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A method for stably expressing secreted human insulin in mesenchymal stem cells

A technology of stem cells and human insulin, which can be applied to insulin, cells modified by introducing foreign genetic material, animal cells, etc., which can solve the problems of non-secretion, poor repeatability and instability.

Active Publication Date: 2022-07-01
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

Method used

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  • A method for stably expressing secreted human insulin in mesenchymal stem cells
  • A method for stably expressing secreted human insulin in mesenchymal stem cells
  • A method for stably expressing secreted human insulin in mesenchymal stem cells

Examples

Experimental program
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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 channels unless otherwise specified.

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

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

[0036] 2. The mCherry-2A-insulin-his fusion gene lentiviral expression vector was transfected and the mesenchymal stable cell line expressing the fusion gene was screened.

[0037] 3. Identification of mesenchymal stem cell stable cell lines expressing the fusion gene.

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

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

[0040] 1) Search the literat...

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Abstract

The invention discloses a method for stably expressing secretable human insulin in mesenchymal stem cells, comprising the following steps: (1) constructing an mCherry-2A-insulin-His fusion gene lentiviral expression vector; ‑2A‑insulin‑His fusion gene lentiviral expression vector is expressed in human mesenchymal stem cells to obtain extracellular human insulin. After transfection of cells, a stable expression cell line is obtained by screening. The cells express the fusion polypeptide and under the action of self-cleaving polypeptide 2A, human insulin and mCherry red fluorescent gene are separated, and human insulin can be secreted outside the cell. This inventive method solves the current dilemma 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 a new discipline developed in the contemporary biomedical field, 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 in tissue regeneration engineering and cell therapy, and it also brings hope for cell therapy of diabetes. Although the work in this area 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 experimenta...

Claims

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Application Information

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Patent Type & Authority Patents(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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