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Olfm4 in nonalcoholic fatty liver disease (NAFLD)

A specific, animal model technology, applied in the field of therapeutic drugs, construction of NAFLD animal models, and preparation of detection reagents

Active Publication Date: 2021-12-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research report on the relationship between OLFM4 and non-alcoholic fatty liver disease. This technical plan firstly proposes that OLFM4 may be related to NAFLD, and through preliminary experiments, it is found that the expression level of OLFM4 is significantly up-regulated in NAFLD animal and cell models, suggesting that OLFM4 plays an important role in NAFLD. Potential modulatory effects in

Method used

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  • Olfm4 in nonalcoholic fatty liver disease (NAFLD)
  • Olfm4 in nonalcoholic fatty liver disease (NAFLD)
  • Olfm4 in nonalcoholic fatty liver disease (NAFLD)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: NAFLD animal model based on OLFM4 gene knockout background

[0051] The construction method of Olfactomedin-4 (OLFM4) gene knockout animal model in nonalcoholic fatty liver disease (NAFLD) is realized through the following steps:

[0052] Step 1: Construct NAFLD animal model;

[0053] Step 2: Construct OLFM4 gene knockout animal model;

[0054] Step 3: Based on the OLFM4 gene knockout background NAFLD animal model;

[0055] Step 4: Verify the successful construction of the OLFM4 gene knockout animal model in NAFLD.

[0056] details as follows:

[0057] Step 1-1: Randomize 8-week-old wild-type C57BL / 6J mice into groups as follows:

[0058] Normal diet group (SCD): wild-type C57BL / 6J mice, normal drinking water, normal diet;

[0059] High-fat diet group (HFD): wild-type C57BL / 6J mice, normal drinking water, including high-fat diet for 4 weeks, 8 weeks, 12 weeks.

[0060] Wherein, the high-fat feed used: purchased from Research Diets, article number D12492...

Embodiment 2

[0076] Example 2: Construction of OLFM4 gene knockout animal model

[0077] Step 2-1: Design mouse OLFM4 gene CRISPR target and select highly active gRNA target.

[0078] According to the protein conservation region and genome structure of the target gene OLFM4, design the CRISPR target on exon7 or exon4 of the transcript OLFM4-201, such as figure 2 The location indicated by the arrow. According to the target, design the corresponding saCas9 CRISPR target sequence as follows:

[0079] sequence name sequence PAM OLFM4-e4g1 GACTCCAGCTTCTCTACCAAAGAGGGT (SEQ ID NO. 1) GAGGGT OLFM4-e4g2 GACCCTCTTGGTAGAGAAGCTGGAGT (SEQ ID NO. 2) TGGAGT OLFM4-e7g1 TGTGATTCAGCTCAACTGGCTGGGGT (SEQ ID NO. 3) TGGGGT OLFM4-e7g2 GGTTCTCTCTGGATGCTGAGGAGAGT (SEQ ID NO. 4) GAGAGT OLFM4-e7g3 CACCCAGTACAGGGTTTCTCTCTGGAT (SEQ ID NO. 5) CTGGAT

[0080] The gRNA target activity of OFM4 was detected using the saCas9 / gRNA Target Efficiency Detection K...

Embodiment 3

[0124] Example 3: Application of OLFM4 as a detection target for NAFLD.

[0125] Step 1: After feeding C57BL / 6J mice with a high-fat diet in Step 1 of Example 1 for 8 weeks, extract liver tissue RNA, and detect the expression of OLFM4 mRNA in mouse liver by qPCR. The primer sequences required for detection are as follows:

[0126] F (SEQ ID NO. 9): GTTAGGGTGAAGGAGGAATGA

[0127] R (SEQ ID NO. 10): CTCCTAGACTTCCCTGAAGC

[0128] The result is as Figure 7 As shown in A, the expression of OLFM4 in the liver of mice fed a high-fat diet was significantly upregulated at the transcriptional level.

[0129] Step 2: Human liver cell line HepG2 cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were subcultured with DMEM medium (purchased from Gibco, USA) containing 10% calf serum and 1% penicillin-streptomycin until the cells reached 70% to 80% fusion.

[0130] Step 3: Stimulate the cells with palmitic acid (PA) or free fatty acid (FFA) to construct a NAFLD cell...

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Abstract

The invention discloses an application of OLFM4 in non-alcoholic fatty liver disease (NAFLD), specifically including the application of OLFM4 in the preparation of NAFLD detection reagents and the application of OLFM4 in the preparation of NAFLD treatment drugs, and the expression change of OLFM4 has a regulating effect on NAFLD , if the therapeutic drug contains human OLFM4 sequence recombinant protein to specifically up-regulate liver OLFM4 expression, it can alleviate liver steatosis in NAFLD patients. The invention also discloses an animal model of NAFLD. The OLFM4 gene is knocked out in the animal model, aiming to solve the deficiency of the prior art on the function and evaluation method of OLFM4 in the detection and treatment of NAFLD occurrence and development. The present invention provides a research method and application of OLFM4 in NAFLD, which is convenient for in-depth analysis of the role of OLFM4 in NAFLD. At the same time, the application of the technical solution facilitates the elucidation of the mechanism of action of OLFM4, and provides a new perspective for the study of OLFM4.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically the application of OLFM4 in Olfactomedin-4 (OLFM4) in non-alcoholic fatty liver disease (NAFLD), specifically including the preparation of detection reagents, therapeutic drugs and the construction of NAFLD animal models. Background technique [0002] Nonalcoholic fatty liver disease (NAFLD) refers to a clinicopathological syndrome characterized by hepatic steatosis and lipid accumulation caused by alcohol and other definite liver damage factors. In recent years, with the changes in people's living habits and dietary structure, the prevalence of NAFLD in adults in my country has increased rapidly, reaching 29.2%, and even higher than 30% in some developed countries in the West. It has become the most common clinical chronic liver disease. According to pathological changes and clinical manifestations, the natural history of NAFLD can be divided into simple NAFLD and nonalcoholic steatohep...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883A61K48/00A61K38/17A61P1/16A01K67/027
CPCC12Q1/6883A61K48/005A61K38/1709A61P1/16A01K67/0276C12Q2600/158A01K2217/075A01K2227/105A01K2267/03
Inventor 徐承富王馨雨陈盛晖
Owner ZHEJIANG UNIV