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Preparation method, product and application of alpha cell

A technology of alpha cells and somatic cells, applied in the field of biomedicine

Inactive Publication Date: 2017-05-31
刘天津
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, partially differentiated cells from ES and iPS may pose an unacceptable risk of tumor formation when used clinically

Method used

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  • Preparation method, product and application of alpha cell
  • Preparation method, product and application of alpha cell
  • Preparation method, product and application of alpha cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Inducing fibroblast differentiation to prepare α cells

[0062] 1. Molecular cloning and lentivirus construction

[0063] The multiple cloning site (shown as SEQ ID NO: 21 in the sequence listing) was inserted into the PmeI restriction site of the lentiviral vector pWPI (purchased from Addgene, Cambridge, MA, USA). The cDNA of the promoter (candidate gene) was cloned into the modified pWPI plasmid. Then, the plasmid was introduced into 293FT cells together with the packaging plasmid psPAX2 (purchased from Addgene) and the envelope plasmid pMD2.G (purchased from Addgene). For specific methods, please refer to the relevant plasmid product instructions. After 48 hours, the medium containing the lentivirus was collected and filtered through a 0.45-mm filter. The gene information of the promoter (candidate gene) is: the gene bank accession number (Gene Bank ID) of Hhex is NM_008245; the Gene Bank ID of Foxa3 is NM_008260; the Gene Bank ID of Gata4 is NM_008092; t...

Embodiment 2

[0072] Functional analysis and results of the obtained alpha cells prepared in embodiment 2

[0073] 1. Animal model

[0074] p19Arf - / - Mice (provided by the Research Institute of Molecular Pathology in Austria, reference: Cell.1997Nov 28; 91 (5): 649-59) and nude mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.) were maintained in a specific pathogen-free environment. under feeding conditions. Nude mice were used as recipients of iAlpha cells to reduce immune rejection after transplantation.

[0075] 2. Immunofluorescence staining analysis and results

[0076] The immunofluorescence staining method included the following steps: cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, and then incubated with PBS containing 0.2% Triton X-100 (purchased from Sigma) for 15 minutes. Cells were then washed three times with PBS. After blocking with 3% bovine serum albumin (BSA) in PBS for 60 minutes at room temperature, cells were incubate...

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Abstract

The invention provides a preparation method, a product and application of an alpha cell. The preparation method of the alpha cell comprises the following steps of over-expressing the following transcription factors in a somatic cell: Hhex, Foxa3, Gata4, Pdx1 and Pax4. The invention also provides the alpha cell prepared and obtained by the method and the application of the alpha cell. The invention discloses a brand-new method for obtaining the alpha cell; the alpha cell has important effects on the research and the treatment of diabetes mellitus and the application of relevant regenerative medicine; the invention also optimizes a method for preparing the alpha call; the better combination of the transcription factors is found out. The alpha cell prepared and obtained by utilizing the method provided by the invention not only has the function of a natural islet alpha cell, but also is a fungible clinical resource for treating the diabetes mellitus, which is wide in source range and is high-efficiency, stable and safe.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of alpha cells and its product and application. Background technique [0002] Alpha cells are a type of cell in the pancreatic islets that produce glucagon, which has the effect of raising blood sugar. Energy homeostasis in pancreatic islets is maintained by insulin-secreting β-cells and glucagon-secreting α-cells, which regulate blood glucose homeostasis in response to a variety of trophic, neural, and hormonal factors. After an overnight fast, plasma glucagon levels rise markedly once glucose falls below a threshold of 7 and gradually decrease until plasma glucose rises above the normal range. [0003] Type I diabetes, formerly known as insulin-dependent diabetes, mostly occurs in children and adolescents, and can also occur in various ages. The onset is relatively sharp, and there is absolutely insufficient insulin in the body, and ketoac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/39A61P3/10
CPCC12N5/0602A61K35/39C07K14/47C12N5/0656C12N15/86C12N2740/15043
Inventor 刘天津
Owner 刘天津
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