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New application of DEFA4 for diagnosis of hepatic failure, kit, and application of kit for diagnosis of hepatic failure

A technology of liver failure and kits, which is applied in the fields of clinical medicine, biomedicine and medical testing, can solve the problems of liver metabolism, detoxification function reduction, systemic cytokine storm, liver cell necrosis, etc., and achieve easy repeatability, simple method, and trauma small effect

Inactive Publication Date: 2019-09-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Significant abnormalities of these indicators usually occur in patients with obvious liver cell necrosis, systemic cytokine storm, liver metabolism and detoxification function are significantly reduced, and the disease enters a stage of rapid progression, which is difficult to control and causes great difficulties in treatment.

Method used

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  • New application of DEFA4 for diagnosis of hepatic failure, kit, and application of kit for diagnosis of hepatic failure
  • New application of DEFA4 for diagnosis of hepatic failure, kit, and application of kit for diagnosis of hepatic failure
  • New application of DEFA4 for diagnosis of hepatic failure, kit, and application of kit for diagnosis of hepatic failure

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] experimental method

[0045] 1. Peripheral blood mononuclear cell isolation

[0046] 1. Draw 8mL of whole blood from the patient, let it stand for about 30 minutes, and pre-cool the centrifuge to 4°C.

[0047]2. Using a density gradient centrifuge, centrifuge at 4°C and 3000 rpm for 5 minutes. The blood cell pellet was fully resuspended with 5mL sterile 0.1M PBS buffer, mixed evenly to form a blood cell suspension, and then slowly added to the centrifuge tube of Ficoll-paqueTM PLUS medium lymphocyte separation medium to form layers naturally. Set the temperature of the centrifuge to 20°C, the speed to 2500 rpm, speed up to 9, speed down to 1, and centrifuge for 20 minutes.

[0048] 3. Aspirate the middle buffy coat layer, add 10 mL of PBS buffer, centrifuge at 1200 rpm at 4°C for 4 minutes, discard the supernatant. This step was repeated 3 times.

[0049] 4. Blot the residual PBS buffer with a pipette gun, and add 1 mL of cell lysis solution TRIzol to completely lys...

Embodiment 2

[0065] Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the expression of DEFA4 mRNA in liver failure, high-risk groups of liver failure, liver cirrhosis, chronic hepatitis B and healthy controls.

[0066] Experimental procedure

[0067] qRT-PCR uses a two-step method for experimental operation. Separation of peripheral blood mononuclear cells and extraction of total RNA were carried out in the same manner as in Example 1, and the next step of the experimental operation was carried out after the mRNA concentration was tested.

[0068] 1. Reverse transcription: QuantiTect Reverse Transcription kit (Qiagen, CA, USA) was used for reverse transcription, and cDNA was obtained by following the instructions of the kit.

[0069] 2. Take the above cDNA template for PCR amplification: the PCR reagent uses TB Green reagent from Takara Company, and the operation is according to the kit instructions.

[0070] figure 2 Peripheral blood mononuclear samples from liver f...

Embodiment 3

[0071] Example 3 The kit includes: DEFA4 amplification primers such as the primer pair shown in the front primer of SEQ ID NO.1 and the rear primer of SEQ ID NO.2, and the internal reference such as the front primer of SEQ ID.NO.3 and the rear primer of SEQ ID NO.4 The primer pair shown: 2.5U / μl PolyA polymerase, reverse transcriptase, 5× reverse transcription buffer, RT mixture, 5ml RNase-free water, qPCR mixture, adsorption column, cell lysate. Application of DEFA4 mRNA kit in the diagnosis of pigs with liver failure

[0072] Animal model: 30 male Chinese young pigs (8-10 kg) were randomly divided into two groups, 15 in each group.

[0073] Experimental group: Each young pig was injected with D-gal 1.5g / kg into the jugular vein to create a liver failure model.

[0074] Control group: inject the same amount of saline without DEFA4.

[0075] The peripheral blood mononuclear cells and serum samples of the experimental group and the control group were separated, and the total ...

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Abstract

The invention discloses new application of DEFA4 for diagnosis of hepatic failure, a kit, and application of the kit for diagnosis of hepatic failure. Samples in the invention are selected from patients with hepatic failure or high-risk populations of hepatic failure, the body fluid sample is preferably a blood sample, the blood sample is a serum sample or a plasma sample, the cell sample is a peripheral blood mononuclear cell sample, and the tissue sample is a hepatic tissue sample. The DEFA4 mRNA diagnosis kit disclosed by the invention has a simple and convenient method, detection processesare simple and easy to repeat, can be completed by professional technicians and have strong feasibility, and the DEFA4 mRNA diagnosis kit can provide an accurate prediction result, timely reflect thedisease state of a patient with hepatic failure, early diagnose the hepatic failure, and guide doctors to treat the patient with hepatic failure early to reduce the mortality.

Description

technical field [0001] The invention relates to the technical fields of clinical medicine, biomedicine and medical testing, in particular to a new application of detecting DEFA4 molecules for diagnosing liver failure, a kit and an application of the kit for diagnosing liver failure. Background technique [0002] Liver failure is a kind of disease caused by extensive necrosis of the liver caused by various reasons. Except for orthotopic liver transplantation, there is currently no specific treatment. However, due to the severe shortage of donor livers, a large number of patients died while waiting for liver transplantation. The current clinical method for diagnosing liver failure is based on the traditional coagulation indicators of total bilirubin in biochemical indicators, such as the international normalized ratio of coagulation, prothrombin activity and so on. Significant abnormalities of these indicators usually occur in patients with obvious liver cell necrosis, syste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6851C12Q1/6883C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101
Inventor 李君陈新李江梁茜李佳琪江静
Owner ZHEJIANG UNIV
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