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Method for inducing stem cells to differentiate to blood vessel smooth muscle cells

A technology for vascular smooth muscle and stem cell differentiation, which is applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., and can solve the problems of easy aging and poor in vitro proliferation ability.

Active Publication Date: 2007-11-14
SHANGHAI TISSUE ENG LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these cells are terminally differentiated cells, prone to aging, and have poor proliferation ability in vitro

Method used

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  • Method for inducing stem cells to differentiate to blood vessel smooth muscle cells
  • Method for inducing stem cells to differentiate to blood vessel smooth muscle cells
  • Method for inducing stem cells to differentiate to blood vessel smooth muscle cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Method for inducing and differentiating bone marrow mesenchymal stem cells into vascular smooth muscle cells

[0052] (1) Extract 5ml of bone marrow from the anterior superior iliac spine of healthy adults, transfer it into a 5ml sterile centrifuge tube containing the same amount of DMEM-LG culture medium (containing 10% FBS, heparin 100U / ml), centrifuge at 300g for 5 minutes, and suck off the upper layer Fat and part of the supernatant;

[0053] (2) Add on 1.073g / ml Percoll separation solution (purchased from Pharmacia, Sweden) at 1:2, and centrifuge at 900g for 30 minutes;

[0054] (3) Take the cloudy mononuclear cell layer in the middle layer, rinse with PBS, centrifuge at 300g for 5min, and discard the supernatant;

[0055] (4) Cells were collected, resuspended in low-glucose essential medium (DMEM-LG, purchased from Gibco, USA), and mixed with 2×10 5 / cm 2 Density inoculation culture, first change the medium after 48 hours of culture, wash once with PBS, remove ...

Embodiment 2

[0058] Detect the cells obtained in Example 1

[0059] 1. Immunofluorescent staining of cells:

[0060]Cells were subcultured for 14 days, fixed with ethanol:acetic acid (99:1) for 20 minutes, rinsed with PBS for 5 minutes x 3 times; added 0.25% Triton X-100 for 10 minutes, rinsed with PBS for 5 minutes x 3 times; 0.5 % goat serum was blocked at room temperature for 30 minutes; monoclonal mouse anti-human smooth muscle actin (MonoclonalMouse Anti-Human Smooth Muscle Actin Clone 1A4, 1:100, DakoCytomation Company), goat polyclonal anti-SM 22 α (Goat polyclonal to SM22 alpha , 1:100, American Abcam Company), mouse anti-smooth muscle myosin heavy chain monoclonal antibody (Mouse anti-smooth musclemyosin, heavy chain monoclonal anti ibody, 1:100, Canada CHEMICON Company), mouse monoclonal anti-Calponin antibody ( Mouse monoclonal to Calponin ab700, Abcam, UK) was incubated overnight at 4°C, rinsed with PBS, fluorescein isothiocyanate (FITC) mouse anti-rat IgG (1:100, Invitrogen, ...

Embodiment 3

[0081] Prepare vascular graft with the vascular smooth muscle cells obtained in Example 1

[0082] 1. Inoculation of vascular smooth muscle cells: within the second to third passage, the total number is 10×10 7 Add 2ml of DMEM culture medium to make cell suspension, inoculate the above cell suspension evenly on a piece of prefabricated PGA fiber, place at 37℃, 5% CO 2 In the incubator, add DMEM culture solution after 4 hours, place it in the incubator to continue culturing, and replace the culture solution every day.

[0083] 2. Construction of cell-PGA tubular complexes in vascular bioreactors.

[0084] (1) After 2 weeks of in vitro culture, the sheet-shaped cell-PGA complex was wrapped around a pre-sterilized silicone tube with an outer diameter of 6 mm, so that the cell-PGA complex formed a tubular structure.

[0085] (2) Set reactor parameters: flow rate: 200ml / min, after acting for 160ms, relax for 200ms, so that the pulsation reaches 167 times / min.

[0086] (3) In vit...

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Abstract

A body method of serving as quality stem cell between outside guide marrow to become the smooth muscle cell of blood vessel of the growth factor that joins the source of blood platelet, Is concerned with the method that gets seed cell in organization engineering science. Form studies change and cell immunity fluorescence etc. to prove guide rear cell to have the table type of the smooth muscle cell of blood vessel.

Description

technical field [0001] The invention relates to a method for obtaining seed cells in tissue engineering. Background technique [0002] At present, the research on the formation of vascular smooth muscle in vivo is still relatively shallow, not to mention the induction of differentiation to form vascular smooth muscle cells in vitro. At present, the vascular smooth muscle cells used in the construction of tissue engineering blood vessels come from the mature smooth muscle cells of the umbilical artery or the internal carotid artery of large animals. . However, these cells are terminally differentiated cells, which are prone to aging and have poor proliferation ability in vitro. [0003] Therefore, there is an urgent need in this field for a method of obtaining vascular smooth muscle cell seed cells in vitro, so as to find a new source of vascular smooth muscle cell seed cells that is convenient to obtain, less traumatic, and can be expanded on a large scale. Contents of th...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N5/08C12N5/077
Inventor 崔磊尹烁李纲曹谊林刘伟
Owner SHANGHAI TISSUE ENG LIFE SCI
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