBS, add 5ml PBS and 50μl dithizone working solution (V / V, 1%), incubate at 37°C for 10 minutes, observe and take pictures under an inverted microscope.

[0035] The cytoplasm of pancreatic β-cells is rich in zinc ions, which are essential components for the formation of 2-zinc-insulin hexam...

Embodiment 3

[0044] Example 3 Transplantation experiment of pancreatic islet β-like cells obtained by this method under the capsule of mouse kidney

[0045] Physiological saline, uninduced hFLMSCs and islet β-like cells induced for 10 days were transplanted under the renal capsule of diabetic mice. Observe the effect of different cells on the blood sugar level of diabetic mice after transplantation. Figure 8 It suggested that the blood glucose of diabetic mice decreased significantly after transplantation of islet β-like cells, and it could be maintained for a long time, while normal saline and uninduced hFLMSCs had no hypoglycemic effect on diabetic mice after transplantation.

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Abstract

The invention relates to a method for inducing the differentiation of mesenchymal stem cells of human embryo livers into islet beta-like cells and stably expressing insulin and special induced liquid thereof. The method comprises the following steps: separating, purifying and amplifying mesenchymal stem cells of human embryo livers; then taking out cells formed after the third generation and inducing the cells by the special induced liquid to obtain islet beta-like cell mass capable of expressing the insulin stably. The induced liquid can be obtained by adding TAT-PDX1 fusion protein in cell culture fluid. The method can realize in-vitro induction of mesenchymal stem cells of human embryo livers into islet beta-like cells capable of secreting the insulin stably. When transplanted in the body of a diabetic patient, the obtained islet beta-like cells can further adjust blood sugar level so as to realize cell therapy of diabetes. Therefore, research in the field has broad clinic application prospect and can bring greater economic benefit and social benefit.

Description

technical field [0001] The invention relates to the technical field of inducing stem cell differentiation, in particular to a method for inducing human fetal liver mesenchymal stem cells to differentiate into islet β-like cells and stably expressing insulin and a special inducing solution thereof. Background technique [0002] Diabetes has become the main killer threatening human life and health in the 21st century. At present, taking drugs and injecting insulin is a commonly used method in various treatment measures for the etiology and pathology of diabetes, but taking drugs and injecting insulin will not only bring a heavy economic burden, but also cause serious complications; in recent years, transplantation Pancreatic islets are initially effective in treating diabetes, but they face two major problems: insufficient tissue sources and immune rejection. Gene / cell therapy has attracted more and more attention. Pancreatic-duodenal homeobox 1 gene (PDX1) is a major regula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12P21/02
Inventor 张洹姜铧李东升
Owner JINAN UNIVERSITY
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