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Small interfering RNA targeting human irak1 gene and its application

A small interference, gene technology, applied in DNA/RNA fragments, applications, gene therapy and other directions, can solve problems such as poor prognosis, easy damage to healthy dental tissue, irritable dental pulp tissue, etc., to reduce expression and promote growth. Effect of dentin differentiation and inhibition of the development of inflammation

Active Publication Date: 2019-11-08
江苏国辰医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional mechanical caries removal method is currently the main method of clinical tooth treatment. The cutting action of the diamond drill bit is mainly used to remove the carious tissue, but it is easy to damage the healthy tooth tissue during the application, and the heat generated by the cutting of the drill bit and the cooling of the liquid are harmful to the cold. Stimulation easily irritates the pulp tissue, resulting in poor prognosis

Method used

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  • Small interfering RNA targeting human irak1 gene and its application
  • Small interfering RNA targeting human irak1 gene and its application
  • Small interfering RNA targeting human irak1 gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Obtaining specific interference sequences targeting human IRAK1 gene

[0024] Design the siRNA sequence according to the human IRAK1 gene sequence (Genbank: NM_001025243) disclosed by NCBI, and refer to the RNAi online design software provided by the website of Invitrogen Corporation of the United States (its website http: / / rnaidesigner.invitrogen.com / rnaiexpress / sort.do) for specific design, After comparison and screening, the target gene sequence containing 19 bases was obtained: 5'-CCGAAGAAAGTGATGAATT-3' (SEQ ID NO: 1), and the corresponding siRNA interference sequence was synthesized by Shanghai Jikai Gene Chemical Technology Co., Ltd., as follows:

[0025] Sense strand: 5'-GCCCGAAGAAAAGUGAUGAAUU-3' (SEQ ID NO:2)

[0026] Antisense strand: 5'-AAUUCAUCACUUUCUUCGGGC-3'(SEQ ID NO:3)

Embodiment 2

[0027] Example 2: Dental pulp stem cell culture and Western blot detection of the effect of siRNA on IRAK1 protein expression

[0028] 1) During the experiment, the dental pulp cells were derived from clinically normal and carious third molars and second premolars that needed to be extracted due to orthodontics. The patients were 15-25 years old and in good general condition. Informed consent was obtained from the participants, and it was approved by the Medical Ethics Committee of the Affiliated Hospital of Nantong University. The extracted teeth were immediately put into the basic DMEM culture medium (containing 100 U / mL penicillin / 100 μg / mL streptomycin) pre-cooled by ice packs. After rinsing the surface of the tooth with povidone iodine on the ultra-clean bench, rinse the surface of the tooth with sterile normal saline until there is no blood stain, use a sterilized rongeur to open the pulp cavity along the enamel-cementum junction, take out the pulp with tweezers, and cut...

Embodiment 3

[0032] Example 3: Western blot detection of siRNA on the odontogenic differentiation ability of dental pulp stem cells (DPSS, DMP-1 protein expression)

[0033] 1) Digest the dental pulp stem cells in the logarithmic growth phase with 0.25% trypsin to make a single cell suspension (cell number 1×10 5 ), inoculated in 6-well plate, 200uL per well. 37°C, 5% CO 2 After culturing in the incubator for 24 hours, discard the supernatant, add 400 uL of serum medium to each well, and transfect for 4 hours (the experiments were divided into blank group, control group, and siRNA group as described above).

[0034] 2) After 24 hours of transfection of dental pulp stem cells, remove the medium, wash the cells with PBS 1-2 times, add 100uL RIPA lysate to the cells, collect the cells into a 1.5mL tube, let stand on ice for 30min, and centrifuge at 12000rpm / min. The supernatant was taken after 30 min and prepared as a protein sample for analysis. Add the prepared protein into the loading b...

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Abstract

The invention relates to the technical field of biology, in particular to a small interfering RNA sequence of the people-targeted IRAK1 gene for curing dental caries and application of the RNA sequence, the small interfering RNA comprises a sense strand and an antisense strand, and the nucleotide sequence of the sense strand is shown by the SEQ ID NO:2; the nucleotide sequence of the antisense strand is shown by the SEQ ID NO:3. The obtained small interfering RNA doesn't appear in disclosed reports, and interfering can be performed after transcription of the IRAK1 gene, so that the IRAK1 protein expression can be specifically reduced, NF-kB expression in inflammatory dental pulp stem cells can be reduced, generation of the inflammatory dental pulp stem cell inflammatory factor-tumor necrosis factor is reduced, the inflammation is resisted, the odontoblast differentiation of the dental pulp stem cells is reduced, and a new curing scheme is provided for the dental caries.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a small interfering RNA sequence targeting human IRAK1 gene and its application. Background technique [0002] Dental caries (dental caries) is a chronic and progressive lesion of dental hard tissue caused by multiple factors in the oral cavity. Its incidence rate is high and the population is widely distributed. The traditional mechanical caries removal method is currently the main method of clinical dental treatment. It mainly uses the cutting action of the diamond drill bit to remove carious tissue, but it is easy to damage healthy tooth tissue during the application, and the heat generated by the drill bit cutting and the cooling of the liquid are harmful to the cold. Stimulation easily irritates the pulp tissue, resulting in a poor prognosis. Therefore, finding a new alternative therapy is of great significance for the treatment of dental caries. [0003] IRAK1 (interleukin-1 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/113A61K31/713A61P1/02
CPCC12N9/12C12N15/1137C12N2310/141C12N2320/30
Inventor 张冬梅刘晓娟宋亦华张烨冯兴梅
Owner 江苏国辰医疗科技有限公司
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