Application of AGAP9 in preparation of osteoarthritis diagnosis product

A technology for osteoarthritis and products, applied in the field of disease diagnosis

Active Publication Date: 2019-11-26
PEOPLES HOSPITAL OF DEYANG CITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The experiment has not yet reached that step in the future, but it is just trying its feasibility with the help of more mature and easier to popularize clinical detection methods

Method used

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  • Application of AGAP9 in preparation of osteoarthritis diagnosis product
  • Application of AGAP9 in preparation of osteoarthritis diagnosis product
  • Application of AGAP9 in preparation of osteoarthritis diagnosis product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Screening of differentially expressed genes

[0063] 1. Materials:

[0064] Synovial tissues from 5 OA patients (OA group) were obtained from OA patients undergoing knee arthroplasty or synovectomy. The cases used were in line with the diagnostic criteria for OA proposed by Altam. 3 cases of normal synovial tissue (Nor group) came from joint synovial tissue of patients undergoing trauma surgery. All clinical samples used in this study were informed to the patients and approved by the ethics committee of the hospital.

[0065] The clinical information of the selected subjects is shown in Table 1.

[0066] Table 1 Clinical Information

[0067]

[0068] 2. Obtaining tissue RNA

[0069] Total RNA was extracted from tissue samples, and the concentration and purity of the extracted RNA were detected by Nanodrop2000, the integrity of RNA was detected by agarose gel electrophoresis, and the RIN value was determined by Agilent2100. A single library constructio...

Embodiment 2

[0087] Example 2 Large sample verification screened out differentially expressed genes

[0088] Based on the results of previous sequencing and according to the size of the P value, we selected the AGAP9 gene for verification.

[0089] 1. Sample collection

[0090] According to the method of Example 1, 42 cases of osteoarthritis synovial tissue and 42 normal synovial tissue were collected.

[0091] 2. Validation at the mRNA level

[0092] 2.1 Extract tissue RNA

[0093] Step is with embodiment 1.

[0094] 2.2 Reverse transcription

[0095] Reverse transcription using Primescript 1 st strand cDNA synthesis kit kit, the operation steps are as follows:

[0096] (1) Add the following reaction liquid in the microcentrifuge tube, as shown in Table 2:

[0097] Table 2 Reaction liquid

[0098] Reagent dose RNA 2.0μg dNTP 1.0μl Oligo(dT) 2.0μl RNase free dH 2 o

Add to 10.0μl

[0099] (2) Incubate at 70°C for 5 minutes, then rapi...

Embodiment 3

[0124] Example 3 Effect of AGAP9 gene on the proliferation of osteoarthritis synoviocytes

[0125] 1. Interference with AGAP9 gene expression

[0126] According to the related sequence of AGAP9 gene, siRNA against AGAP9 (siRNA-AGAP9) was designed and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. The sequence of siRNA-AGAP9 is as follows: sense strand 5'-UUACUUAGUUUACGAAAAGUATT-3' (SEQ ID NO.5), antisense strand 5'-CUUUUCGUAAACUAAGUAAAUTT-3' (SEQ ID NO.6).

[0127] 2. Cell culture

[0128] Human osteoarthritis synoviocytes (Cat. No.: 408OA-05A, Cell applications, INC.) were grown in synoviocyte growth medium (Cat. No.: 415-500, Cell applications, INC.) containing 10% FBS and penicillin-streptomycin. ), placed at 37°C, 5% CO 2 cultured in a humid incubator.

[0129] 3. Cell transfection and expression level detection

[0130] According to 2*10 5 Cells per well were seeded in a 6-well plate, and transfected with siRNA-AGAP9 and siRNA-NC (general negative ...

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Abstract

The invention discloses a diagnostic tool for osteoarthritis. The diagnostic tool achieves the purpose of diagnosis by detecting an AGAP9 gene and an expression product of the AGAP9 gene. The study proves that compared with normal synovial tissue, the mRNA expression level of the AGAP9 gene of an osteoarthritis patient is remarkably up-regulated. According to correlation between the AGAP9 gene andosteoarthritis, a kit for diagnosing osteoarthritis can be prepared, and can be widely applied in clinic. Besides, synoviocyte cell proliferation can be inhibited by inhibiting expression of the AGAP9 gene, it is indicated that the AGAP9 gene can be used for researching and developing a drug for treating osteoarthritis, and the research result provides theoretical support for development of a newclinical drug.

Description

technical field [0001] The present invention relates to the field of disease diagnosis, more specifically, the present invention relates to a disease diagnosis method by means of detecting AGAP9 abnormality. Background technique [0002] Osteoarthritis (OA) is a common chronic and progressive joint disease among middle-aged and elderly people. Synovial inflammatory response is the main pathological feature, its pathogenesis is not completely clear so far, and early diagnosis is difficult. The commonly used clinical diagnostic methods are often aimed at middle and late patients. The impact is more direct and obvious. [0003] Osteoarthritis is considered a chronic disease that can remain "silent" for long periods of time, and its early stages are difficult to diagnose clinically; diagnosis is usually based on clinical symptoms and imaging changes. However, X-ray has a poor sensitivity and precision, only provides a cumulative damage, and the error is relatively large, so it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883G01N33/68
CPCC12Q1/6883C12Q2600/178G01N33/6893G01N2800/105
Inventor 刘跃洪周宇陈曦王志聪张建军汪红杨灵江伟
Owner PEOPLES HOSPITAL OF DEYANG CITY
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