Application of USP4 as autoimmune liver disease biomarker

A technology of autoimmunity and markers, applied in the field of medical testing, can solve problems such as limited means and unclear results, and achieve the effect of obvious technical effect and important clinical application value

Pending Publication Date: 2021-04-13
THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of limited means and unclear results for the diagnosis of autoimmune liver disease in the prior art, the present invention provides the application of USP4 as a biomarker for autoimmune liver disease

Method used

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  • Application of USP4 as autoimmune liver disease biomarker
  • Application of USP4 as autoimmune liver disease biomarker
  • Application of USP4 as autoimmune liver disease biomarker

Examples

Experimental program
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Effect test

preparation example Construction

[0042] 3.2 Preparation of peripheral blood mononuclear cells

[0043] (1) Take fresh anticoagulated blood from healthy subjects or patients with autoimmune liver disease described in step 3.1, centrifuge at 3000rpm for 10min, and keep the supernatant.

[0044] (2) Absorb the supernatant, that is, the plasma, with a rubber-tipped dropper.

[0045](3) Transfer the remaining peripheral blood after removing plasma to a 15ml centrifuge tube, add PBS for dilution, and the ratio of peripheral blood to diluent is 1:2.

[0046] (4) Add Ficoll to the 50ml centrifuge tube, slowly add the peripheral blood diluted in step (3) along the inclined tube wall, the ratio of Ficoll to diluted blood is 1:1.

[0047] (5) 20°C, 1800rpm (500-1100g), centrifuge for 30min.

[0048] (6) After centrifugation, the centrifuge tube is divided into four layers from top to bottom. The first layer is the plasma layer, the second layer is the ring-shaped milky white lymphocyte layer, the third layer is the t...

Embodiment 1

[0053] Embodiment 1 is used to detect the kit of USP4 mRNA

[0054] The kit is used for the detection of the relative expression of USP4 gene for clinical testing purposes. Real-time fluorescent quantitative PCR technology is used to measure the expression level of USP4 gene in peripheral blood mononuclear cells of patients with liver disease. At the same time, it is helpful for the diagnosis of autoimmune liver disease.

[0055] Kit components include:

[0056] (1) Sample total RNA extraction reagents: TRIZOL, chloroform, ethanol, DEPC water;

[0057] (2) Total RNA reverse transcription reagent: Thermo Fisher's Revert First Strand cDNA Synthesis Kit.

[0058] (3) Real-time fluorescent quantitative PCR reaction reagent: upstream and downstream standard primers of USP4 and GAPDH, SYBR Green (Thermo Fisher). The primer sequences are shown in Table 1 below,

[0059] Table 1

[0060]

Embodiment 2

[0061] Embodiment 2 Real-time fluorescence quantitative PCR detects the method for the mRNA level of USP4

[0062] 1. RNA extraction

[0063] ①Take 50 mg of mouse liver tissue, healthy subjects or human peripheral blood mononuclear cells from patients with autoimmune liver disease prepared in sample preparation, add 1 ml of Trizol, grind / pip and beat sufficiently, and let stand at room temperature for 5 minutes to completely separate the nuclei Protein complex; for AML12 hepatocytes cultured in six-well plates, discard the medium and wash with PBS for 1 to 2 times, then add 1ml of Trizol to the wells, pipette fully and let stand at room temperature for 5 minutes to completely separate nucleoproteins Complex;

[0064] ②Add 0.2ml chloroform respectively, shake vigorously by hand for 15 seconds, wait at room temperature for 2 minutes, centrifuge at 4°C and 13000rpm for 15 minutes, it can be seen that the solution is divided into three layers, from top to bottom are transparent w...

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Abstract

The invention discloses an application of USP4 as an autoimmune liver disease biomarker, and belongs to the field of medical detection. USP4 is applied to preparation of the autoimmune liver disease marker. The USP4 is also applied to preparation of a fluorescent quantitative detection kit for diagnosing or predicting the autoimmune liver disease, and the kit is used for detecting the expression level of the USP4 in peripheral blood mononuclear cells. Experiments prove for the first time that the expression level of USP4 in peripheral blood mononuclear cells of patients with autoimmune liver diseases is significantly increased compared with the expression level of USP4 in healthy subjects. The USP4 expression quantity in the peripheral blood provided by the invention is used as a molecular marker, so that noninvasive detection can be carried out on the autoimmune liver disease, noninvasive dynamic monitoring can be carried out, and the kit has important clinical application value.

Description

technical field [0001] The invention relates to the field of medical detection, in particular to the application of USP4 as a biomarker for autoimmune liver disease. Background technique [0002] Autoimmune liver disease (AILD) is a group of chronic inflammatory diseases of the hepatobiliary system mediated by autoimmune reactions, mainly involving liver damage, mainly including primary biliary cirrhosis (primary biliary cirrhosis, PBC), autoimmune hepatitis (autoimmune hepatitis, AIH) and primary sclerosing cholangitis (primary sclerosing cholangitis, PSC), etc. There is no clear clinical standard for the diagnosis of autoimmune liver disease. However, if the disease is not controlled, it can lead to progressive liver fibrosis and cirrhosis after long-term development. Therefore, early diagnosis of autoimmune liver disease is very necessary. [0003] Deubiquitination is a reversible process of ubiquitination by the removal of ubiquitin protein groups by deubiquitinating e...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883G01N33/573G01N33/564
CPCC12Q1/6883G01N33/573G01N33/564C12Q2600/158G01N2800/085G01N2800/24G01N2333/96466
Inventor 陈永平徐洁金兰灵陈正康蔡超陈达之
Owner THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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