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Use of carageen polysaccharide in inhibiting mesenchymal stem cell adipogenic transformation

A technology of mesenchymal stem cells, carrageen, applied in the field of stem cells

Active Publication Date: 2020-07-28
湖南南华爱世普林生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the function of carrageenan in stem cells is unknown

Method used

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  • Use of carageen polysaccharide in inhibiting mesenchymal stem cell adipogenic transformation
  • Use of carageen polysaccharide in inhibiting mesenchymal stem cell adipogenic transformation
  • Use of carageen polysaccharide in inhibiting mesenchymal stem cell adipogenic transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Wash the dried carrageen, dry in a blast dryer at 60°C, and soak in 95% ethanol for 12 hours;

[0036] (2) Dried in a blast dryer at 60°C, crushed, and passed through a 50-mesh sieve;

[0037] (3) Transfer 1000g of dried carrageen to ultra-high pressure biological extraction equipment, add 15 times the amount of water, and extract for 3 minutes under 200MPa pressure;

[0038] (4) Transfer to a centrifuge, centrifuge at 3500r / min for 15 minutes, remove the precipitate, and keep the supernatant to obtain the crude polysaccharide extract;

[0039] (5) Concentrate the crude polysaccharide extract under reduced pressure, add it to absolute ethanol, and ethanol precipitation for 20 hours;

[0040] (6) Transfer to a centrifuge, centrifuge at 3500r / min for 15 minutes, remove the supernatant and keep the precipitate;

[0041] (7) The precipitate was washed 3 times with absolute ethanol, and freeze-dried to obtain carrageenan.

Embodiment 2

[0043] 1. Induction of Adipogenic Transformation

[0044] (1) Divide 5×10 4 Bone marrow mesenchymal stem cells were inoculated in the culture plate, and cultured by adding complete medium;

[0045] (2) When the cell confluency reaches 70%, replace the medium with adipogenic induction medium + carrageenan (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL), and induce adipogenicity for 14 days , change the medium every 3 days, and set 3 repetitions in each group;

[0046] 2. RNA extraction

[0047] (1) After 14 days of culture, remove the medium, add 500 μL Trizol to each well, and repeatedly pipette the cells until a clear and non-viscous liquid is formed;

[0048] (2) Transfer the mixture to a 1.5ml EP tube, add 100μL chloroform, shake vigorously on the shaker for 15 seconds, and let stand at room temperature for 5 minutes;

[0049] (3) Centrifuge in a low-temperature high-speed centrifuge at 12,000 r / min for 15 minutes at 4°C, and carefully transfer the upper transparent RNA aqueo...

Embodiment 3

[0069] Western blot detection

[0070] 1. Protein extraction

[0071] (1) The process of adipogenic induction is the same as in Example 2. After 14 days of adipogenic induction, add 100 μL of RIPA lysate to the culture plate, scrape off the cells with a cell scraper, and collect the cell lysate into an EP tube;

[0072] (2) Break the cells with a cell ultrasonic breaker, centrifuge at 4°C, 12000r / min, for 15min;

[0073] (3) Transfer the supernatant to a new EP tube, and use the BCA method to measure the protein concentration;

[0074] (4) Use 5× loading buffer to adjust the protein concentration to 2 μg / μL, and cook in boiling water for 5 minutes to obtain protein samples.

[0075] 2.Western blot experiment

[0076] (1) Configure 12% separating gel and 5% stacking gel, and add 10 μL protein sample to each well;

[0077] (2) Electrophoresis conditions: the stacking gel is at a constant voltage of 90V, and the time is about 20 minutes; the separation gel is at 130V, and the...

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Abstract

The invention provides a use of carageen polysaccharide in inhibiting the mesenchymal stem cell adipogenic transformation, and belongs to the technical field of stem cells. The carageen polysaccharideis used to treat bone marrow mesenchymal stem cells, the result finds that the carageen polysaccharide can inhibit the expression of mRNA and protein of adipogenic transformation-related genes PPAR-gamma and C / EBP-alpha, and thereby inhibits a number of fat droplets produced in a bone marrow mesenchymal stem cell adipogenic transformation process, and indicates that the carageen polysaccharide can play a role in inhibiting the bone marrow mesenchymal stem cell adipogenic transformation.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to an application of carrageenan in inhibiting adipogenic transformation of mesenchymal stem cells. Background technique [0002] Polysaccharides, also known as polymers, are a class of biological macromolecules that are ubiquitous in biological organisms. Polysaccharides are not only involved in the formation of the cytoskeleton, but also an important component of a variety of endogenous bioactive molecules. Polysaccharides participate in life processes in free form, and also form conjugates with proteins, peptides, and lipids, which play an important role in regulating the balance and stability of physiological functions. [0003] Carrageenan is a low-fat, high-protein macroalgae. Carrageenan is not only one of the important algae for the production of carrageenan, but also contains a large amount of active substances in the body of carrageenan. The main types are ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C08B37/00A61K31/731A61P19/10
CPCC12N5/0663C08B37/0003C12N2501/90
Inventor 刘金凤
Owner 湖南南华爱世普林生物技术有限公司
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