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Application of Caspase recruitment domain protein 6 (Card6) in hepatic ischemia reperfusion injury

A technology for reperfusion injury and liver ischemia, applied in the field of gene function and application, can solve problems such as failure to properly solve liver and injury, and achieve the effect of inhibiting liver tissue ischemia-reperfusion injury

Active Publication Date: 2017-01-11
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 hepatic ischemia reperfusion injury
  • Application of Caspase recruitment domain protein 6 (Card6) in hepatic ischemia reperfusion injury
  • Application of Caspase recruitment domain protein 6 (Card6) in hepatic ischemia reperfusion injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0037] (1) Construction of hepatocyte-specific Card6 gene knockout mice (see figure 1 A in

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

[0039] Card6-sRNA1: GgAGAGTAGGCAACATGACT TGG;

[0040] Card6-sRNA2: gGTGAAAGCAGTTATCAGTAGGG.

[0041] In addition, a donor vector (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: respectively fuse 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 endonuclease BsaI . There is a T7 promoter upstream of the...

Embodiment 2

[0061] Example 2 Obtainment of mouse liver ischemia-reperfusion injury (ischemia / reperfusion injury, I / R) model

[0062](1) Grouping of experimental animals: male C57BL / 6 strain wild-type mice, liver cell-specific Card6 knockout mice, Card6 transgenic mice, and non-transgenic mice, established by liver ischemia-reperfusion (I / R) Liver ischemia-reperfusion injury model. They were randomly divided into 8 groups: C57BL / 6J strain wild-type mice sham operation group (WT Sham) and I / R operation group (WT I / R), liver cell-specific Card6 gene 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) I / R model surgery of hepatic ischemia-reperfusion injury (using non-invasive vascular clips to clamp the portal vein and hepatic artery in the middle lobe and left lobe, so that...

Embodiment 3

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

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

[0071] (1) Take materials

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

[0073] Separation of serum: the EP tube where the blood was collected was left at room temperatu...

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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 invention belongs to the field of gene function and application, and relates to the application of caspase recruiting domain protein 6 (Card6) in liver ischemia-reperfusion injury, in particular to the application of Card6 in the screening or preparation of preventing, alleviating and / or treating liver ischemia-reperfusion injury. Application of Perfusion Injury Drugs. Background technique [0002] Clinically, during liver resection, treatment of severe liver trauma, and liver transplantation, it is usually necessary to block the hepatic portal blood flow. The damaged liver cells that were originally damaged during ischemia will not be relieved after the blood supply is restored, but will be aggravated. , known as hepatic ischemia reperfusion injury (hepatic ischemia reperfusion injury, HIRI) [1]. HIRI can reduce the metabolism and detoxification function of the liver, and cause liver failure in severe cases, which directly affects the prognosis o...

Claims

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Application Information

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Patent Type & Authority Applications(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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