Adipsin gene modified adipose-derived stem cells as well as preparation method and application thereof

An adipose stem cell, gene modification technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, and botanical equipment and methods, etc. It can improve the vitality and increase the secretion of insulin.

Active Publication Date: 2020-06-12
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] ADSCs are co-cultured with pancreatic β cells. During the culture process, the cells tend to form clusters and cell growth is inhibited, resulting in a decrease in insulin secretion from pancreatic β cells and the inability to maintain blood sugar balance for a long time.
At the same time, adipose-derived stem cells are not effective in promoting the proliferation of islet cells and the secretion of insulin by islet cells.

Method used

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  • Adipsin gene modified adipose-derived stem cells as well as preparation method and application thereof
  • Adipsin gene modified adipose-derived stem cells as well as preparation method and application thereof
  • Adipsin gene modified adipose-derived stem cells as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Containing the construction of adipsin gene expression vector

[0048] The fusion gene for modifying fat stem cells, including signal nucleic acid artificial sequence (SEQ ID NO.1) and adipsin nucleic acid artificial sequence (SEQ ID NO.2), respectively commissioned according to the nucleic acid coding sequences of SEQ ID NO.1 and SEQ ID NO.2 Sangon Bioengineering (Shanghai) Co., Ltd. synthesized the entire expression cassette and inserted it into the BamHI-EcoRI site of the adenovirus expression vector pAAV-IRES-ZsGreen1 (SEQ ID NO.3) (see attached figure 1 ), transformed into E.coli (DH5α), after the correct sequencing, the plasmid was extracted and purified using Invitrogen’s plasmid purification kit to obtain a high-quality plasmid of the recombinant expression vector.

[0049] In more detail, the preparation method provided in this embodiment includes the following steps:

[0050] The nucleic acid artificial sequence of the fusion gene adipsin was e...

Embodiment 2

[0054] Example 2: Preparation of fat stem cells

[0055] Adipose tissue was extracted from the abdomen of volunteers, washed with PBS solution under aseptic conditions to remove red blood cells, cut into fine particles with a scalpel, added trypsin and collagenase (v / v 1:1), and collected liquid after shaking and digesting, 1500rpm / 10min, resuspend the sediment in the medium, filter through a 200 mesh fine sieve, 1500rpm / 10min, resuspend the obtained cell extract with 10% FBS+DMEM culture medium, and place at 37°C, 5% CO 2 Cultured in the incubator, after 5 days, the medium was changed for the first time. Afterwards, the medium was changed every 3 days. When the confluence of the primary cells was 80%, they were digested and passed on to the P3 generation for subsequent experiments. Cell growth and morphology were observed under an optical microscope (see figure 2 ). The flow cytometer surface markers were detected on the P3 generation human adipose stem cells, and the cel...

Embodiment 3

[0056] Example 3: Adenovirus packaging and titer detection

[0057] (1) Recovery of 293T cells

[0058] 1) Take out the frozen 293T cells from the liquid nitrogen tank, quickly throw them into a 37°C water bath and shake them quickly until the cell solution is completely dissolved.

[0059] 2) Transfer the cell solution to a 50mL centrifuge tube, add 10 mL fresh complete medium to it, mix well and centrifuge at 1500 rpm for 5 min.

[0060] 3) Remove the supernatant, add 3 mL of fresh DMEM medium to resuspend the cells, transfer them evenly to a six-well plate, and make up 3 mL of medium in each well.

[0061] 4) Put the six-well plate in 37 ℃, 5%CO steadily 2 and cultured in an incubator with 95% relative humidity.

[0062] 5) Observe the cell survival rate the next day and replace with fresh medium. Afterwards, the growth of the cells was observed every day. When the cells covered the bottom of the well, they were subcultured, and were used for experiments when they cove...

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Abstract

The invention provides adipsin gene modified adipose-derived stem cells, the adipsin gene modified adipose-derived stem cells comprise an adipsin recombinant gene, and the adipsin recombinant gene expresses an adipsin protein; the invention further provides a preparation method and application of the adipose-derived stem cells. The adipsin gene modified adipose-derived stem cells are adopted, theactivity of pancreatic islet cells can be improved, and after the adipsin gene modified adipose-derived stem cells and the pancreatic islet cells are co-cultured, the activity of the pancreatic isletcells is improved by one time compared with common co-culture of the adipose-derived stem cells and the pancreatic islet cells. The adipsin gene modified adipose-derived stem cells are adopted, various growth factors are secreted by utilizing the paracrine effect and the regeneration and repair effect of the adipose-derived stem cells, so that the multiplication capacity of pancreatic beta cells is doubled, and meanwhile, the secreted adipsin proteins can increase insulin secretion of the pancreatic beta cells.

Description

technical field [0001] The invention relates to adipose stem cells modified with adipsin gene, a preparation method and application thereof, belonging to the technical field of biomedicine. Background technique [0002] With the improvement of people's living standards and changes in diet structure, the incidence of diabetes in my country is increasing rapidly. The latest research data shows that about 114 million adults in my country suffer from diabetes, and the prevalence rate is as high as 11.6%, ranking first in the world. The conventional treatment of diabetes is oral or injection antidiabetic drugs, which can temporarily improve hyperglycemia and insulin sensitivity, but cannot reverse insulin resistance (decreased insulin-regulated glucose metabolism), nor can it repair the failure of pancreatic β cells, and Long-term use of insulin can easily lead to drug resistance. Allogeneic pancreas transplantation is an alternative treatment for diabetes, but its application i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/861C12N15/62C12N5/071
CPCC12N5/0667C12N15/86C12N9/1077C12Y204/02012C12N5/0676C12N2510/00C12N2710/10343C12N2800/107C07K2319/02C12N2502/99C12N2501/724
Inventor 刘明录冯建海金海锋王立新卢永灿强邦明张传鹏王亮许淼
Owner SHANDONG XINRUI BIOTECH CO LTD
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