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Method for building lipopolysaccharide induced piglet liver cell programmed necrosis model

A technology of programmed necrosis and liver cells, which is applied in the field of establishment of lipopolysaccharide-induced programmed necrosis model of piglet liver cells, can solve the problems of cumbersome steps and achieve good repeatability, reliable measurement results, and simple and easy operation methods

Inactive Publication Date: 2018-04-20
WUHAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, inducers commonly used to induce programmed necrosis include fusion proteins, virus transfection systems, and millet grain protein, but they involve complex inducer preparation processes, including many steps such as constructing vectors, transfection, and induction, as well as desalination. , cation exchange chromatography, gel chromatography and other cumbersome steps to obtain the preparation process of purified protein, therefore, the use of such inducers to construct the programmed cell necrosis model has the disadvantage of cumbersome steps

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  • Method for building lipopolysaccharide induced piglet liver cell programmed necrosis model
  • Method for building lipopolysaccharide induced piglet liver cell programmed necrosis model
  • Method for building lipopolysaccharide induced piglet liver cell programmed necrosis model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Experimental Design and Tissue Sample Collection

[0053] (1) Select 12 Duroc×Landrace×Large White weaned piglets (8.9±0.2kg, 35±1d), and randomly distribute them to 2 treatment groups (control group and treatment group) according to the principle of similar body weight, each treatment group 6 repetitions. The test piglets were raised in a 1.80×1.10m 2 Stainless steel cages were kept at a temperature of 22-25°C during the feeding period, and food and water were provided ad libitum. After feeding for 10 days, the treatment group was injected with 100 μg of lipopolysaccharide (LPS, Escherichia coli serotype O55: B5, purity >99%, Sigma Company) per kg body weight of piglets, and the control group was injected with an equivalent amount of 0.9% normal saline (injection Stop feeding 12h before).

[0054] (2) 4 hours after the test piglets were injected with LPS (treatment group) or normal saline (control group) peritoneally, 10 mL of blood was collected from t...

Embodiment 2

[0056] Example 2 Effect of LPS stimulation on the morphology and structure of piglet liver tissue

[0057] The fresh liver tissue dissected out after slaughter was fixed in 4% paraformaldehyde for more than 24 hours, and the effect of LPS stimulation on the morphology and structure of liver tissue was observed by using a high-definition color pathology graphic report analysis system. The specific method is as follows:

[0058] (1) Take the liver tissue out of the fixative, use a scalpel in the fume hood to flatten the tissue at the target site, put the trimmed tissue and the corresponding label in the dehydration box, and put the dehydration box into the hanging basket Dehydration in the dehydrator with gradient alcohol: 75% alcohol (4h), 85% alcohol (2h), 90% alcohol (2h), 95% alcohol (1h), absolute ethanol I (30min), absolute ethanol II (30min), alcohol benzene (5-10min), xylene I (5-10min), xylene II (5-10min), wax I (1h), wax II (1h), wax III (1h).

[0059] (2) Embed the ...

Embodiment 3

[0064] Example 3 Effect of LPS stimulation on the ultrastructure of piglet liver cells

[0065] The fresh liver tissue dissected after slaughter was fixed in 2.5% glutaraldehyde for more than 4 hours, and Tecnai G was used to 2 20 TWIN transmission electron microscope to observe the effect of LPS stimulation on the ultrastructure of liver cells, the specific method is as follows:

[0066] (1) Cut 2mm 3 The liver samples were added with 2.5% glutaraldehyde, stored and fixed at 4°C for 4 hours, rinsed three times with 0.1M phosphate buffer (pH value 7.4), 15min each time; then pre-cooled at 4°C 1% osmic acid, fixed at 20°C for 2-3 hours; then rinsed with 0.1M phosphate buffer (pH 7.4) for 3 times, 15 minutes each time.

[0067] (2) Dehydrate the liver sample treated in step (1) through graded alcohol (30%, 50%, 70%, 80%, 85%, 90%, 95%, 100% twice), each time for 15-20min . Then the dehydrated liver tissue was infiltrated in a 37°C incubator. The infiltrating agent was aceton...

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Abstract

The invention discloses a method for building a lipopolysaccharide induced piglet liver cell programmed necrosis model. The method includes: performing lipopolysaccharide induction on a test piglet toobtain a to-be-measured piglet; sampling blood from the precaval vein of the to-be-measured piglet, separating serum, measuring biochemical indexes in the serum, and judging the influence of the lipopolysaccharide induction on the liver function of the piglet; taking the liver sample of the to-be-measured piglet, observing the shape structure of liver tissue and the ultrastructure of liver cells,measuring the key genes of liver cell programmed necrosis and the protein expression of the key genes, and analyzing the effect of the lipopolysaccharide induction on the piglet liver cell programmednecrosis. The method has the advantages that lipopolysaccharides are used as the induction agent, the liver cells are used as the model cells of the cell programmed necrosis model, and the piglet isused as the modeling object to successfully build the liver cell programmed necrosis model, and the method is simple and practical to operate; by measuring the key genes of the liver cell programmed necrosis and the protein expression of the key genes, the method is reliable in measuring result and good in repeatability.

Description

technical field [0001] The invention relates to the technical field of cell programmed necrosis research, in particular to a method for establishing a lipopolysaccharide-induced programmed necrosis model of piglet liver cells. Background technique [0002] Necroptosis is a caspase-independent mode of cell death mediated by death receptors that usually occurs when apoptosis is inhibited, and dying cells have pronounced necrosis feature. Studies have shown that programmed cell necrosis plays an important role in tissue (such as liver) damage or necrosis caused by various factors such as ischemia-reperfusion and inflammatory reactions, and effective inhibition of programmed cell necrosis has a positive effect on tissue damage or necrosis induced by these factors. Important preventive and therapeutic effects. [0003] At present, inducers commonly used to induce programmed necrosis include fusion proteins, virus transfection systems, and millet grain protein, but they involve ...

Claims

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Application Information

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IPC IPC(8): A01K67/027
CPCA01K67/027A01K2207/20A01K2227/108A01K2267/03
Inventor 刘玉兰康萍汪龙梅朱惠玲王秀英
Owner WUHAN POLYTECHNIC UNIVERSITY
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