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Method for enhancing proliferation and post-transplantation survival ability of adipose derived stem cells

An adipose stem cell and capacity technology, which is applied in the direction of cell culture active agents, animal cells, vertebrate cells, etc., can solve the problems of high fusion rate and cell function maintenance, large number of stem cells, etc., and achieve good seed cell source, protein The effect of increased expression and accelerated proliferation

Inactive Publication Date: 2017-02-22
山东海斯福生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of stem cells required in clinical applications is huge, and the requirements for the fusion rate and cell function maintenance after stem cell transplantation are quite high

Method used

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  • Method for enhancing proliferation and post-transplantation survival ability of adipose derived stem cells
  • Method for enhancing proliferation and post-transplantation survival ability of adipose derived stem cells
  • Method for enhancing proliferation and post-transplantation survival ability of adipose derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Extraction of adipose stem cells

[0024] 1. Solid Fats

[0025] (1) The human adipose tissue was repeatedly washed with double-antibody physiological saline for 2-3 times, and then with sterile physiological saline for 3-4 times.

[0026] (2) Put the rinsed fat in a sterile dish, observe whether there is spoilage, whether the color of the fat is abnormal, etc., and record it in time.

[0027] (3) Use sterile scissors to cut the fat into small particles of 2×2×2 mm, wash the cut fat small pieces with normal saline 3 times, filter with a 100-micron filter during washing, and wash and discard the red blood cell components as much as possible;

[0028] (4) Take the cut tissue block and add it to 0.3% collagenase type I / PBS digestion solution at a volume ratio of 1:5 (1:3), for example, add 20 mL of fat to 100 mL of digestion solution;

[0029] (5) place this tube in a 37°C constant temperature air bath rotary shaking box, and shake at a constant temperature of...

Embodiment 2

[0039] Example 2: TGF-β1 promotes the proliferation of adipose stem cells

[0040] Passage 3 passages of adipose stem cells at 6,000 cells / cm 2 The density of TGF-β1 was inoculated into 24-well plates, and after 24 h, different concentrations of TGF-β1 were added to make the final concentrations of 0.1, 0.5, 1, 5, 10 and 20 ng / mL, respectively. There were 3 replicate wells in each group, and were cultured for 24 h and 48 h respectively. The cells were collected after 72 hours, and the CCK-8 cytotoxicity detection kit was used to detect each experimental group. The results showed that 0.1ng / ml was the optimum concentration to promote the proliferation of adipose stem cells.

Embodiment 3

[0041] Example 3: TGF-β1 does not affect the immunophenotype of adipose stem cells

[0042] The control and TGF-β1-treated adipose stem cells were collected, labeled with antibodies against human CD45, CD31, CD34, CD29, CD44, CD73, CD90 and CD105, respectively, and analyzed by flow cytometry. Like other sources of mesenchymal stem cells, they express CD29, CD44, CD73, CD90 and CD105; they do not express CD31, CD34 and CD45. And after adding TGF-β1, the expression of each molecule did not change significantly.

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PUM

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Abstract

The invention discloses a method for enhancing proliferation and post-transplantation survival ability of adipose derived stem cells. TGF-[beta]1 at a special concentration is added to culture of the adipose derived stem cells, and the TGF-[beta]1, after binding with a TGF-[beta]II type receptor (TGF-[beta]RII), activates and recruits TGF-[beta]I receptor (TGF-[beta]RI) composition so as to form a receptor complex in the form of a dimer. The TGF-[beta]RII can phosphorylate a glycine-serine enriched region (GS sequence) of the TGF-[beta]RI and activate serine / threonine activity of the TGF-[beta]RI. The activated TGF-[beta]RI can phosphorylate receptor related smad2 and smad3 proteins, and then the smad2 / smad3 form a complex and further bind with smad4; the formed complex is transferred into cell nucleus; the smad3 and the smad4 are bound to a DNA sequence called SBE, while the smad2 reacts with the DNA complex through an interaction with the smad4, so that the expression of an ECM synthetic gene is activated. The proliferation of the adipose derived stem cells, which are processed by virtue of the method provided by the invention, is accelerated and the post-transplantation survival ability is enhanced.

Description

technical field [0001] The invention relates to the cultivation of adipose stem cells in the technical field of regenerative medicine and cell therapy, and the content of the invention is a method for enhancing the proliferation and survival ability of adipose stem cells after transplantation Background technique [0002] Adipose derived stem cells (ADSCs) are pluripotent stem cells developed from the mesoderm, a kind of adipose tissue-derived mesenchymal stem cells (MSCs), which can be induced to differentiate into a variety of cells. Including the same mesoderm derived osteoblasts, adipocytes, chondrocytes. It can also differentiate into endoderm-derived hepatocytes, pancreatic islet β-like cells, ectoderm-derived nerve cells, cardiomyocytes, epidermal cells, etc. Compared with other mesenchymal stem cells, adipose stem cells are rich in sources, easy to obtain and easy to culture, and are excellent seed cells for tissue engineering and cell therapy (Park Andrew, et al. 2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2501/11C12N2501/113C12N2501/115C12N2501/117C12N2501/119C12N2501/135C12N2501/15C12N2501/165C12N2501/998
Inventor 邓长江李栋印海峰李安娜吴燕
Owner 山东海斯福生物科技有限公司
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