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Solution of chitosan, preparation method thereof and using method thereof

A chitosan solution, chitosan technology, applied in the field of its preparation, chitosan solution, promotion of gene transduction efficiency mediated by adenovirus vector

Active Publication Date: 2013-10-16
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] To sum up, these existing methods all have certain limitations, and they cannot be non-receptor-dependent, efficient, non-toxic and simple at the same time. and many other advantages

Method used

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  • Solution of chitosan, preparation method thereof and using method thereof
  • Solution of chitosan, preparation method thereof and using method thereof
  • Solution of chitosan, preparation method thereof and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1. chitosan sodium bicarbonate solution (chitosan / NaHCO 3 ) preparation

[0038] (1) Preparation method 1 of pH7.4 chitosan solution:

[0039] Get a 50ml test tube, add 100 μl of glacial acetic acid, then add 10ml of distilled water, shake to make an acetic acid solution, add 0.03g of chitosan (high, medium and low molecular weight chitosan can be used, chitosan is purchased from Sigma- Aldrich, the degree of deacetylation of high molecular weight chitosan is 75%, the viscosity in 1% acetic acid is 800-2000cP; the degree of deacetylation of medium molecular weight chitosan is 75%-85%, the viscosity in 1% acetic acid is 200-800cP ; The deacetylation degree of low molecular weight chitosan is 75-85%, and the viscosity in 1% acetic acid is 20-200cP.), the shock is fully dissolved, and the NaHCO of 1mol / L is added in the shock process 3 Solution 3.6ml, at this time, a large amount of foam can be seen, and the pH value of the solution rises sharply at the same t...

Embodiment 2

[0062] Embodiment 2. Chitosan sodium bicarbonate solution promotes recombinant adenovirus vector transduction efficiency

[0063] Human embryonic kidney cell 293, Chinese hamster ovary cell (CHO), mouse dendritic cell (DC2.4), mouse melanoma cell (B16), human embryonic rhabdomyosarcoma cell (RD), human laryngeal squamous cell carcinoma Cells (Hep II), human lung adenocarcinoma cells (A549) were purchased from ATCC.

[0064] 293 cells, B16 cells, RD cells, Hep II cells, and A549 cells were cultured in high-glucose DMEM medium, DC2.4 cells were cultured in RPMI 1640 medium, and CHO cells were cultured in DMEM / F12 medium (both added 2 mM L -Glutamine, 100U / ml penicillin and streptomycin 0.075% NaHCO 3 and 10% FBS), culture conditions are: 37 ° C, 5% CO2 environment, culture in 96-well plate. All the above media were purchased from Hyclone Company.

[0065] Dissolve 4 μl of adenoviral vector (Ad5-LacZ) containing β-galactosidase reporter gene (Ad5-LacZ) in 0.1M PBS (the titer o...

Embodiment 3

[0068] Embodiment 3. The chitosan sodium bicarbonate solution of different molecular weight promotes the transduction efficiency of recombinant adenovirus vector on CHO cells

[0069] Dissolve 4 μl of the adenoviral vector containing the galactosidase reporter gene (Ad5-LacZ) in 0.1M PBS (the titer of the adenovirus is 2x10 8 PFU / ml) solution was added to 149.1, 148.13, 146.25, 142.5, 135, 120, 90μl 0.1M PBS, vortexed for 5 seconds and then added 0.9, 1.87, 3.75, 7.5, 15, 30, 60μl 0.2% Chitosan / NaHCO with different molecular weight 3 (Example 1 (1)) solution (being divided into chitosan / NaHCO of high, medium and low three molecular weights 3 ), vortex for 5 seconds and let stand for 20 minutes, add 450μl DMEM / F12 containing 2% FBS, the final concentration of chitosan is 3.125μg / ml, 6.25μg / ml, 12.5μg / ml, 25μg / ml, 50μg / ml ml, 100 μg / ml, and 200 μg / ml, the mixture was vortexed for 5 seconds to mix. The medium in the original 96-well plate was discarded, and the mixture was add...

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Abstract

The invention relates to the fields of macromolecular substances, physical chemistry, molecular biology and virology, in particular to solution of chitosan, preparation method thereof and a method for using the same to improve the efficiency of gene transduction mediated by an adenoviral vector.

Description

technical field [0001] The invention relates to the fields of macromolecular substances, physical chemistry, molecular biology and virology. Specifically, the present invention relates to a chitosan solution, a preparation method thereof and a method for using it to promote gene transduction efficiency mediated by an adenovirus vector. Background technique [0002] Chitosan is the product of deacetylation of chitin, and the degree of deacetylation can be 65% to 100%. Chitosan is the only known cationic polysaccharide, insoluble in water and organic solvents, soluble in dilute acids with pH<6.5, including adipic acid, hydrochloric acid, acetic acid, formic acid, lactic acid, malic acid, propionic acid and succinic acid Acid etc. Chitosan has the characteristics of non-toxicity, good biocompatibility and biodegradability, so it is widely used in the fields of food, medicine and biology. [0003] Although chitosan has the advantages of non-toxicity, good biocompatibility ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08L5/08C08J3/02C12N15/861
Inventor 王健伟钟辉
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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