Small interfering RNA of people-targeted IRAK1 gene and application of RNA

A small interference, gene technology, applied in DNA/RNA fragments, applications, gene therapy and other directions, can solve the problems of irritable pulp tissue, poor prognosis, easy damage to healthy tooth tissue, etc., and promote odontoblast differentiation. , the effect of reducing expression and inhibiting the development of inflammation

Active Publication Date: 2016-12-14
江苏国辰医疗科技有限公司
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  • 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 of people-targeted IRAK1 gene and application of RNA
  • Small interfering RNA of people-targeted IRAK1 gene and application of RNA
  • Small interfering RNA of people-targeted IRAK1 gene and application of RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Acquisition of specific interfering sequences targeting human IRAK1 gene

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

[0025] Sense strand: 5'-GCCCGAAGAAAGUGAUGAAUU-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) The dental pulp cells in the experiment are derived from clinically normal and carious third molars and second premolars that need to be extracted for orthodontic treatment. The patients are 15-25 years old and their general condition is good. Informed consent was obtained from the participants and approved by the Medical Ethics Committee of the Affiliated Hospital of Nantong University. Immediately put the extracted teeth into ice-packed basal DMEM medium (containing 100 U / mL penicillin / 100 μg / mL streptomycin). After rinsing the tooth surface with iodophor on the ultra-clean bench, rinse the tooth surface with sterile saline until there is no blood stain, use the sterilized rongeur to open the pulp cavity along the cementoenamel boundary, take out the pulp, and cut it off. The pulp tissue of the root. Digest with 3 mg / mL type I collagenas...

Embodiment 3

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

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

[0034] 2) 24h after dental pulp stem cells were transfected, the medium was aspirated, the cells were washed 1-2 times with PBS, 100uLRIPA lysis solution was added to the cells, the cells were collected into a 1.5mL tube, left standing on ice for 30min, centrifuged at 12000rpm / min, Take the supernatant for 30 min and prepare a protein sample for analysis. The prepared protein was ad...

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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 is a chronic and progressive damage to the dental hard tissue caused by the combined action of various factors in the oral cavity. The traditional mechanical caries removal method is the main method of clinical dental treatment at present. It mainly uses the cutting action of the emery bit to remove the carious tissue. However, it is easy to damage the healthy tooth tissue in the application, and the heat generated by the cutting of the drill and the cooling of the liquid are very cold. Stimulation easily irritates the pulp tissue, resulting in a poor prognosis. Therefore, finding new alternative treatments is of great significance for the treatment of caries. [0003] IRAK1 (interleukin-1 receptor-associated kinase 1) is a member of the IRAK family. Through ...

Claims

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Application Information

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Patent Type & Authority Applications(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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