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Bletilla striata polysaccharide hydrogel, culture medium and application thereof as well as method of inducing differentiation of umbilical cord mesenchymal stem cells to corneal epithelial cells

A technology of bletilla striata polysaccharides and mesenchymal stem cells, applied in the field of bletilla striata polysaccharide hydrogel to induce umbilical cord mesenchymal stem cells to differentiate into corneal epithelial cells, can solve the problem of cell affinity, poor adhesion of hydrophilic cells, lack of cell recognition signal sites, etc. problem, to achieve the effect of vigorous cell growth, simple operation, and good porosity

Active Publication Date: 2015-11-25
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many shortcomings have been found in practical applications: synthetic materials usually have defects such as poor cell affinity, hydrophilicity and cell adhesion, and lack of cell recognition signal sites.

Method used

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  • Bletilla striata polysaccharide hydrogel, culture medium and application thereof as well as method of inducing differentiation of umbilical cord mesenchymal stem cells to corneal epithelial cells
  • Bletilla striata polysaccharide hydrogel, culture medium and application thereof as well as method of inducing differentiation of umbilical cord mesenchymal stem cells to corneal epithelial cells
  • Bletilla striata polysaccharide hydrogel, culture medium and application thereof as well as method of inducing differentiation of umbilical cord mesenchymal stem cells to corneal epithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] The preparation of embodiment 1 bletilla striata polysaccharide

[0080] Bletilla striata decoction pieces were ground into powder, dispersed and dissolved in distilled water at 80°C for 4 hours, and then filtered to remove impurities.

[0081] The filtrate was soaked overnight in 3 volumes of 95% ethanol, and the precipitate was resuspended with distilled water.

[0082] Add 1 / 3 volume of a mixture of chloroform and butanol (volume ratio 5:1.) Mix evenly and centrifuge to extract protein components, and extract the liquid in the aqueous phase layer.

[0083] Dialyzed through a 30000D-50000D dialysis column (the dialysate is distilled water), made into freeze-dried powder, and obtained the crude product of Bletilla striata polysaccharide. The crude Bletilla striata polysaccharide was dissolved and further purified by cross-linked dextran G-100 to obtain the Bletilla striata polysaccharide.

Embodiment 2

[0084] The sulfation of embodiment 2 bletilla striata polysaccharide

[0085] The Bletilla striata polysaccharide prepared in Example 1 was modified by sulfation through the chlorosulfonic acid-pyridine method. The specific steps are:

[0086] Dissolve 0.2g of polysaccharide powder in 35ml of anhydrous N,N-dimethylformamide, stir at room temperature for 30min, add 17.5ml of sulfation reagent (volume ratio of chlorosulfonic acid to pyridine is 1:6), place in 60 ℃ water bath stirring reaction for 3h. Cool to room temperature after the reaction, add 100ml of ice water to the reaction solution, neutralize it with 1mol / L NaOH to pH 7.5, concentrate under reduced pressure to a certain concentration, dialyze with distilled water for 3 days, concentrate the dialysate and freeze-dry to obtain sulfated polysaccharide.

Embodiment 3

[0087] The preparation of embodiment 3 bletilla striata polysaccharide gel

[0088] The sulfated Bletilla striata polysaccharide prepared in Example 2 was dissolved in PBS to prepare a polysaccharide solution with a concentration of 200 μg / ml. Dissolve ε-polylysine in PBS with a mass fraction of 3% to prepare a cross-linking agent. Mix the polysaccharide solution and cross-linking agent at a volume ratio of 1:1, and let it stand at room temperature for 30 seconds to form a translucent film-like Bletilla striata polysaccharide hydrogel.

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Abstract

The invention relates to the field of cell culture, in particular relates to letilla striata polysaccharide hydrogel, a culture medium and application thereof as well as a method of inducing differentiation of umbilical cord mesenchymal stem cells (hUC-MSCs) to corneal epithelial cells. The method comprises the steps of sulfating letilla striata polysaccharide and then crosslinking with Epsilon-polylysine carrying polyfunctional groups to prepare the polysaccharide hydrogel. The letilla striata polysaccharide hydrogel is of a semi-transparent membrane, has good porosities, and is suitable for the growth and the differentiation of cells. The experiment shows that by taking the letilla striata polysaccharide hydrogel provided by the invention as a scaffold, after the hUC-MSCs and amniotic epithelial cells are subjected to co-culture for 7 days, the differentiation rate of the hUC-MSCs can be up to 31.96%. The differentiation rate (p is less than 0.01) is obviously superior to that of a contrasting example which does not adopt the hydrogel scaffold. In addition, the hydrogel provided by the invention can promote the cells to grow vigorously, and shorten the induction time.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a bletilla striata polysaccharide hydrogel, a culture substrate and its application, and a method for inducing differentiation of umbilical cord mesenchymal stem cells into corneal epithelial cells. Background technique [0002] Sound corneal epithelial cells are an important condition for the cornea to resist damage from various external harmful factors, and limbal stem cells with sound structure and function are the main source of corneal epithelial cell regeneration. A variety of pathogenic factors, such as: ocular trauma, surgical trauma, inflammation, and drug toxicity can cause corneal limbal damage, resulting in limbal stem cell dysfunction and loss of epithelial cells, resulting in increased risk of corneal infection, perforation, and neovascularization . Therefore, the problem of obtaining a large number of epithelial cells for corneal repair through in vitro culture needs t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08J3/075C08J3/24C08L5/00C08L77/02C08B37/00C12N5/071
Inventor 王一飞陈海佳葛啸虎戚康艺
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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