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Application of TNF (tumor necrosis factor) receptor associated factor 5 (Traf5) to ischemia reperfusion injury

A reperfusion injury, 1.traf5 technology, applied in the field of gene function and application, can solve problems such as unclear effect, and achieve the effect of inhibiting liver tissue ischemia-reperfusion injury

Inactive Publication Date: 2017-05-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the role of Traf5 in liver ischemia-reperfusion injury is unclear

Method used

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  • Application of TNF (tumor necrosis factor) receptor associated factor 5 (Traf5) to ischemia reperfusion injury
  • Application of TNF (tumor necrosis factor) receptor associated factor 5 (Traf5) to ischemia reperfusion injury
  • Application of TNF (tumor necrosis factor) receptor associated factor 5 (Traf5) to ischemia reperfusion injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of hepatocyte-specific Traf5 knockout mice and Traf5 transgenic mice

[0035] (1) Construction of hepatocyte-specific Traf5 gene knockout mice (see figure 1 A)

[0036] According to the information of Traf5 gene, CRISPR Design was used to design a CRISPR targeting site in intron 2 and 3 respectively. The target sequences are:

[0037] Traf5-sRNA1: GgAGGCTGTAGTACAGTGTGACGGC CGG;

[0038] Traf5-sRNA2: gGTTTTAAGGGTTATTCTAGACTAGA AGG.

[0039] In addition, a donor vector (Donor Vector) for homology repair was designed, which includes homology arms on both sides, exon 3 in the middle and two loxp sequences in the same direction.

[0040] 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 vector, which can be used...

Embodiment 2

[0060] Example 2 Obtainment of mouse liver ischemia-reperfusion injury model (ischemia / reperfusion injury, I / R)

[0061] (1) Grouping of experimental animals: male C57BL / 6 strain wild-type mice, hepatocyte-specific Traf5 knockout mice, Traf5 transgenic mice, and non-transgenic mice. Liver ischemia-reperfusion was established through liver ischemia-reperfusion Injury (I / R) 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), Traf5 gene knockout mouse sham operation group (KO Sham) and I / R operation group R operation group (KO I / R), non-transgenic mouse sham operation group (NTGSham) and I / R operation group (NTG I / R), liver cell-specific Traf5 transgenic mouse sham operation group (TG Sham) and I / R surgery group (TG I / R).

[0062] (2) I / R surgery in the model of hepatic ischemia-reperfusion injury (using non-invasive vascular clips to clamp the portal vein and hepatic artery in the mid...

Embodiment 3

[0068] Example 3 Determination of liver necrosis area and liver function indexes (AST, ALT)

[0069] The evaluation indicators of the severity of liver ischemia-reperfusion injury mainly include the area of ​​liver necrosis and liver function indicators (AST, ALT), all of which are positively correlated with the severity of liver ischemia-reperfusion injury.

[0070] (1) Take materials

[0071] Mice in the sham operation group (Sham) and the ischemia / reperfusion group were collected at 1 h, 3 h, 6 h, and 24 h after operation, and sacrificed by cervical dislocation. Blood was collected 1 mL 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.

[0072] Separation of serum: the EP tube where the blood was collected was left at room temperature for...

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Abstract

The invention discloses an application of a TNF (tumor necrosis factor) receptor associated factor 5 (Traf5) to ischemia reperfusion injury, and belongs to the field of gene functions and applications. Liver cell specific Traf5 gene knock-out mice and Traf5 transgenic mice serve as experimental objects, functions of Traf5 genes are researched by an ischemia reperfusion injury model, and the Traf5 has the function of improving ischemia reperfusion injury according to discovery, so that the Traf5 has the following applications: an application of the Traf5 serving as a drug target to screening of drugs for preventing, relieving and / or treating ischemia reperfusion injury; an application of the Traf5 to preparation of the drugs for preventing, relieving and / or treating ischemia reperfusion injury, wherein screening of the drugs for preventing, relieving and / or treating ischemia reperfusion injury refers to screening of drugs capable of promoting Traf5 expression.

Description

technical field [0001] The invention belongs to the field of gene function and application, and relates to the application of TNF receptor-related factor 5 (Traf5) in hepatic ischemia-reperfusion injury, and specifically relates to the application of Traf5 in screening or preparing to prevent, alleviate and / or treat hepatic ischemia-reperfusion Application in injury medicine. Background technique [0002] Hepatic ischemia-reperfusion injury (ischemia reperfusion injury, IRI) is the hepatic ischemia caused by hepatic blood flow occlusion or extracorporeal perfusion of the liver donor during liver surgery or liver transplantation. Physiological and pathological process of aggravated liver injury and even dysfunction [1] . Hepatic IR is a complex process involving multiple cell types and molecular mediators of different pathways. Cell injury can occur during ischemia and reperfusion, eventually leading to cell death through apoptosis and necrosis. Warm ischemic injury occur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K49/00A61K48/00A61P1/16A61P9/10C12Q1/52C12Q1/02
CPCA61K48/005A61K49/0008C12Q1/52G01N33/5008
Inventor 李红良王晓占毛文哲
Owner WUHAN UNIV
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