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Simulation method of chronic atrophic gastritis lesion and mouse modeling identification method

A technology of atrophic gastritis and simulation methods, applied in the field of life sciences, can solve problems such as unstable modeling, expensive intervention reagents, and lack of inflammation in the gastric mucosa

Active Publication Date: 2022-01-18
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, DMP-777, L635 intervention methods, intervention reagents are expensive and difficult to obtain; tamoxifen modeling not only lacks inflammation of the gastric mucosa, but also recovers on its own after 3 weeks, and cannot form a stable model; Hp is a class I carcinogen defined by WHO , is the most common inducing factor for the formation of CAG and even gastric cancer, but its molecular target is still unclear, and the model has certain limitations in terms of modeling cycle, pathway and biosafety

Method used

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  • Simulation method of chronic atrophic gastritis lesion and mouse modeling identification method
  • Simulation method of chronic atrophic gastritis lesion and mouse modeling identification method
  • Simulation method of chronic atrophic gastritis lesion and mouse modeling identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of Gastric Parietal Cell-specific GRIM-19 Gene Knockout Mouse Model

[0041] 1. Construct the GRIM-19 gene knockout vector, such as figure 1 As shown in A, the GRIM-19 gene knockout vector was constructed by using the conditional gene targeting method, that is, the GRIM-19 target vector contains a LoxP site, and the LoxP-neo fragment replaces the third exon of the GRIM-19 gene, which is equivalent to Knockout of exon 3.

[0042] 2. Construct GRIM-19 gene knockout mice. After the GRIM-19 gene knockout vector is constructed, electroporation is used to transfect the GRIM-19 gene knockout vector into mouse embryonic stem cells, and then, the Embryonic stem cells of GRIM-19 gene knockout vector were injected into embryo sacs of C57 / B6 background, implanted into the uterus of pseudopregnant mother mice, and chimeric mice were generated, and chimeric mice were crossed with flp mice to knock out NEO The resistance gene was used to generate the F1 genera...

Embodiment 2

[0058] Example 2 Analysis of expression of GRIM-19 in mouse gastric mucosal tissue

[0059] The expression of GRIM-19 in mouse gastric mucosa was analyzed by Western blot and immunofluorescence techniques.

[0060] Western blotting: extract mouse gastric mucosal tissue protein, lyse with Western and IP cell lysate (Beiyuntian), homogenate, lyse on ice for at least 30 minutes, centrifuge at 12000g 4°C for 3 minutes, collect supernatant into Ep tubes, and take part Use the BCA protein concentration determination kit (Biyuntian) to determine the protein concentration, add the corresponding volume of SDS-PAGE protein loading buffer (5×), mix well, heat at 100°C for 10min, use immediately after centrifugation or store in a -80°C refrigerator . SDS-PAGE electrophoresis, gel preparation and sample loading, using 12.5% ​​SDS-PAGE separating gel and 5% SDS-PAGE stacking gel, running the stacking gel at 80V constant pressure, and running the separating gel at 120V constant pressure unt...

Embodiment 3

[0063] Example 3 HE staining analysis of mouse gastric mucosa tissue

[0064]Mice in the 8-month-old control group, heterozygous group, and homozygous group were harvested to obtain mouse gastric mucosal tissues. (1) Fix tissue samples with 4% paraformaldehyde solution for at least 24 hours. (2) After dehydration with different concentrations of ethanol, xylene becomes transparent. After embedding in paraffin, slice into 4 μm slices, and bake the slices at 60°C for 24-48h. (3) Dewaxing: Xylene I dewaxed for 15 minutes, and Xylene II dewaxed for 15 minutes. (4) Hydration: 100% ethanol for 5 minutes, 95% ethanol for 3 minutes, 80% ethanol for 3 minutes, 75% ethanol for 3 minutes, rinse with tap water for 2 minutes, and drain the water. (5) Soak in hematoxylin dye solution for 3 minutes, and rinse with tap water for 1 minute. (6) Soak in 1% hydrochloric acid ethanol for 3-5 seconds, and rinse with tap water for 1 minute. (7) Soak in saturated lithium carbonate for 5-10 second...

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Abstract

The invention provides a simulation method of chronic atrophic gastritis lesion. The simulation method comprises the following steps: (1) adopting a metamorphic lesion stage as a simulation object; (2) adopting a simulation form of spasmolysis polypeptide expression metaplasia; and (3) conditionally knocking out a GRIM-19 gene from gastric mucosa wall cells. Initialized response after gastric mucosa damage is successfully simulated, IM and even gastric cancer can be developed under continuous stimulation of chronic inflammation, simulation of pathology formation provides a basis for research on early prevention and control of intestinal gastric cancer and effective restraint of precancerous lesions of gastric cancer, a research basis can be provided for screening and development of drugs for preventing and treating CAG, and an important experimental tool is provided for anti-inflammatory and anti-cancer drug tests. The method has the characteristics of high simulation degree, stability, durability, specific phenotype and high efficiency.

Description

technical field [0001] The invention belongs to the field of life sciences, in particular to a method for simulating chronic atrophic gastritis lesions. Background technique [0002] Gastric cancer is a malignant tumor of the digestive tract with a high incidence in my country, among which intestinal type gastric cancer is the most common subtype of gastric cancer, which seriously threatens human health. Chronic atrophic gastritis (CAG) is recognized as a precancerous lesion of intestinal type gastric cancer. There are many theories about the formation of gastric cancer. In 1975, Correa proposed a gradual evolution model: normal gastric mucosa-chronic superficial gastritis- Chronic Atrophic Gastritis (CAG)-intestinal metaplasia-dysplasia-gastric cancer, the accumulation of many gene and molecular changes is closely related to this evolution process. CAG is a precancerous disease of gastric cancer, especially those associated with intestinal metaplasia and dysplasia. CAG in ...

Claims

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Application Information

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IPC IPC(8): A01K67/02C12N15/85C12N15/89C12N15/12C12Q1/6888
CPCC12N15/8509C12N15/89C12Q1/6888C07K14/47A01K67/0275C12N2015/8536A01K67/0276A01K67/0271A01K67/027A01K2207/12A01K2217/075A01K2227/105A01K2267/0331
Inventor 黄轶曾欣冯金梅徐小惠
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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