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Application of caspase recruitment domain protein 6 (card6) in liver ischemia-reperfusion injury

A reperfusion injury, 1.card6 technology, applied in the field of gene function and application, can solve the problems of failing to properly solve the liver, injury, etc., and achieve the effect of inhibiting liver tissue ischemia-reperfusion injury

Active Publication Date: 2021-02-02
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, the above strategies have not been able to properly solve the problem of liver damage caused by HIRI in clinical practice.

Method used

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  • Application of caspase recruitment domain protein 6 (card6) in liver ischemia-reperfusion injury
  • Application of caspase recruitment domain protein 6 (card6) in liver ischemia-reperfusion injury
  • Application of caspase recruitment domain protein 6 (card6) in liver ischemia-reperfusion injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Example 1 Construction of hepatocyte-specific Card6 gene knockout mice and Card6 transgenic mice

[0037](1) Construction of hepatocyte-specific Card6 gene knockout mice (see the construction strategyfigure 1 A)

[0038]According to the Card6 gene information, use CRISPR Design to design a CRISPR target site in the non-coding region 4 of intron 3 and exon 4. The target sequences are:

[0039]Card6-sRNA1: GgAGAGTAGGCAACATGACT TGG;

[0040]Card6-sRNA2: gGTGAAAGCAGTTATCAGTA GGG.

[0041]In addition, a Donor Vector for homology repair was designed, which includes homology arms on both sides, exon 4 in the middle, and two loxp sequences in the same direction.

[0042]1) Construction of targeting vector: fusion of the two primers corresponding to sgRNA1 and sgRNA2 into double-stranded DNA, and then use T4 DNA ligase to ligate into pUC57-sgRNA (Addgene 51132) vector treated with restriction enzyme BsaI . There is a T7 promoter upstream of the vector, which can be used for subsequent in vitro transcrip...

Embodiment 2

[0061]Example 2 Obtaining a mouse liver ischemia / reperfusion injury (I / R) model

[0062](1) Experimental animal grouping: male C57BL / 6 strain wild-type mice, hepatocyte-specific Card6 knockout mice, Card6 transgenic mice, non-transgenic mice, established by liver ischemia-reperfusion (I / R) Liver ischemia-reperfusion injury model. Randomly divided into 8 groups: C57BL / 6J wild-type mouse sham operation group (WT Sham) and I / R operation group (WT I / R), hepatocyte-specific Card6 knockout mouse sham operation group (LKO Sham) ) And I / R operation group (LKO I / R), non-transgenic mouse sham operation group (NTG Sham) and I / R operation group (NTG I / R), Card6 transgenic mouse sham operation group (TG Sham) and I / R surgery group (TG I / R).

[0063](2) Hepatic ischemia-reperfusion injury I / R model surgery (using non-invasive vascular clips to clamp the portal vein and hepatic artery of the middle and left lobe to make about 70% of the liver ischemia) Operation process:

[0064]1) The mice were fasted 12h...

Embodiment 3

[0069]Example 3 Measurement of liver necrosis area and liver function indexes (AST, ALT)

[0070]The evaluation indicators of the severity of liver ischemia-reperfusion injury mainly include liver necrosis area and liver function indicators (AST, ALT), which are all positively correlated with the severity of liver ischemia-reperfusion injury.

[0071](1) Taking materials

[0072]Mice in the sham operation group (Sham) and the ischemia-reperfusion group were taken at 1h, 3h, 6h, 12h, and 24h after operation, and sacrificed by cervical dislocation. 1 mL of blood was immediately taken from the inferior vena cava and the serum was separated. At the same time, the left liver lobe tissues in the ischemic area were taken from the ischemic area about 1.5cm×1cm×0.2cm, fixed in 10% neutral formalin for 24 hours, dehydrated, embedded, and paraffin sectioned for HE staining.

[0073]Separation of serum: The EP tube for collecting blood is allowed to stand for 1-2 hours at room temperature to allow the bloo...

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Abstract

The invention discloses an application of Caspase recruitment domain protein 6 (Card6) in hepatic ischemia reperfusion injury and belongs to the field of functions and applications of genes. According to the application, hepatic-cell specific Card6 gene knockout mice and Card6 transgenic mice serve as experimental subjects, functions of a Card6 gene are researched through a hepatic ischemia reperfusion injury model, and the condition that Card6 plays a role in improving the hepatic ischemia reperfusion injury is discovered, so that the Card6 has the applications as follows: an application of the Card6 in screening of drugs for preventing, relieving and / or treating the hepatic ischemia reperfusion injury as a drug target, wherein the screening of the drugs for preventing, relieving and / or treating the hepatic ischemia reperfusion injury means screening the drugs capable of promoting Card6 expression; an application of the Card6 in preparation of drugs for preventing, relieving and / or treating the hepatic ischemia reperfusion injury.

Description

Technical field[0001]The present invention belongs to the field of gene function and application, and relates to the application of caspase recruitment domain protein 6 (Card6) in hepatic ischemia-reperfusion injury, and specifically relates to the screening or preparation of Card6 in the prevention, alleviation and / or treatment of liver ischemia and reperfusion injury. Application of perfusion injury drugs.Background technique[0002]Clinically, in the process of liver resection, treatment of severe liver trauma, and liver transplantation, it is usually necessary to block the blood flow of the hepatic portal. After the blood supply is restored, the damage of the liver cells that were damaged during ischemia is not alleviated, but worsened. It is called hepatic ischemia reperfusion injury (HIRI) [1]. HIRI can reduce liver metabolism and detoxification. In severe cases, it can cause liver failure, which directly affects the prognosis of the disease, the success rate of surgery and the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61K48/00A61P1/16A61P9/10
CPCA61K38/1709A61K48/00
Inventor 李红良王晓占毛文哲
Owner WUHAN UNIV
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