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Application of siRNA targeting FGF5 to treatment of alopecia

A targeted and hair loss technology, applied in the field of hair growth promoters, can solve the problem of low siRNA transfection efficiency

Active Publication Date: 2020-12-18
WENZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method of administration can cause excruciating pain, and the transfection efficiency of siRNA is not high

Method used

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  • Application of siRNA targeting FGF5 to treatment of alopecia
  • Application of siRNA targeting FGF5 to treatment of alopecia
  • Application of siRNA targeting FGF5 to treatment of alopecia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039]Example 1 Design, synthesis and cholesterol modification of siRNA

[0040]According to the mRNA sequence of mouse FGF5 in Gene Bank, online open software is used for siRNA design. As different design software uses different algorithms and their design principles are biased, as an example to improve the accuracy of siRNA design, we compared the characteristics of each software and finally selected 3 types with strong technical support and continuous updates. The software SiDirect, Oligowalk and SiRNA Design are used for siRNA design, and select siRNA with high repetition rate from the output sequence as candidate siRNA for further cell biology experiment screening to confirm its gene interference activity.

[0041]The synthesis of candidate siRNAs and the covalent modification of 5'-end cholesterol were entrusted to Shanghai Jima Pharmaceutical Technology Co., Ltd. to complete.

Embodiment 2

[0042]Example 2 Screening of siRNA and evaluation of target gene interference effect of different siRNA delivery methods

[0043]As the cell line expressing FGF5 is currently not available, this study constructed a eukaryotic expression plasmid of mouse FGF5, transfected NIH / 3T3 cells, and used NIH / 3T3 cells overexpressing mouse FGF5 to screen candidate siRNAs. The eukaryotic expression plasmid of mouse FGF5 can be prepared according to conventional methods in the art.

[0044](1) The NIH / 3T3 cells that have been transfected with the FGF5 expression plasmid are divided into 1×105The density of cells / well is inoculated into a 6-well plate and cultured in 2ml complete medium containing serum and double antibodies. When the cell density reaches 80%, replace it with 2ml serum-free cell culture medium;

[0045](2) Dilute 200pmol of siRNA in 100μl of serum-free medium without dual antibodies, and add another 100μl of serum-free medium without dual antibodies to 4μl lipofectamine 2000, mix gently,...

Embodiment 3

[0048]Example 3 Screening of siRNA transfection delivery system

[0049]1. Cholesterol-modified siRNA transfection

[0050](1) The NIH / 3T3 cells that have been transfected with the FGF5 expression plasmid are divided into 1×105The density of cells / well is inoculated into a 6-well plate and cultured in 2ml of cell culture medium containing serum and double antibodies. When the cell density reaches 80%, replace with fresh complete medium;

[0051](2) Dilute 200 pmol of cholesterol-modified siRNA (entrusted by Zima Gene Co., Ltd. to modify according to conventional technical means in the field) and dilute it in 200 μl serum-free and anti-bibial-free medium, mix gently, and let it stand for 5 min, and add it to 6 wells In the plate, put 5% CO2, Cultivate 24h in 37℃ cell incubator;

[0052](3) Collect the cells and detect the mRNA level of FGF5 gene in the cells by qPCR method.

[0053]2. siRNA transfection mediated by penetrating peptide

[0054](1) The NIH / 3T3 cells that have been transfected with the ...

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Abstract

The invention provides an siRNA combination for treating alopecia. The siRNA combination has a significant inhibitory effect on FGF5 in skin tissue. In addition, the invention also provides a deliverysystem for delivering the siRNA combination.

Description

Technical field[0001]The present invention relates to a hair growth promoter, a hair regeneration promoter or a hair loss treatment agent containing a substance that inhibits the growth of hair follicles, and a method for screening these substances.Background technique[0002]With the accelerating pace of life and increasing work pressure, more and more people are suffering from hair loss. Taking the most common androgen-induced alopecia (abbreviated as androgenic alopecia) as an example, the incidence rate in men over 50 years old is as high as About 50%, and every 10 years of age increases, the incidence of androgenetic alopecia increases by 10%. The upward shift of the hairline and thinning of the hair will bring psychological burden and mental pressure to people, and affect the quality of life of patients with baldness. Therefore, there is a high demand for hair loss prevention and hair growth treatment. At present, the main drugs for the treatment of androgenetic alopecia are fin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7105A61K48/00A61P17/14
CPCA61K31/7105A61P17/14
Inventor 高爽许诺李校堃胡振林陈毓赵文刚
Owner WENZHOU UNIVERSITY
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