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Method for stably expressing secretable human insulin in mesenchymal stem cells

A technology of stem cells and human insulin, applied in the direction of insulin, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problems of poor repeatability, non-secretion, and difficulty in expressing human insulin.

Active Publication Date: 2021-08-03
WUHAN UNIV OF SCI & TECH
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  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is: aiming at the problems that human insulin (insulin) is difficult to express in mesenchymal stem cells, does not secrete, has poor reproducibility, and is unstable, the present invention provides a reproducible insulin that can be stably expressed in mesenchymal stem cells and the way insulin is secreted

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  • Method for stably expressing secretable human insulin in mesenchymal stem cells
  • Method for stably expressing secretable human insulin in mesenchymal stem cells
  • Method for stably expressing secretable human insulin in mesenchymal stem cells

Examples

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Embodiment Construction

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from commercial sources unless otherwise specified.

[0034] This method mainly includes three main stages of work:

[0035] 1. Construct the mCherry-2A-insulin-His fusion gene lentiviral expression vector containing 2A based on the self-cleaving polypeptide 2A technology.

[0036] 2. Transfection of mCherry-2A-insulin-his fusion gene lentiviral expression vector and screening of mesenchymal stable cell lines expressing the fusion gene.

[0037] 3. Identification of a stable cell line of mesenchymal stem cells expressing the fusion gene.

[0038] The steps involved in each stage of work are described below

[0039] 1. Construct the mCherry-2A-insulin-his fusion gene lentiviral expression vector containing 2A based on the self-cleaving polypeptide 2A technology.

[0040] 1) The sequence of self-cleavi...

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Abstract

The invention discloses a method for stably expressing secretable human insulin in mesenchymal stem cells, wherein the method comprises the following steps: (1) constructing a lentiviral expression vector of an mCherry-2A-inslin-His fusion gene, and (2) expressing the constructed lentiviral expression vector of the mCherry-2A-inslin-His fusion gene in the human mesenchymal stem cells, so as to obtain the human insulin secreted outside cells. After cells are transfected, a stable expression cell line is obtained through screening, and the human insulin is separated from a mCherry red fluorescent gene under the action of a self-cleavage polypeptide 2A when the cells express fusion polypeptide, so that the human insulin can be secreted outside the cells. The method provided by the invention solves the current difficulty of expressing human insulin in mesenchymal stem cells, and has great practical significance and potential medical application value.

Description

technical field [0001] The invention belongs to the field of tissue regeneration engineering, and in particular relates to a method for stably expressing and secreting human insulin in mesenchymal stem cells. Background technique [0002] Tissue regeneration engineering and stem cells and cell therapy are new disciplines developed in the field of contemporary biomedicine, bringing hope for the treatment of many major diseases including cancer, heart disease, diabetes, neurodegeneration and other diseases. Using embryonic stem cells or mesenchymal stem cells to differentiate into insulin-secreting islet cells or islet-like cells is an important research direction for tissue regeneration engineering and cell therapy, and it also brings hope for cell therapy of diabetes. Although the work on this aspect has been carried out for decades, the progress is relatively slow, because the islet cells obtained through the stem cell differentiation pathway are very unstable and the exper...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C12N5/10C12N15/86
CPCC07K14/62C12N15/86C12N5/0662C12N2510/02C12N2740/15043
Inventor 邓文生周翔宇张程
Owner WUHAN UNIV OF SCI & TECH
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