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Application of miR-146a in preparing medicine for curing gastricism

A 1. mir-146a, gastritis technology, applied in gene therapy, drug combination, pharmaceutical formulations, etc., can solve the problems of miRNAs and H.

Inactive Publication Date: 2010-03-17
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching the domestic and foreign literature of the prior art, there has been no research report on the diagnosis and treatment of miRNAs and Hp infection-related inflammation.

Method used

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  • Application of miR-146a in preparing medicine for curing gastricism
  • Application of miR-146a in preparing medicine for curing gastricism
  • Application of miR-146a in preparing medicine for curing gastricism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Establishment of gastric epithelial cell model of Hp infection

[0018] The Hp 26695 standard strain was cultured to the logarithmic growth phase, the bacteria were collected by centrifugation at 5000 × g, and the Hp bacterial suspension was prepared with antibiotic-free RPMI 1640 medium, and the bacterial concentration was adjusted by measuring the A600 absorbance value (1A600=1×108cfu / ml). The human gastric epithelial cell line GES-1 was cultured with RPMI 1640 (100 U / ml penicillin; 100 U / ml streptomycin) containing 10% calf serum as the complete medium. When the adherent growth reached 80%, the medium was discarded. . Add Hp according to the multiplicity of infection (MOI) (number of bacteria: number of cells) at 100:1, 37°C, 5% CO 2 co-cultured for 24 hours.

Embodiment 2

[0019] Example 2: miRNAs microarray detects the changes of miRNAs expression profile in gastric epithelial cells infected with Hp infection

[0020] Total RNA was extracted by Trizol method, 1×10 7 1ml Trizol was added to the cells, vigorously pipetted repeatedly with a pipette tip, fully vortexed on a vortexer to completely lyse the cells, and allowed to stand at room temperature for 5 minutes; chloroform was added at a ratio of 0.2ml chloroform / 1ml Trizol, vortexed sufficiently, and allowed to stand at room temperature for 10 minutes; 4°C, 12000 Centrifuge at ×g for 15min, take the upper colorless liquid into a new 1.5ml centrifuge tube; add isopropanol at the ratio of 0.5ml isopropanol / 1ml Trizol, mix well, and leave at room temperature for 5-10min to form RNA precipitation; 4 ℃, centrifuge at 12000×g for 10min, discard the supernatant; add at least 1ml 75% ethanol / 1ml Trizol to suspend the RNA precipitate; 4℃, centrifuge at 7500×g for 5min, discard the supernatant; after...

Embodiment 3

[0025] Example 3: Real-time PCR technique to detect the expression of miRNAs

[0026] In this example, the TaqMan miRNA Assay kit (Applied Biosystems) was used for Real-time PCR detection of miRNAs.

[0027] First, extract the total cell RNA according to the Trizol method described in Example 1, take 10 ng of each of the above total RNAs as templates, and then use the miRNAs-specific primers that come with the kit to synthesize cDNA. The reaction system is as follows: 0.15 μl of dNTPs (100 mM); MultiScribe Reverse Transcriptase 1 μl; 10× RT Buffer 1.5 μl; RNase Inhibitor (20U / μl) 0.19 μl; RT Primer 3 μl; Total RNA 5 μl; DEPC H 2 O 4.16 μl. Reaction conditions: 30 minutes at 16°C, 30 minutes at 42°C, 5 minutes at 85°C, and storage at 4°C.

[0028] Then, using the cDNA as the template, the TaqMan probe method Real-time PCR was performed using the specific primers and reverse primers of the miRNAs that came with the kit. The system is as follows: 2×PCR mix 10 μl; cDNA 2 μl; Pr...

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Abstract

The invention relates to an application of a non-coding small RNA gene miR-146a in preparing a medicine for curing gastricism. The gastricism can be caused by helicobacter pylori infection. The miR-146a is highly expressed in helicobacter pylori infected gastric epithelial cells and human gastric mucosa tissues; furthermore, after the gastric epithelial cells GES-1 are transfected by a miR-146a simulator or a miR-146a inhibitor respectively, the miR-146a simulator can obviously reduce the protein secretion level of inflammatory factor lnterleukin 8, macrophage inflammatory protein 3Alpha and growth-related oncogene Alpha and the protein secretion level of the macrophage inflammatory protein 3Alpha, thus indicating that the miR-146a can effectively inhibit inflammatory response related to the helicobacter pylori infection.

Description

technical field [0001] The invention relates to the application of the non-coding small RNA gene miR-146a, in particular to the application of miR-146a in the preparation of a drug for treating gastritis, especially the application of miR-146a in the preparation of a drug for treating gastritis caused by Helicobacter pylori infection. Background technique [0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNA molecules with a length of about 20-25 nt, which are ubiquitous in eukaryotes. ) The complementary binding of the 3' untranslated region induces the degradation of the target mRNA or inhibits the translation of the target mRNA, thereby achieving post-transcriptional gene regulation. Many evidences show that miRNAs play important regulatory roles in various biological processes such as cell proliferation, development, and differentiation, and are closely related to tumorigenesis. [0003] In recent years, more and more attention has been paid to the relati...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P1/04
Inventor 肖斌邹全明刘真毛旭虎黎伯胜唐彬朱恩东李娜郭刚
Owner ARMY MEDICAL UNIV
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