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Human JNK1 (c-Jun N-terminal kinase) gene targeting small interfering RNA (ribonucleic acid) and application thereof

A small interference, gene technology, applied in DNA/RNA fragmentation, application, gene therapy and other directions, can solve the problems of easy fracture, limited widespread use, increased tooth fragility, etc., to reduce apoptosis and promote odontoblast differentiation. Effect

Active Publication Date: 2016-10-26
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

Caries is one of the main causes of pulpitis. Bacterial infection and infiltration of inflammatory cells cause dentin damage. At this time, the body can form restorative dentin to prevent the pulp from being stimulated by bacteria and toxins, so as to maintain the tooth Structural integrity, but because the repair speed of dentin is slower than the destruction speed of dentin, resulting in the destruction of tooth structure, eventually leading to the occurrence of pulpitis
Root canal therapy is currently the most commonly used clinical treatment method, but the increased fragility and fracture of teeth after treatment limits its widespread use

Method used

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  • Human JNK1 (c-Jun N-terminal kinase) gene targeting small interfering RNA (ribonucleic acid) and application thereof
  • Human JNK1 (c-Jun N-terminal kinase) gene targeting small interfering RNA (ribonucleic acid) and application thereof
  • Human JNK1 (c-Jun N-terminal kinase) gene targeting small interfering RNA (ribonucleic acid) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Obtaining specific interference sequences targeting human JNK1 gene

[0023] Design the siRNA sequence according to the human JNK1 gene sequence (GenBank: JN257262.1) published by NCBI, and refer to the RNAi online design provided by the website of Invitrogen Corporation of the United States (its website http: / / rnaidesigner.invitrogen.com / rnaiexpress / sort.do) Software, after comparison and screening, the target gene sequence containing 19 bases was obtained: 5'-GTCGGTTAGTCATTGATAG-3' (SEQ ID NO: 1), and the corresponding siRNA interference sequence was synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd., as follows:

[0024] Sense strand: 5'-GAGUCGGUUAGUCAUUGAUAG-3' (SEQ ID NO:2)

[0025] Antisense strand: 5'-CUAUCAAUGACUAACCGACUC-3'(SEQ ID NO:3)

Embodiment 2

[0026] Example 2: Dental pulp stem cell culture and Western blot detection of the effect of siRNA on JNK1 protein expression

[0027]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 i...

Embodiment 3

[0031] Example 3: Western blot detection of siRNA on the odontogenic differentiation ability of dental pulp stem cells (DPSS, DMP-1 protein expression)

[0032] 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, the supernatant was discarded, and 400ul of serum medium was added to each well, and transfected for 4 hours (the experiments were divided into blank group, control group and siRNA group as described above).

[0033] 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 them stand on ice for 30min, centrifuge at 12000rpm, and take them for 30min. Supernatant, prepared as a protein sample for analysis. Add the prepared protein into the l...

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Abstract

The invention relates to the technical field of biology, in particular to a human JNK1 (c-Jun N-terminal kinase) gene targeting small interfering RNA (ribonucleic acid) and application thereof. The small interfering RNA comprises a sense strand and an anti-sense strand, a nucleotide sequence of the sense strand is as shown in SEQ ID NO:2, and a nucleotide sequence of the anti-sense strand is as shown in SEQ ID NO:3. The sequences of the small interfering RNA are not publically reported yet, interference after transcription of the JNK1 gene can be realized to reduce expression of p-JNK in inflamed dental pulp stem cells, apoptosis of the dental pulp stem cells can be specifically reduced, odontoblast differentiation of the dental pulp stem cells is promoted, and a novel treatment scheme is provided for pulpitis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a small interfering RNA sequence targeting human JNK1 gene for treating pulpitis and its application. Background technique [0002] Pulpitis is one of the most common oral diseases. The main symptom is pain. If it is not treated in time, it can cause apical periodontitis, jaw cyst, etc., which will affect the patient's oral function, appearance, pronunciation and psychological state, and even become a lesion, causing Low-grade fever, nephritis, myocarditis, iridocyclitis, rheumatoid arthritis and other diseases affect the health of the whole body. Caries is one of the main causes of pulpitis. Bacterial infection and infiltration of inflammatory cells cause dentin damage. At this time, the body can form restorative dentin to prevent the pulp from being stimulated by bacteria and toxins, so as to maintain the tooth The integrity of the structure, but because the repair speed of dentin...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/113A61K31/713A61P1/02
CPCC12N9/12C12N15/1137C12N2310/141C12N2320/30C12Y207/11024
Inventor 张冬梅刘晓娟肖静雯冯兴梅
Owner 江苏国辰医疗科技有限公司
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