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Method for preparing hepatic stem cell decorated by insulin gene and application and preparation thereof

A technology of liver stem cells and gene modification, which is applied in the field of preparation of diabetes preparations, preparations, and preparations of liver stem cells, and can solve problems such as the treatment of liver stem cells that have not been taught

Inactive Publication Date: 2011-08-17
沈达青
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prior art only discloses how to obtain relevant hepatic stem cells but does not teach how to process the obtained hepatic stem cells

Method used

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  • Method for preparing hepatic stem cell decorated by insulin gene and application and preparation thereof
  • Method for preparing hepatic stem cell decorated by insulin gene and application and preparation thereof
  • Method for preparing hepatic stem cell decorated by insulin gene and application and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Umbilical cord tissue comes from umbilical cords collected from pregnant women after giving birth. An informed consent was signed with the patient to have her umbilical cord harvested after delivery. Cut it into about 1mm with ophthalmic scissors 3 of small pieces. The resulting chopped tissue pieces were digested with 0.25% trypsin solution at a weight-to-volume ratio of 1 g: 0.5 mL for 5 minutes, and then calf serum (U.S. Hyclone Company), which was one-fifth of the volume of the trypsin solution, was added to stop. Centrifuge at 1000rpm to collect the digested tissue fragments, pour off the supernatant of the trypsin solution, and then use 0.01% DNAse (DNase, Shanghai Sangong) in HBSS (Hans buffer solution, Shanghai Sangong) at a weight volume of 1 g: 1 mL After washing three times, the resulting centrifuged pellet was mixed with 0.25% trypsin solution at a volume ratio of 1 g: 1 mL for 10 minutes at 37°C, and then centrifuged at 1000 rpm for 5 min to collect the c...

Embodiment 2

[0062] The processing process of bone marrow or umbilical cord blood: After separating single cells by Ficoll-Hypaque density gradient centrifugation, count the cells, add complete medium, and cultivate into complete medium, containing epidermal growth factor 5ng / ml-50ng / ml , basic epidermal growth factor 5ng / ml-50ng / ml, and DMEM / F12 (1:1) containing 5%-15% fetal bovine serum. Plant in a 75cm2 culture bottle at a density of 1-5×105 cells / cm2, and add 10ml of culture solution to each bottle. Tighten the cap of the culture bottle, then turn it inward for 1 turn, put it into the patient-specific incubator, culture at 370C, 5% CO2, and change the medium in half on the third day of culture. When changing the medium, suck out the culture medium slowly, add fresh complete medium, add 5ml to each bottle, cultivate at 370C, 5% CO2. When the cell adherence reaches 80-90%, the cells are digested with trypsin or acutis enzyme, collected by centrifugation and cultivated. It is a complete ...

Embodiment 3

[0065] The constructed insulin gene cloning adenovirus (Introgen Corporation, USA) and the hepatic stem cells were taken at a ratio of 1:2, and the insulin gene cloning adenovirus was implanted into the hepatic stem cells by electroporation for 1 hour.

[0066] After electroporation, the insulin gene-modified hepatic stem cells were cultured in DMEM / F12 (1:1) complete medium (containing epidermal growth factor 5ng / ml-50ng / ml, basic epidermal growth factor 5ng / ml-50ng / ml , and cultured in 5%-15% of fetal bovine serum), so that the cell adherent growth can reach 80-90%. Insulin expression by insulin gene-modified hepatic stem cells was detected by insulin ELISA kit (R&D Company, USA).

[0067] Example 4: Application of Insulin Gene Modified Hepatic Stem Cells in Diabetes Model

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Abstract

The invention provides a method for preparing hepatic stem cell decorated by an insulin gene, application of the method to preparation of a diabetes preparation and a preparation comprising the hapatic stem cell decorated by the insulin gene. The invention also discloses the method for preparing the hepatic stem cell decorated by the insulin gene and the application of the method. By the preparation prepared from the hapatic stem cell decorated by the insulin gene, which is provided by the invention, pancreatic island beta cell damaged in the diabetes can be repaired in vivo to secrete insulin so as to provide the possibility of overcoming the defect that the diabetes depends on insulin therapy and radically treating the diabetes.

Description

technical field [0001] The invention relates to hepatic stem cells and a preparation method thereof, the application of the hepatic stem cells in the preparation of diabetes preparations, and the preparations comprising the hepatic stem cells. In particular, the present invention relates to the preparation method and application of insulin gene-modified hepatic stem cells, as well as the insulin gene-modified hepatic stem cells and the preparation method thereof, the application of the insulin gene-modified hepatic stem cells in the preparation of diabetes preparations, the preparation of the insulin gene Preparation of modified hepatic stem cell antigen-loaded hepatic stem cells. Background technique [0002] The current treatment methods for diabetes mainly include traditional methods such as insulin injection, oral hypoglycemic drugs, and diet control, etc., but often end up causing insulin dependence in patients, and there is no suitable treatment method at present. In ...

Claims

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Application Information

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IPC IPC(8): C12N5/10A61K48/00A61K35/44A61K35/28A61K35/14A61P3/10A61K35/407
Inventor 沈达青
Owner 沈达青
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